EP1194593A2 - Amplification d'acides nucleiques avec detection electronique - Google Patents

Amplification d'acides nucleiques avec detection electronique

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Publication number
EP1194593A2
EP1194593A2 EP00950511A EP00950511A EP1194593A2 EP 1194593 A2 EP1194593 A2 EP 1194593A2 EP 00950511 A EP00950511 A EP 00950511A EP 00950511 A EP00950511 A EP 00950511A EP 1194593 A2 EP1194593 A2 EP 1194593A2
Authority
EP
European Patent Office
Prior art keywords
probe
sequence
probes
preferred
target
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00950511A
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German (de)
English (en)
Inventor
Jon Falz Kayyem
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Clinical Micro Sensors Inc
Original Assignee
Clinical Micro Sensors Inc
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Filing date
Publication date
Application filed by Clinical Micro Sensors Inc filed Critical Clinical Micro Sensors Inc
Publication of EP1194593A2 publication Critical patent/EP1194593A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry

Definitions

  • the present invention is a continuing application of U.S.S.N.s 60/144,698, filed July 20, 1999; 09/238,351 , filed January 27, 1999; 09/014,304, filed January 27, 1998; 60/073,011 , filed January 29, 1998; 60/028,102, filed March 16, 1998; 60/084,425, filed May 6, 1998; 60/084,509, filed May 6, 1998; and 09/135,183, filed August 17, 1998, all of which are expressly incorporated herein in their entirety.
  • the invention relates to compositions and methods useful in the detection of nucleic acids using a variety of amplification techniques, including both signal amplification and target amplification. Detection proceeds through the use of an electron transfer moiety (ETM) that is associated with the nucleic acid, either directly or indirectly, to allow electronic detection of the ETM using an electrode.
  • ETM electron transfer moiety
  • Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
  • a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)).
  • the requirement for sensitivity i.e. low detection limits
  • PCR polymerase chain reaction
  • other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis as outlined below (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
  • Target amplification involves the amplification (i.e. replication) of the target sequence to be detected, resulting in a significant increase in the number of target molecules.
  • Target amplification strategies include the polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), and transcription mediated amplification (TMA).
  • amplification strategies include the ligase chain reaction (LCR), cycling probe technology (CPT), InvaderTM, Q-beta replicase (QBR), and the use of "amplification probes” such as "branched DNA” that result in multiple label probes binding to a single target sequence.
  • LCR ligase chain reaction
  • CPT cycling probe technology
  • QBR Q-beta replicase
  • PCR polymerase chain reaction
  • PCR there are a number of variations of PCR which may also find use in the invention, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or “AP-PCR” , “immuno-PCR”, “Alu-PCR”, “PCR single strand conformational polymorphism” or “PCR- SSCP”, “reverse transcriptase PCR” or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”. “panhandle PCR”, and “PCR select cDNA subtration”, among others.
  • SDA Strand displacement amplification
  • Nucleic acid sequence based amplification is generally described in U.S. Patent No. 5,409,818; Sooknanan et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp. 261-285) of Molecular Methods for Virus Detection, Academic Press, 1995; and "Profiting from Gene-based
  • TMA Transcription mediated amplification
  • Cycling probe technology is a nucleic acid detection system based on signal or probe amplification rather than target amplification, such as is done in polymerase chain reactions (PCR). Cycling probe technology relies on a molar excess of labeled probe which contains a scissile linkage of RNA. Upon hybridization of the probe to the target, the resulting hybrid contains a portion of RNA:DNA. This area of RNA:DNA duplex is recognized by RNAseH and the RNA is excised, resulting in cleavage of the probe. The probe now consists of two smaller sequences which may be released, thus leaving the target intact for repeated rounds of the reaction. The unreacted probe is removed and the label is then detected. CPT is generally described in U.S. Patent Nos. 5,011 ,769, 5,403,711 , 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416, and WO
  • LCR ligation chain reaction
  • QBR Q-beta replicase
  • InvaderTM technology is based on structure-specific polymerases that cleave nucleic acids in a site- specific manner. Two probes are used: an "invader” probe and a “signalling” probe, that adjacently hybridize to a target sequence with a non-complementary overlap. The enzyme cleaves at the overlap due to its recognition of the "tail”, and releases the "tail” with a label. This can then be detected.
  • the InvaderTM technology is described in U.S. Patent Nos. 5,846,717; 5,614,402; 5,719,028; 5,541 ,311 ; and 5,843,669, all of which are hereby incorporated by reference.
  • Rolling circle amplification is based on extension of a circular probe that has hybridized to a target sequence. A polymerase is added that extends the probe sequence. As the circular probe has no terminus, the polymerase repeatedly extends the circular probe resulting in concatamers of the circular probe. As such, the probe is amplified. Rolling-circle amplification is generally described in Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991 ) Proc. Natl. Acad. Sci. USA 88:189-193; Lizardi et al. (1998) Nat. Genet. 19:225-232; Zhang et al., Gene 211 :277 (1998); and
  • Branched DNA signal amplification relies on the synthesis of branched nucleic acids, containing a multiplicity of nucleic acid "arms” that function to increase the amount of label that can be put onto one probe. This technology is generally described in U.S. Patent Nos. 5,681 ,702, 5,597,909, 5,545,730,
  • the present invention provides methods for detecting a target nucleic acid sequence.
  • the methods comprise hybridizing at least a first primer nucleic acid to the target sequence to form a first hybridization complex, and contacting the first hybridization complex with a first enzyme to form a modified first primer nucleic acid.
  • the first hybridization complex is then disassociated. These steps may be repeated a plurality of times.
  • a first assay complex is then formed comprising at least one ETM and the modified first primer nucleic acid.
  • the assay complex is covalently attached to an electrode. Electron transfer is then detected between the ETM and the electrode as an indication of the presence of the target sequence.
  • the method can include the same method on a second target sequence substantially complementary to the the first target sequence.
  • the method utilizes a DNA polymerase and the modification to the primer is an extension of the primer such that the polymerase chain reaction (PCR) occurs.
  • PCR polymerase chain reaction
  • the method utilizes a ligase and the modification to the primer comprises a ligation of the first primer which hybridizes to a first domain of the first target sequence to a third primer which hybridizes to a second adjacent domain of the first target sequence, such that the ligase chain reaction (LCR) occurs.
  • LCR ligase chain reaction
  • the method utilizes a first primer comprising a first probe sequence, a first scissile linkage and a second probe sequence.
  • the enzyme will cleave the first scissile linkage resulting in the separation of the first and the second probe sequences and the disassociation of the hybridization complex while leaving the first target sequence intact, such that the cycling probe technology (CPT) reaction occurs.
  • CPT cycling probe technology
  • the method utilizes a first enzyme that is a polymerase that extends the first primer to form a modified first primer comprising a first newly synthesized strand, and said method further comprises the addition of a second enzyme comprising a nicking enzyme that nicks the extended first primer leaving the first target sequence intact.
  • the method additionally comprises extending from the nick using the polymerase, thereby displacing the first newly synthesized strand and generating a second newly synthesized strand, such that strand displacement amplification (SDA) occurs.
  • SDA strand displacement amplification
  • the method utilizes a first target sequence that is a RNA target sequence, a first primer nucleic acid that is a DNA primer comprising an RNA polymerase promoter, and the first enzyme is a reverse-transcriptase that extends the first primer to form a first newly synthesized DNA strand.
  • the method further comprises the addition of a second enzyme comprising an RNA degrading enzyme that degrades the first target sequence.
  • a third primer is then added that hybridizes to the first newly synthesized DNA strand.
  • a third enzyme is added comprising a DNA polymerase that extends the third primer to form a second newly synthesized DNA strand, to form a newly synthesized DNA hybrid.
  • a fourth enzyme is then added comprising an RNA polymerase that recognizes the RNA polymerase promoter and generates at least one newly synthesized RNA strand from the DNA hybrid, such that nucleic acid sequence-based amplification (NASBA) occurs.
  • NASBA nucleic acid sequence-based amplification
  • the invention provides methods for detecting a target nucleic acid sequence comprising forming a first hybridization complex comprising an amplifier probe and a target sequence, wherein the amplifier probe comprises at least two amplification sequences and hybridizing a first portion of at least one label probe to all or part of at least one amplification sequence.
  • a second portion of the label probe is then hybridized to a detection probe covalently attached to an electrode via a conductive oligomer to form a second hybridization complex that contains at least a first electron transfer moiety (ETM).
  • ETM electron transfer moiety
  • the invention provides methods for detecting a target nucleic acid sequence comprising forming a first hybridization complex comprising an amplifier probe and a target sequence, wherein the amplifier probe comprises at least two amplification sequences and wherein the first hybridization complex is covalently attached to an electrode comprising a monolayer comprising conductive oligomers.
  • At least one label probe comprising at least one electron transfer moiety (ETM) is hybridized to all or part of at least one amplification sequence, and the label probe is detected by measuring electron transfer between said first ETM and said electrode.
  • ETM electron transfer moiety
  • the invention provides kits for the detection of a first target nucleic acid sequence comprising at least a first nucleic acid primer substantially complementary to at least a first domain of the target sequence and at least a first enzyme that will modify the first nucleic acid primer.
  • the kits additionally comprise an electrode comprising at least one detection probe covalently attached to the electrode via a conductive oligomer.
  • the invention provides methods of detecting target sequences comprising providing a rolling circle probe (RCP) comprising a first ligation sequence substantially complementary to a first domain of said target sequence, a second ligation sequence substantially complementary to a second domain of said target sequence; and a priming sequence.
  • RCP rolling circle probe
  • the methods further comprise hybridizing the first ligation sequence to said first domain and the second ligation sequence to the second domain to form a first hybridization complex and ligating the first and second ligation sequences together.
  • a primer substantially complementary to said priming sequence, a polymerase, dNTPs and an ETM are added to the first hybridization complex under conditions whereby a rolling circle concatamer is formed, and the ETM is detected as an indicator of the presence of the target sequence.
  • the RCP may further optionally comprise a cleavage site and a capture sequence.
  • the invention provides methods for detecting a first target nucleic acid sequence comprising hybridizing an invader primer and a signaling primer to form a first hybridization complex.
  • the signaling primer comprises a first portion comprising a sequence that will hybridize to a first portion of the target sequence; a cleavage site and a detection sequence that does not hybridize with the target sequence.
  • the first hybridization complex is contacted with a structure specific cleavage enzyme such that the signaling primer is cleaved and the detection sequence is released.
  • the released detection sequence is contacted with an electrode comprising a capture probe to form a second hybridization complex, wherein the second hybridization complex comprises at least one ETM.
  • the ETM is detected as an indication of the presence of said target sequence.
  • Figures 1 A-10 depict depict a number of different compositions of the invention; the results are shown in Example 1 and 2.
  • Figure 1A depicts I, also referred to as P290.
  • Figure 1 B depicts II, also referred to as P291.
  • Figure 1 C depicts III, also referred to as W31.
  • Figure 1 D depicts IV, also referred to as N6.
  • Figure 1 E depicts V, also referred to as P292.
  • Figure 1 F depicts II, also referred to as C23.
  • Figure 1G depicts VII, also referred to as C15.
  • Figure 1 H depicts VIII, also referred to as C95.
  • Figure 1A depicts I, also referred to as P290.
  • Figure 1 B depicts II, also referred to as P291.
  • Figure 1 C depicts III, also referred to as W31.
  • Figure 1 D depicts IV, also referred to as N6.
  • Figure 1 E depicts V, also referred to as P292.
  • Figure 1 F depict
  • Figure 11 depicts Y63.
  • Figure 1J depicts another compound of the invention.
  • Figure 1 K depicts N11.
  • Figure 1 L depicts C131 , with a phosphoramidite group and a DMT protecting group.
  • Figure 1 M depicts W38, also with a phosphoramidite group and a DMT protecting group.
  • Figure 1 N depicts the commercially available moiety that enables "branching" to occur, as its incorporation into a growing oligonucleotide chain results in addition at both the DMT protected oxygens.
  • Figure 10 depicts glen, also with a phosphoramidite group and a DMT protecting group, that serves as a non-nucleic acid linker.
  • Figures 1A to 1G and 1 J are shown without the phosphoramidite and protecting groups (i.e. DMT) that are readily added.
  • Figure 2 depicts the synthetic scheme of a preferred attachment of an ETM, in this case ferrocene, to a nucleoside (in this case adenosine) via an oxo linkage to the ribose, forming the N6 compound of the invention.
  • ETM in this case ferrocene
  • nucleoside in this case adenosine
  • Figure 3 is similar to Figure 2 except that the nucleoside is cytidine, forming the W38 compound of the invention.
  • Figure 4 depicts the synthetic scheme of a preferred attachment of an ETM, in this case ferrocene, to a nucleoside via the phosphate, forming the Y63 compound of the invention.
  • Figure 5 depicts the synthetic scheme of a triphosphate nucleotide, in this case adenosine, with an attached ETM, in this case ferrocene, via an oxo linkage to the ribose.
  • Figure 6 depicts the use of an activated carboxylate for the addition of a nucleic acid functionalized with a primary amine to a pre-formed SAM.
  • Figure 7 depicts a schematic of the use of "universal" type gene chips, utilizing restriction endonuclease sites.
  • Figures 8A and 8B depicts two phosphate attachments of conductive oligomers that can be used to add the conductive oligomers at the 5' position, or any position.
  • Figure 9 depicts the synthesis of an insulator (C109) to the ribose of a nucleoside for attachment to an electrode.
  • Figure 10 depicts the synthetic scheme of ethylene glycol terminated conductive oligomers.
  • Figures 11 A, 11 B and 11 C depict the synthesis of three different "branch" points (in this case each using adenosine as the base), to allow the addition of ETM polymers.
  • Figure 11 A depicts the synthesis of the N17 compound of the invention.
  • Figure 11 B depicts the synthesis of the W90 compound, and
  • Figure 11C depicts the synthesis of the N38 compound.
  • Figure 12 depicts a schematic of the synthesis of simultaneous incorporation of multiple ETMs into a nucleic acid, using the N17 "branch" point nucleoside.
  • Figure 13 depicts a schematic of an alternate method of adding large numbers of ETMs simultaneously to a nucleic acid using a "branch" point phosphoramidite, in this case utilizing three branch points (although two branch points are also possible; see for example Figure 1 N) as is known in the art.
  • each end point can contain any number of ETMs.
  • Figure 14 shows a representative hairpin structure.
  • 500 is a target binding sequence
  • 510 is a loop sequence
  • 520 is a self-complementary region
  • 530 is substantially complementary to a detection probe
  • 530 is the "sticky end", that is, a portion that does not hybridize to any other portion of the probe, that contains the ETMs.
  • Figures 15A, 15B and 15C depict three preferred embodiments for attaching a target sequence to the electrode.
  • Figure 15A depicts a target sequence 120 hybridized to a capture probe 100 linked via a attachment linker 106, which as outlined herein may be either a conductive oligomer or an insulator.
  • the electrode 105 comprises a monolayer of passivation agent 107, which can comprise conductive oligomers (herein depicted as 108) and/or insulators (herein depicted as 109).
  • n is an integer of at least 1 , although as will be appreciated by those in the art, the system may not utilize a capture probe at all (i.e. n is zero), although this is generally not preferred.
  • Figure 15B depicts the use of a single capture extender probe 110 with a first portion 111 that will hybridize to a first portion of the target sequence 120 and a second portion that will hybridize to the capture probe 100.
  • Figure 15C depicts the use of two capture extender probes
  • the first capture extender probe 110 has a first portion 111 that will hybridize to a first portion of the target sequence 120 and a second portion 112 that will hybridize to a first portion 102 of the capture probe 100.
  • the second capture extender probe 130 has a first portion 132 that will hybridize to a second portion of the target sequence 120 and a second portion 131 that will hybridize to a second portion 101 of the capture probe 100.
  • any of these attachment configurations may be used with any of the other systems, including the embodiments of Figure 16.
  • Figures 16A, 16B, 16C, 16D, 16E, 16F and 16G depict some of the embodiments of the invention. All of the monolayers depicted herein show the presence of both conductive oligomers 108 and insulators 107 in roughly a 1 :1 ratio, although as discussed herein, a variety of different ratios may be used, or the insulator may be completely absent. In addition, as will be appreciated by those in the art, any one of these structures may be repeated for a particular target sequence; that is, for long target sequences, there may be multiple assay complexes formed. Additionally, any of the electrode- attachment embodiments of Figure 15 may be used in any of these systems.
  • Figures 16A, 16B and 16D have the target sequence 120 containing the ETMs 135; as discussed herein, these may be added enzymatically, for example during a PCR reaction using nucleotides modified with ETMs, resulting in essentially random incorporation throughout the target sequence, or added to the terminus of the target sequence.
  • Figure 16C depicts the use of two different capture probes 100 and 100", that hybridize to different portions of the target sequence 120. As will be appreciated by those in the art, the 5'-3' orientation of the two capture probes in this embodiment is different.
  • Figure 16C depicts the use of label probes 145 that hybridize directly to the target sequence 120.
  • Figure 16C shows the use of a label probe 145, comprising a first portion 141 that hybridizes to a portion of the target sequence 120, a second portion 142 comprising ETMs 135.
  • Figures 16E, 16F and 16G depict systems utilizing label probes 145 that do not hybridize directly to the target, but rather to amplifier probes that are directly (Figure 16E) or indirectly ( Figures 16F and 16G) hybridized to the target sequence.
  • Figure 16E utilizes an amplifier probe 150 has a first portion 151 that hybridizes to the target sequence 120 and at least one second portion 152, i.e. the amplifier sequence, that hybridizes to the first portion 141 of the label probe.
  • Figure 16F is similar, except that a first label extender probe 160 is used, comprising a first portion 161 that hybridizes to the target sequence 120 and a second portion 162 that hybridizes to a first portion 151 of amplifier probe 150.
  • a second portion 152 of the amplifier probe 150 hybridizes to a first portion 141 of the label probe 140, which also comprises a recruitment linker 142 comprising ETMs 135.
  • Figure 16G adds a second label extender probe 170, with a first portion 171 that hybridizes to a portion of the target sequence 120 and a second portion that hybridizes to a portion of the amplifier probe.
  • Figure 16H depicts a system that utilizes multiple label probes.
  • the first portion 141 of the label probe 140 can hybridize to all or part of the recruitment linker 142.
  • Figures 17A, 17B, 17C, 17D and 17E depict different possible configurations of label probes and attachments of ETMs.
  • the recruitment linker is nucleic acid; in Figures 17D and E, is is not.
  • A nucleoside replacement;
  • B attachment to a base;
  • C attachment to a ribose;
  • D attachment to a phosphate;
  • E metallocene polymer (although as described herein, this can be a polymer of other ETMs as well), attached to a base, ribose or phosphate (or other backbone analogs);
  • Figure 18 depicts an improvement utilizing a stem-loop probe. This can be desirable as it creates torsional strain on the surface-bound probe, which has been shown to increase binding efficiency and in some cases thermodynamic stability.
  • the surface bound probe comprises a capture probe 100, a first stem-loop sequence 550, a target binding sequence 560, and a second stem-loop sequence 570 that is substantially complementary to the first stem-loop sequence.
  • the target sequence 120 which can contain the ETMs 135 either directly or indirectly using a label probe 145, the effective concentration of the target at the surface increases.
  • Figures 19A-19AA depict some of the sequences used in Example 1.
  • Figures 20A - 20O depict representative scans from the experiments outlined in Example 1. Unless otherwise noted, all scans were run at initial voltage -0.11 V, final voltage 0.5 V, with points taken every 10 mV, amplitude of 0.025, frequency of 10 Hz, a sample period of 1 sec, a quiet time of 2 sec.
  • Figure 20A has a peak potential of 0.160 V, a peak current of 1.092 X 10 "8 A, and a peak A of 7.563 X 10 "10 VA.
  • Figure 20C has a peak potential of 0.190 V, a peak current of 2.046 X 10 '7 A, and a peak area of 2.046 X 10 "8 VA.
  • Figure 20d has a peak potential of 0.190 V, a peak current of 3.552 X 10 "8 A, and a peak A of 3.568 X 10 "9 VA.
  • Figure 20E has a peak potential of 0.190 V, a peak current of 2.3762
  • Figure 20F has a peak potential of 0.180 V, a peak current of 2.992 X 10 '8 A, and a peak area of 2.709 X 10 "9 VA.
  • Figure 20G has a peak potential of 0.150 V, a peak current of 1.494 X 10 "7 A, and a peak area of 1.1 X 10 "8 VA.
  • Figure 20H has a peak potential of 0.160 V, a peak current of 1.967 X 10 '8 A, and a peak area of 1.443 X 10 "9 VA.
  • Figure 20I has a peak potential of 0.150 V, a peak current of 8.031 X 10 "8 A, and a peak area of 6.033 X 10 "9 VA.
  • Figure 20J has a peak potential of 0.150 V, a peak current of 8.871 X 10 "9 A, and a peak area of 5.51 X 10 '10 VA.
  • Figure 20L has a peak potential of 0.140 V, a peak current of 2.449 X 10 '8 A, and a peak area of 1.706 X 10 -9 VA.
  • Figure 20M has a peak potential of 0.150 V, a peak current of 6.637 X 10 "8 A, and a peak area of 7.335 X 10 '9 VA.
  • Figure 20N has a peak potential of 0.140 V, a peak current of 2.877 X 10- 9 A, and a peak area of 2.056 X 10 "10 VA.
  • Figure 21 depicts the ligation chain reaction (LCR) experiment of Example 13.
  • Figures 22A and 22B depicts the results of Example 12.
  • the "hybrid code” refers to the system number; + and - refer to the presence or absence of the rRNA target.
  • Figures 23A, 23B, 23C, 23D, 23E and 23F depict the compositions and results of Example 13.
  • Figures 24A and 24B depict the compositions and results from Example 13.
  • FIGS 25A and 25B depict the set up of two of the experiments of Example 8.
  • Figure 26 shows the results of a PCR experiment as outlined in Example 9.
  • Figures 27A, 27B, 27C, 27D, 27E and 27F depict some "mechanism-1" detection systems.
  • Figure 27A shows portions of target sequence 120 hybridized to detection probes 300 linked via conductive oligomers 108 to electrode 105.
  • the hybridization complex comprises an ETM 135 that can be covalently linked either to the target sequence or the detection probe 300, or non-covalently (i.e. a hybridization indicator).
  • the monolayer is depicted with insulators 107, although as outlined herein, any type of passivation agent may be used.
  • Figure 27B depicts the use of capture probe 100 linked via attachment linker 106 to electrode 105, although as will be appreciated, since the label probe 145 hybridizes to the detection probe 300 this can serve as a type of capture probe.
  • a label probe 145 comprising a first portion 141 that hybridizes to a portion of the target sequence 120 and a second portion 142 that hybridizes to the detection probe 300.
  • Figure 27C depicts the use of branched amplifier probe 150 comprising a first portion 151 that hybridizes to the target sequence 120 (although label extender probes can be used as well) and amplification sequences 152 that hybridize directly to the detection probes 300.
  • the target sequence may be additionally attached to the electrode using capture probes as outlined in Figure 15.
  • Figure 27D depicts the same thing utilizing a linear amplification probe 150.
  • Figure 27E depicts a similar system, but uses label probes 145 comprising a first portion 141 that hybridizes to the amplification sequence 152 and a second portion 142 that hybridizes to a detection probe 300.
  • Figure 27F depicts the same thing but using a linear amplification probe 150.
  • Figure 28 depicts an LCR embodiment of the invention.
  • a first probe nucleic acid 200 and a second probe nucleic acid 210 are hybridized to a a first and second target domains of a single-stranded target sequence 120.
  • the probes are attached, generally through ligation, to form a ligated probe 220.
  • the first hybridization complex 225 is denatured, and the process is repeated, to generate a pool of ligated probes 220.
  • the unligated probes 200 and 210 are removed as is known in the art, and then the ligated probes 220 are hybridized to detection probes 300 and detected as outlined herein.
  • the target domains depicted are directly adjacent, i.e. contiguous, but gaps that are then filled using nucleotides and polymerase can also be used.
  • the probes are depicted with attached ETMs 135, although as will be appreciated, non-covalently attached ETMs may also be used.
  • Figure 29 depicts an alternate LCR embodiment of the invention.
  • a first probe nucleic acid 200 and a second probe nucleic acid 210 are hybridized to a a first and second target domains of a single- stranded target sequence 120.
  • a third probe nucleic acid 200 and a fourth probe nucleic acid 210 are hybridized to a third and fourth target domains of the complementary single-stranded target sequence 125.
  • the probes are attached, generally through ligation, to form ligated probes 220 and 221.
  • the hybridization complexes 225 and 226 are denatured, and the process is repeated to generate a pool of ligated probes 220 and 221.
  • the unligated probes 200, 201, 210 and 211 are removed as is known in the art, and then the ligated probes 220 and 221 are hybridized to detection probes 300 and detected as outlined herein.
  • the target domains depicted are directly adjacent, i.e. contiguous, but gaps that are then filled using nucleotides and polymerase can also be used.
  • the probes are depicted with attached ETMs, although as will be appreciated, non- covalently attached ETMs may also be used.
  • Figure 30 depicts a preferred CPT embodiment of the invention;
  • Figure 30 depicts a "mechanism-1 " system, but as will be appreciated by those in the art, "mechanism-2" systems may be used as well.
  • a primary probe 1 comprising a first probe sequence 10, a scissile linkage 11 , and a second probe sequence 12, with two covalently attached ETMs 13, is added to a target sequence 120, to form a hybridization complex, 6. While Figure 30 depicts two ETMs, only one of the probe sequences may be covalently labeled with an ETM. Furthermore, additional probe sequences, and additional scissile linkages, may also be used.
  • the scissile linkage 11 Upon subjection to cleavage conditions, the scissile linkage 11 is cleaved, leaving the two probe sequences 10 and 12 hybridized to the target sequence 120. These probe sequences then disassociate, leaving the probe sequences 10 and 12, and the target sequence 120. The target sequence is then free to hybridize with additional primary probes 1 , and the reaction is repeated, generating a pool of probe sequences.
  • the uncleaved primary probes are removed as outlined herein, and the pool of probe sequences is added to an electrode 105, with an optional layer of passivation agent 107, and detection probes 300 and 302 covalently attached via conductive oligomers 108. The detection probes and the probe sequences form a hybridization complex, 30.
  • the detection probes may be mixed on the electrode, or may be at separate addresses 101 and 102. Additional ETMs in the form of hybridization indicators may optionally be added. Electron transfer is then initiated as is outlined herein. In addition, while this figure depicts a soluble reaction, the primary probes may be bound to a solid support as is described herein.
  • Figure 31 depicts an additional CPT embodiment utilizing a single capture sequence.
  • a primary probe 1 comprising a first capture sequence 14, first probe sequence 10, a scissile linkage 11, and a second probe sequence 12, with a covalently attached ETM 135, is added to a target sequence 124, to form a hybridization complex, 6.
  • the capture sequence 14 does not hybridize to the target, although it can.
  • Figure 31 depicts only one covalently attached ETM, the other probe sequence 12 may be covalently labeled with an ETM.
  • additional probe sequences, and additional scissile linkages may also be used.
  • the scissile linkage 11 Upon subjection to cleavage conditions, the scissile linkage 11 is cleaved, leaving the two probe sequences 10 and 12 hybridized to the target sequence 120. These probe sequences then disassociate, leaving the probe sequences 14-10 and 12, and the target sequence 5. The target sequence is then free to hybridize with additional primary probes 1 , and the reaction is repeated, generating a pool of probe sequences.
  • the uncleaved primary probes are removed as outlined herein, and the pool of probe sequences is added to an electrode 105, with an optional layer of passivation agent 107, and a detection probe, comprising the substantial complement of the capture sequence 24 and the substantial complement of the first probe sequence 20, covalently attached via conductive oligomers 108.
  • detection probes 304 that only comprise probes for the capture sequence.
  • the detection probes and the probe sequences form a hybridization complex, 30. Additional ETMs in the form of hybridization indicators may optionally be added. Electron transfer is then initiated as is outlined herein.
  • this figure depicts a soluble reaction, the primary probes may be bound to a solid support as is described herein.
  • Figure 32 depicts the use of two capture sequences in a CPT aplication.
  • a primary probe 1 comprising a first capture sequence 14, a first probe sequence 10, a scissile linkage 11 , a second probe sequence 12, and a second capture sequence 15, with two covalently attached ETMs 135, is added to a target sequence 120, to form a hybridization complex, 6.
  • the capture sequence 14 does not hybridize to the target, although it can.
  • Figure 32 depicts two covalently attached ETMs, there may be only one or none.
  • additional probe sequences, and additional scissile linkages may also be used.
  • the scissile linkage 11 Upon subjection to cleavage conditions, the scissile linkage 11 is cleaved, leaving the two probe sequences with associated capture sequences, 14-10 and 12-15 hybridized to the target sequence 120. These probe sequences then disassociate, leaving the probe/capture sequences 14-10 and 12-15, and the target sequence 120. The target sequence is then free to hybridize with additional primary probes 1 , and the reaction is repeated, generating a pool of probe sequences.
  • the uncleaved primary probes is neutralized by the addition of a substantially complementary neutralization probe 40, comprising sequences that are the substantial complement of the first capture sequence 24, the first probe sequence 20, the scissile linkage 25, the second probe sequence 22, and the second capture sequence 26.
  • the complementary scissile linkage 25 is only required in those embodiments that utilize nucleic acid scissile linkages.
  • the neutralization probe 40 may also be bound to the electrode.
  • the pool of probe sequences is added to an electrode 105, with an optional layer of passivation agent 107, and detection probes, comprising the substantial complement of the capture sequence 24 and the substantial complement of the first probe sequence 20, and the substantial complement of the capture sequence 22 and the substantial complement of the first probe sequence 26, covalently attached via conductive oligomers 108.
  • the detection probes and the probe sequences form a hybridization complex, 30. Additional ETMs in the form of hybridization indicators may optionally be added. Electron transfer is then initiated as is outlined herein.
  • the primary probes may be bound to a solid support as is described herein.
  • Figure 33 depicts a similar reaction, where a separation sequence 210 is used to remove the uncleaved scissile probe, in this case a primary probe 1.
  • a primary probe 1 comprising a first probe sequence 10, a first scissile linkage 11 , a separation sequence 210, a second scissile linkage 11 , and a second probe sequence 12, with two covalently attached ETMs 13, is added to a target sequence 120, to form a hybridization complex, 6.
  • Other configurations include those depicted. While Figure 33 depicts two ETMs, only one of the probe sequences may be covalently labeled with an ETM. Furthermore, additional probe sequences, and additional scissile linkages, may also be used.
  • the scissile linkage 11 Upon subjection to cleavage conditions, the scissile linkage 11 is cleaved, leaving the two probe sequences 10 and 12 and the separation sequence 210 hybridized to the target sequence 120.
  • the separation sequence does not hybridize to the target, for example when it is at a terminus of the probe; this may be preferred as it allows generic "separation beads" to be used with any target.
  • These probe sequences then disassociate, leaving the probe sequences 10 and 12, and separation sequence 210, and the target sequence 120. The target sequence is then free to hybridize with additional primary probes 1, and the reaction is repeated, generating a pool of probe sequences.
  • the uncleaved primary probes are removed by the addition of a solid support bead 200, with the substantial complement of the separation sequence 212 attached, generally via a linker. This bead then binds up the uncleaved probe 1 and the cleaved separation sequences 210, leaving only cleaved probe sequences in solution.
  • the pool of probe sequences is added to an electrode 105, with an optional layer of passivation agent 107, and detection probes 300 and 302 covalently attached via conductive oligomers 108.
  • the detection probes and the probe sequences form a hybridization complex, 30.
  • the detection probes may be mixed on the electrode, or may be at separate addresses 101 and 102. Additional ETMs in the form of hybridization indicators may optionally be added. Electron transfer is then initiated as is outlined herein.
  • Figure 34 depicts the use of solid-support bound primary scissile probes.
  • a solid support bead 200 with attached primary probes 1 is added to the target sequence 120, to form a hybridization complex, 6.
  • the primary probes may comprise an optional additional sequence 16 (which may be used to stabilize the first scissile linkage hybrid, if necessary), a first scissile linkage 11 , a first probe sequence 10, a second scissile linkage 11 , and a second probe sequence 12. While Figure 34 does not utilize covalently attached ETMs, one or more of the probe sequences may be so labelled. Furthermore, additional probe sequences, and additional scissile linkages, may also be used.
  • the scissile linkage 11 Upon subjection to cleavage conditions, the scissile linkage 11 is cleaved, leaving the two probe sequences 10 and 12 hybridized to the target sequence 120. These probe sequences then disassociate, leaving the probe sequences 10 and 12, and the target sequence 120. The target sequence is then free to hybridize with additional primary probes 1 , and the reaction is repeated, generating a pool of probe sequences.
  • the uncleaved primary probes are removed by removing the beads, and the pool of probe sequences is added to an electrode 105, with an optional layer of passivation agent 107, and detection probes 300 and 302 covalently attached via conductive oligomers 108.
  • the detection probes and the probe sequences form a hybridization complex, 30.
  • the detection probes may be mixed on the electrode, or may be at separate addresses 101 and 102. ETMs in the form of hybridization indicators are then added, and electron transfer is then initiated as is outlined herein.
  • Figure 35 depicts the use of bead-bound primary and secondary probes. Two (or more) types of beads are used.
  • the first type is a solid support bead 250 with attached secondary scissile probes 2 comprising a first scissile linkage 11 , a first secondary probe sequence 60, a second scissile linkage
  • a second secondary scissile probe may be preferably used, comprising a first scissile linkage 11 , a first secondary probe sequence 80, a second scissile linkage 11, and a second secondary probe sequence 90. These are depicted in Figure 35 as being on the same bead, although two sets of beads may be preferably used.
  • the primary probe beads 260 have attached primary probes 1 comprising a first scissile linkage 11 , a first probe sequence 10, a second scissile linkage 11 , and a second probe sequence 12.
  • the probes may contain optional additional sequences (depicted herein as 16), or additional probe sequences or scissile linkages.
  • the beads 250 and 260 are added to the target sequence 120.
  • the primary probes form a hybridization complex, 6, with the target 120. While Figure 35 does not utilize covalently attached ETMs, any or all of the probe sequences may be so labelled.
  • the scissile linkages 11 are cleaved, leaving the two primary probe sequences 10 and 12 hybridized to the target sequence 120. These probe sequences then disassociate, leaving the probe sequences 10 and 12, and the target sequence 120.
  • the target sequence is then free to hybridize with additional primary probes 1, and the reaction is repeated, generating a pool of probe sequences.
  • the primary probe sequences are then free to diffuse to the secondary beads 250, where they may serve as the next "target", hybridizing to the secondary probes to form additional hybridization complexes, 7 and 8.
  • Cleavage, followed by secondary probe sequence disassociation from the primary probe sequence "targets” generates a pool of secondary probe sequences which can be detected.
  • the uncleaved probes are removed by removing the beads, and the pool of probe sequences is added to an electrode 105, with an optional layer of passivation agent 107, and detection probes 62, 72, 82, and 92 covalently attached via conductive oligomers 108.
  • FIG. 35 does not show this, there may be detection probes for the primary probe sequences as well.
  • the detection probes and the probe sequences form hybridization complexes.
  • the detection probes may be mixed on the electrode, or may be at separate addresses. ETMs in the form of hybridization indicators are then added, and electron transfer is then initiated as is outlined herein.
  • the present invention is directed to compositions and methods useful in the detection of nucleic acids using a variety of amplification techniques, including both signal amplification and target amplification. Once amplification has occurred, detection proceeds based on electron transfer, as is described below and generally outlined in U.S. Patent Nos. 5,591 ,578, 5,824,473, 5,770,369, 5,705,348, and 5,780,234, and PCT application WO98/20162, all of which are expressly incorporated herein by reference in their entirety.
  • target nucleic acid or “target sequence” or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid.
  • the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others. It may be any length, with the understanding that longer sequences are more specific. In some embodiments, it may be desirable to fragment or cleave the sample nucleic acid into fragments of 100 to 10,000 basepairs, with fragments of roughly 500 basepairs being preferred in some embodiments.
  • the complementary target sequence may take many forms. For example, it may be contained within a larger nucleic acid sequence, i.e. all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others.
  • the sample comprising the target sequence may be virtually any tissue from any organism, including blood, bone marrow, lymph, hard tissues (e.g. organs such as liver, spleen, kidney, heart, lung, etc.) saliva, vaginal and anal secretions, urine, feces, perspiration, tears, and other bodily fluids, as well as cell lysates of bacteria and pathogens, including viruses.
  • the target nucleic acid comprises all or a portion of rRNA.
  • rRNA is a particularly preferred target sequence because of the high levels of rRNA in bacteria; accordingly, in many embodiments, no amplification reaction needs to be done.
  • Suitable rRNA targets include, but are not limited to, those outlined in U.S.
  • helper sequences When rRNA is used as the target sequence, preferred embodiments utilize "helper" sequences as outlined in WO 89/04876, hereby incorporated by reference. As is known in the art, rRNA takes on specific secondary and tertiary structures that can hinder the formation of hybridization complexes with the probes of the invention. Accordingly, helper sequences bind to rRNA sequences and impose different secondary or tertiary structures and thus facilitate the binding of the probes to the target rRNA.
  • rRNA for example when rRNA is used as the target sequence, it may be desirable to utilize a plurality of capture probes or capture probe extenders, to "tack down" large targets periodically.
  • a particularly preferred embodiment of the invention is the formation of assay complexes comprising rRNA target sequences, capture probes and label probes.
  • probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample.
  • the target sequence may also be comprised of different target domains; for example, in "sandwich" type assays as outlined below, a first target domain of the sample target sequence may hybridize to a capture probe or a portion of capture extender probe, a second target domain may hybridize to a portion of an amplifier probe, a label probe, or a different capture or capture extender probe, etc.
  • the target domains may be adjacent (i.e. contiguous) or separated.
  • a first primer may hybridize to a first target domain and a second primer may hybridize to a second target domain; either the domains are adjacent, or they may be separated by one or more nucleotides, coupled with the use of a polymerase and dNTPs, as is more fully outlined below.
  • first and second are not meant to confer an orientation of the sequences with respect to the 5'-3' orientation of the target sequence.
  • the first target domain may be located either 5' to the second domain, or 3' to the second domain.
  • the target sequence is prepared using known techniques.
  • the sample may be treated to lyse the cells, using known lysis buffers, sonication, electroporation, etc., with purification occuring as needed, as will be appreciated by those in the art.
  • the reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
  • the reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g.
  • albumin may be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
  • double stranded target nucleic acids are denatured to render them single stranded so as to permit hybridization of the primers and other probes of the invention.
  • a preferred embodiment utilizes a thermal step, generally by raising the temperature of the reaction to about 95 ° C, although pH changes and other techniques may also be used.
  • a primer nucleic acid is then contacted to the target sequence to form a hybridization complex.
  • primer nucleic acid herein is meant a probe nucleic acid that will hybridize to some portion, i.e. a domain, of the target sequence.
  • Probes of the present invention are designed to be complementary to a target sequence (either the target sequence of the sample or to other probe sequences, as is described below), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention.
  • the sequence is not a complementary target sequence.
  • substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions.
  • hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al, hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
  • stringent conditions are selected to be about 5-10 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 ° C for short probes (e.g.
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • the hybridization conditions may also vary when a non-ionic backbone, i.e. PNA is used, as is known in the art.
  • cross- linking agents may be added after target binding to cross-link, i.e. covalently attach, the two strands of the hybridization complex.
  • the assays are generally run under stringency conditions which allows formation of the hybridization complex only in the presence of target.
  • Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
  • Patent No. 5,681 ,697 Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
  • the probes comprise nucleic acids.
  • nucleic acid or "oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sblul et al., Eur. J. Biochem.
  • nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991 ); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series
  • nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176).
  • nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of ETMs, or to increase the stability and half-life of such molecules in physiological environments.
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids and analogs can be made; for example, at the site of conductive oligomer or ETM attachment, an analog structure may be used.
  • nucleic acid analogs and mixtures of naturally occuring nucleic acids and analogs may be made.
  • PNA peptide nucleic acids
  • These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages.
  • the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4 ° C drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9°C. This allows for better detection of mismatches.
  • hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. This is particularly advantageous in the systems of the present invention, as a reduced salt hybridization solution has a lower Faradaic current than a physiological salt solution (in the range of 150 mM).
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo- nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • nucleoside includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
  • nucleoside includes non-naturally occuring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
  • the size of the primer nucleic acid may vary, as will be appreciated by those in the art, in general varying from 5 to 500 nucleotides in length, with primers of between 10 and 100 being preferred, between 15 and 50 being particularly preferred, and from 10 to 35 being especially preferred, depending on the use and amplification technique.
  • the different amplification techniques may have further requirements of the primers, as is more fully described below.
  • an enzyme sometimes termed an "amplification enzyme" is used to modify the primer.
  • the enzymes may be added at any point during the assay, either prior to, during, or after the addition of the primers.
  • the identification of the enzyme will depend on the amplification technique used, as is more fully outlined below.
  • the modification will depend on the amplification technique, as outlined below, although generally the first step of all the reactions herein is an extension of the primer, that is, nucleotides are added to the primer to extend its length.
  • the hybridization complex is disassociated.
  • the amplification steps are repeated for a period of time to allow a number of cycles, depending on the number of copies of the original target sequence and the sensitivity of detection, with cycles ranging from 1 to thousands, with from 10 to 100 cycles being preferred and from 20 to 50 cycles being especially preferred.
  • the modified primer is incorporated into an assay complex, as is more fully outlined below.
  • the assay complex is covalently attached to an electrode, and comprises at least one electron transfer moiety (ETM), described below. Electron transfer between the ETM and the electrode is then detected to indicate the presence or absence of the original target sequence, as described below.
  • ETM electron transfer moiety
  • the amplification is target amplification.
  • Target amplification involves the amplification (replication) of the target sequence to be detected, such that the number of copies of the target sequence is increased.
  • Suitable target amplification techniques include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), and transcription mediated amplification (TMA).
  • the target amplification technique is PCR.
  • the polymerase chain reaction (PCR) is widely used and described, and involve the use of primer extension combined with thermal cycling to amplify a target sequence; see U.S. Patent Nos. 4,683,195 and 4,683,202, and PCR
  • PCR Generic competitive PCR
  • QC-PCR quantitative competitive PCR
  • AP-PCR AP-PCR
  • Immuno-PCR "Alu-PCR”
  • PCR single strand conformational polymorphism or "PCR-SSCP”
  • RT-PCR reverse transcriptase PCR
  • biotin capture PCR “vectorette PCR”.
  • panhandle PCR and “PCR select cDNA subtration”, among others.
  • the amplification technique is not PCR.
  • PCR may be briefly described as follows.
  • a double stranded target nucleic acid is denatured, generally by raising the temperature, and then cooled in the presence of an excess of a PCR primer, which then hybridizes to the first target strand.
  • a DNA polymerase then acts to extend the primer, resulting in the synthesis of a new strand forming a hybridization complex.
  • the sample is then heated again, to disassociate the hybridization complex, and the process is repeated.
  • a second PCR primer for the complementary target strand, rapid and exponential amplification occurs.
  • PCR steps are denaturation, annealing and extension.
  • the particulars of PCR are well known, and include the use of a thermostabile polymerase such as Taq I polymerase and thermal cycling.
  • the PCR reaction requires at least one PCR primer and a polymerase.
  • the target amplification technique is SDA.
  • Strand displacement amplification (SDA) is generally described in Walker et al., in Molecular Methods for Virus Detection, Academic Press, Inc., 1995, and U.S. Patent Nos. 5,455,166 and 5,130,238, all of which are hereby expressly incorporated by reference in their entirety.
  • SDA may be described as follows.
  • a single stranded target nucleic acid is contacted with an SDA primer.
  • An "SDA primer” generally has a length of 25-100 nucleotides, with SDA primers of approximately 35 nucleotides being preferred.
  • An SDA primer is substantially complementary to a region at the 3' end of the target sequence, and the primer has a sequence at its 5' end (outside of the region that is complementary to the target) that is a recognition sequence for a restriction endonuclease, sometimes referred to herein as a "nicking enzyme” or a "nicking endonuclease", as outlined below.
  • the SDA primer then hybridizes to the target sequence.
  • the SDA reaction mixture also contains a polymerase (an "SDA polymerase”, as outlined below) and a mixture of all four deoxynucleoside-triphosphates (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), at least one species of which is a substituted or modified dNTP; thus, the SDA primer is modified, i.e. extended, to form a modified primer, sometimes referred to herein as a "newly synthesized strand".
  • the substituted dNTP is modified such that it will inhibit cleavage in the strand containing the substituted dNTP but will not inhibit cleavage on the other strand.
  • Suitable substituted dNTPs include, but are not limited, 2'deoxyadenosine 5'-0-(1-thiotriphosphate), 5- methyldeoxycytidine 5'-triphosphate, 2'-deoxyuridine 5'-triphosphate, adn 7-deaza-2'-deoxyguanosine 5'-triphosphate.
  • the substitution of the dNTP may occur after incorporation into a newly synthesized strand; for example, a methylase may be used to add methyl groups to the synthesized strand.
  • the polymerase may have 5' ⁇ 3' exonuclease activity. However, if less than all the nucleotides are substituted, the polymerase preferably lacks 5'-3' exonuclease activity.
  • the recognition site/endonuclease pair can be any of a wide variety of known combinations.
  • the endonuclease is chosen to cleave a strand either at the recognition site, or either 3 * or 5' to it, without cleaving the complementary sequence, either because the enzyme only cleaves one strand or because of the incorporation of the substituted nucleotides.
  • Suitable recognition site/endonuclease pairs are well known in the art; suitable endonucleases include, but are not limited to, Hindi, Hindll, Aval, Fnu4HI, Tthllll, Nell, BstXI, Baml, etc.
  • a chart depicting suitable enzymes, and their corresponding recognition sites and the modified dNTP to use is found in U.S. Patent No. 5,455,166, hereby expressly incorporated by reference.
  • a polymerase (an "SDA polymerase") is used to extend the newly nicked strand, 5'-3', thereby creating another newly synthesized strand.
  • the polymerase chosen should be able to intiate 5'-3' polymerization at a nick site, should also displace the polymerized strand downstream from the nick, and should lack 5' ⁇ 3' exonuclease activity (this may be additionally accomplished by the addition of a blocking agent).
  • suitable polymerases in SDA include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA polymerase.
  • the SDA reaction requires, in no particular order, an SDA primer, an SDA polymerase, a nicking endonuclease, and dNTPs, at least one species of which is modified.
  • SDA does not require thermocycling.
  • the temperature of the reaction is generally set to be high enough to prevent non-specific hybridization but low enough to allow specific hybridization; this is generally from about 37 ° C to about 42°C, depending on the enzymes.
  • a second amplification reaction can be done using the complementary target sequence, resulting in a substantial increase in amplification during a set period of time. That is, a second primer nucleic acid is hybridized to a second target sequence, that is substantially complementary to the first target sequence, to form a second hybridization complex. The addition of the enzyme, followed by disassociation of the second hybridization complex, results in the generation of a number of newly synthesized second strands.
  • the ETM(s) may be incorporated in three ways: (1 ) the primers comprise the ETM(s), for example attached to the base, a ribose, a phosphate, or to analogous structures in a nucleic acid analog; (2) modified nucleosides are used that are modified at either the base or the ribose (or to analogous structures in a nucleic acid analog) with the ETM(s); these ETM modified nucleosides are then converted to the triphosphate form and are incorporated into the newly synthesized strand by a polymerase; or (3) a "tail" of ETMs can be added, as outlined below. Either of these methods result in a newly synthesized strand that comprises ETMs, that can be directly detected as outlined below.
  • indirect detection proceeds as a sandwich assay, with the newly synthesized strands containing few or no ETMs. Detection then proceeds via the use of label probes that comprise the
  • the target amplification technique is nucleic acid sequence based amplification (NASBA).
  • NASBA is generally described in U.S. Patent No. 5,409,818; Sooknanan et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp.
  • TMA and QBR Transcription mediated amplification
  • TMA Transcription mediated amplification
  • NASBA utilizes the addition of RNAse H to effect RNA degradation, and TMA relies on inherent RNAse H activity of the reverse transcriptase.
  • a single stranded target nucleic acid is contacted with a first primer, generally referred to herein as a “NASBA primer” (although “TMA primer” is also suitable).
  • a first primer generally referred to herein as a "NASBA primer” (although “TMA primer” is also suitable).
  • NASBA primer a first primer
  • TMA primer TMA primer
  • the first primer is preferably a DNA primer that has at its 3' end a sequence that is substantially complementary to the 3' end of the first template.
  • the first primer also has an RNA polymerase promoter at its 5' end (or its complement (antisense), depending on the configuration of the system).
  • the first primer is then hybridized to the first template to form a first hybridization complex.
  • the reaction mixture also includes a reverse transcriptase enzyme (an "NASBA reverse transcriptase") and a mixture of the four dNTPs, such that the first NASBA primer is modified, i.e. extended, to form a modified first primer, comprising a hybridization complex of RNA (the first template) and DNA (the newly synthesized strand).
  • RNA-directed DNA polymerase an enzyme capable of synthesizing DNA from a DNA primer and an RNA template.
  • Suitable RNA-directed DNA polymerases include, but are not limited to, avian myloblastosis virus reverse transcriptase ("AMV
  • the reverse transcriptase enzyme further comprises a RNA degrading activity as outlined below.
  • the NASBA reaction also includes an RNA degrading enzyme, also sometimes referred to herein as a ribonuclease, that will hydrolyze RNA of an RNA:DNA hybrid without hydrolyzing single- or double-stranded RNA or DNA.
  • RNA degrading enzyme also sometimes referred to herein as a ribonuclease
  • Suitable ribonucleases include, but are not limited to, RNase H from E. coli and calf thymus.
  • the NASBA reaction also includes a second NASBA primer, generally comprising DNA (although as for all the probes herein, including primers, nucleic acid analogs may also be used).
  • This second NASBA primer has a sequence at its 3' end that is substantially complementary to the 3' end of the second template, and also contains an antisense sequence for a functional promoter and the antisense sequence of a transcription initiation site.
  • this primer sequence when used as a template for synthesis of the third DNA template, contains sufficient information to allow specific and efficient binding of an RNA polymerase and initiation of transcription at the desired site.
  • Preferred embodiments utilizes the antisense promoter and transcription initiation site are that of the T7 RNA polymerase, although other RNA polymerase promoters and initiation sites can be used as well, as outlined below.
  • the second primer hybridizes to the second template, and a DNA polymerase, also termed a "DNA- directed DNA polymerase", also present in the reaction, synthesizes a third template (a second newly synthesized DNA strand), resulting in second hybridization complex comprising two newly synthesized DNA strands.
  • a DNA polymerase also termed a "DNA- directed DNA polymerase”
  • RNA polymerase (ribonucleotides or NTPs) results in the synthesis of an RNA strand (a third newly synthesized strand that is essentially the same as the first template).
  • the RNA polymerase sometimes referred to herein as a "DNA-directed RNA polymerase", recognizes the promoter and specifically initiates RNA synthesis at the initiation site.
  • the RNA polymerase preferably synthesizes several copies of RNA per DNA duplex.
  • RNA polymerases include, but are not limited to, T7 RNA polymerase, and other bacteriophage RNA polymerases including those of phage T3, phage ⁇ ll, Salmonella phage sp6, or Pseudomonase phage gh-1.
  • TMA and NASBA are used with starting DNA target sequences.
  • the NASBA reaction requires, in no particular order, a first NASBA primer, a second NASBA primer comprising an antisense sequence of an RNA polymerase promoter, an RNA polymerase that recognizes the promoter, a reverse transcriptase, a DNA polymerase, an RNA degrading enzyme, NTPs and dNTPs, in addition to the detection components outlined below.
  • the TMA reaction requires, in no particular order, a first TMA primer, a second TMA primer comprising an antisense sequence of an RNA polymerase promoter, an RNA polymerase that recognizes the promoter, a reverse transcriptase with RNA degrading activity, a DNA polymerase, NTPs and dNTPs, in addition to the detection components outlined below.
  • the detection of the newly synthesized strands can proceed in several ways. Direct detection can be done when the newly synthesized strands comprise ETM labels, either by incorporation into the primers or by incorporation of modified labelled nucleotides into the growing strand. Alternatively, as is more fully outlined below, indirect detection of unlabelled strands (which now serve as "targets" in the detection mode) can occur using a variety of sandwich assay configurations. As will be appreciated by those in the art, any of the newly synthesized strands can serve as the "target” for form an assay complex on a surface with a capture probe. In NASBA and TMA, it is preferable to utilize the newly formed RNA strands as the target, as this is where significant amplification occurs.
  • the amplification technique is signal amplification.
  • Signal amplification involves the use of limited number of target molecules as templates to either generate multiple signalling probes or allow the use of multiple signalling probes.
  • Signal amplification strategies include LCR, CPT, InvaderTM, and the use of amplification probes in sandwich assays.
  • the signal amplification technique is LCR, as is generally depicted in Figures 21 , 28 and 29.
  • the method can be run in two different ways; in a first embodiment, only one strand of a target sequence is used as a template for ligation (Figure 28); alternatively, both strands may be used ( Figure 29). See generally U.S. Patent Nos. 5,185,243 and 5,573,907; EP 0 320 308 B1 ; EP 0 336 731 B1 ; EP 0 439 182 B1 ; WO 90/01069; WO 89/12696; and WO 89/09835, and U.S.S.N.s
  • the single-stranded target sequence comprises a first target domain and a second target domain, and a first LCR primer and a second LCR primer nucleic acids are added, that are substantially complementary to its respective target domain and thus will hybridize to the target domains.
  • These target domains may be directly adjacent, i.e. contiguous, or separated by a number of nucleotides. If they are non-contiguous, nucleotides are added along with means to join nucleotides, such as a polymerase, that will add the nucleotides to one of the primers.
  • the two LCR primers are then covalently attached, for example using a ligase enzyme such as is known in the art.
  • This forms a first hybridization complex comprising the ligated probe and the target sequence.
  • This hybridization complex is then denatured (disassociated), and the process is repeated to generate a pool of ligated probes.
  • the detection probes may be desirable to have the detection probes, described below, comprise a mismatch at the probe junction site, such that the detection probe cannot be used as a template for ligation.
  • LCR is done for two strands of a double-stranded target sequence.
  • the target sequence is denatured, and two sets of probes are added: one set as outlined above for one strand of the target, and a separate set (i.e. third and fourth primer robe nucleic acids) for the other strand of the target.
  • the first and third probes will hybridize, and the second and fourth probes will hybridize, such that amplification can occur. That is, when the first and second probes have been attached, the ligated probe can now be used as a template, in addition to the second target sequence, for the attachment of the third and fourth probes. Similarly, the ligated third and fourth probes will serve as a template for the attachment of the first and second probes, in addition to the first target strand. In this way, an exponential, rather than just a linear, amplification can occur.
  • the detection of the LCR reaction can occur directly, in the case where one or both of the primers comprises at least one ETM, or indirectly, using sandwich assays, through the use of additional probes; that is, the ligated probes can serve as target sequences, and detection may utilize amplification probes, capture probes, capture extender probes, label probes, and label extender probes, etc.
  • LCR utilizes a "chemical ligation" of sorts, as is generally outlined in U.S. Patent Nos.
  • each primer has a portion that acts as a first domain of the target and the second primer is substantially complementary to an adjacent second domain of the target (although, as for LCR, if a "gap" exists, a polymerase and dNTPs may be added to "fill in” the gap).
  • Each primer has a portion that acts as a first domain of the target and the second primer is substantially complementary to an adjacent second domain of the target (although, as for LCR, if a "gap" exists, a polymerase and dNTPs may be added to "fill in” the gap).
  • Each primer has a portion that acts as a first domain of the target and the second primer is substantially complementary to an adjacent second domain of the target (although, as for LCR, if a "gap" exists, a polymerase and dNTPs may be added to "fill in” the gap).
  • Each primer has a portion that acts as a first domain of the target and the second primer is substantially complementary to an adjacent second domain of the target (
  • side chain that does not bind the target sequence and acts one half of a stem structure that interacts non-covalently through hydrogen bonding, salt bridges, van der Waal's forces, etc.
  • Preferred embodiments utilize substantially complementary nucleic acids as the side chains.
  • the side chains of the primers upon hybridization of the primers to the target sequence, the side chains of the primers are brought into spatial proximity, and, if the side chains comprise nucleic acids as well, can also form side chain hybridization complexes.
  • At least one of the side chains of the primers comprises an activatable cross-linking agent, generally covalently attached to the side chain, that upon activation, results in a chemical cross-link or chemical ligation.
  • the activatible group may comprise any moiety that will allow cross-linking of the side chains, and include groups activated chemically, photonically and thermally, with photoactivatable groups being preferred.
  • a single activatable group on one of the side chains is enough to result in cross-linking via interaction to a functional group on the other side chain; in alternate embodiments, activatable groups are required on each side chain.
  • the reaction is subjected to conditions to allow for the disassocation of the hybridization complex, thus freeing up the target to serve as a template for the next ligation or cross-linking. In this way, signal amplification occurs, and can be detected as outlined herein.
  • the signal amplification technique is RCA.
  • Rolling-circle amplification is generally described in Baner et al. (1998) N ⁇ c. Acids Res. 26:5073-5078; Barany, F. (1991 ) Proc.
  • RCA may be described as follows. First, as is outlined in more detail below, a single RCA probe is hybridized with a target nucleic acid. Each terminus of the probe hybridizes adjacently on the target nucleic acid (or alternatively, there are intervening nucleotides that can be "filled in” using a polymerase and dNTPs, as outlined below) and the OLA assay as described above occurs. When ligated, the probe is circularized while hybridized to the target nucleic acid. Addition of a primer, a polymerase and dNTPs results in extension of the circular probe. However, since the probe has no terminus, the polymerase continues to extend the probe repeatedly. Thus results in amplification of the circular probe. This very large concatemer can be detected intact, as described below, or can be cleaved in a variety of ways to form smaller amplicons for detection as outlined herein.
  • a single oligonucleotide is used both for OLA and as the circular template for RCA (referred to herein as a "padlock probe” or a "RCA probe”). That is, each terminus of the oligonucleotide contains sequence complementary to the target nucleic acid and functions as an OLA primer as described above. That is, the first end of the RCA probe is substantially complementary to a first target domain, and the second end of the RCA probe is substantially complementary to a second target domain, adjacent (either directly or indirectly, as outlined herein) to the first domain. Hybridization of the probe to the target nucleic acid results in the formation of a hybridization complex.
  • Ligation of the "primers" results in the formation of a modified hybridization complex containing a circular probe i.e. an RCA template complex. That is, the oligonucleotide is circularized while still hybridized with the target nucleic acid. This serves as a circular template for RCA.
  • Addition of a primer, a polymerase and the required dNTPs to the RCA template complex results in the formation of an amplified product nucleic acid. Following RCA, the amplified product nucleic acid is detected as outlined herein.
  • the polymerase may incorporate labelled nucleotides; a labeled primer may be used, or alternatively, a label probe is used that is substantially complementary to a portion of the RCA probe and comprises at least one label is used.
  • the present invention provides RCA probes (sometimes referred to herein as “rolling circle probes (RCPs) or “padlock probes” (PPs)).
  • the RCPs may comprise any number of elements, including a first and second ligation sequence, a cleavage site, a priming site, a capture sequence, nucleotide analogs, and a label sequence.
  • the RCP comprises first and second ligation sequences.
  • the ligation sequences are substantially complementary to adjacent domains of the target sequence.
  • the domains may be directly adjacent (i.e. with no intervening bases between the 3' end of the first and the 5' of the second) or indirectly adjacent, with from 1 to 100 or more bases in between.
  • the RCPs comprise a cleavage site, such that either after or during the rolling circle amplification, the RCP concatamer may be cleaved into amplicons. In some embodiments, this facilitates the detection, since the amplicons are generally smaller and exhibit favorable hybridization kinetics on the surface.
  • the cleavage site can take on a number of forms, including, but not limited to, the use of restriction sites in the probe, the use of ribozyme sequences, or through the use or incorporation of nucleic acid cleavage moieties.
  • the padlock probe contains a restriction site.
  • the restriction endonuclease site allows for cleavage of the long concatamers that are typically the result of RCA into smaller individual units that hybridize either more efficiently or faster to surface bound capture probes.
  • the product nucleic acid is contacted with the appropriate restriction endonuclease. This results in cleavage of the product nucleic acid into smaller fragments.
  • the fragments are then hybridized with the capture probe that is immobilized resulting in a concentration of product fragments onto the detection electrode.
  • these fragments can be detected in one of two ways: either labelled nucleotides are incorporated during the replication step, for example either as labeled individual dNTPs or through the use of a labeled primer, or an additional label probe is added.
  • the restriction site is a single-stranded restriction site chosen such that its complement occurs only once in the RCP.
  • the cleavage site is a ribozyme cleavage site as is generally described in Daubendiek et al., Nature Biotech. 15:273 (1997), hereby expressly incorporated by reference.
  • RCPs that encode catalytic RNAs, NTPs and an RNA polymerase, the resulting concatamer can self cleave, ultimately forming monomeric amplicons.
  • the cleavage site comprises one or more labile bases such as UTP or dUTP, and cleavage is effected either chemically (e.g. using basic conditions) or enzymatically.
  • labile bases such as UTP or dUTP
  • cleavage is effected either chemically (e.g. using basic conditions) or enzymatically.
  • uracil-N-glycosylase cleaves at uracil groups; similarly, RNAse H cleaves ribose-containing bases in RNA/DNA hybrids.
  • cleavage is accomplished using DNA cleavage reagents.
  • DNA cleavage reagents for example, as is known in the art, there are a number of intercalating moieties that can effect cleavage, for example using light.
  • the RCPs do not comprise a cleavage site.
  • the size of the RCP is designed such that it may hybridize "smoothly" to many capture probes on a surface.
  • the reaction may be cycled such that very long concatamers are not formed.
  • the RCPs comprise a priming site, to allow the binding of a DNA polymerase primer.
  • a DNA polymerase primer As is known in the art, many DNA polymerases require double stranded nucleic acid and a free terminus to allow nucleic acid synthesis. However, in some cases, for example when RNA polymerases are used, a primer may not be required (see Daubendiek, supra). Similarly, depending on the size and orientation of the target strand, it is possible that a free end of the target sequence can serve as the primer; see Baner et al., supra.
  • the padlock probe also contains a priming site for priming the RCA reaction. That is, each padlock probe comprises a sequence to which a primer nucleic acid hybridizes forming a template for the polymerase.
  • the primer can be found in any portion of the circular probe. In a preferred embodiment, the primer is located at a discrete site in the probe. In this embodiment, the primer site in each distinct padlock probe is identical, although this is not required. Advantages of using primer sites with identical sequences include the ability to use only a single primer oligonucleotide to prime the RCA assay with a plurality of different hybridization complexes. That is, the padlock probe hybridizes uniquely to the target nucleic acid to which it is designed. A single primer hybridizes to all of the unique hybridization complexes forming a priming site for the polymerase. RCA then proceeds from an identical locus within each unique padlock probe of the hybridization complexes.
  • the primer site can overlap, encompass, or reside within any of the above-described elements of the padlock probe. That is, the primer can be found, for example, overlapping or within the restriction site or the identifier sequence. In this embodiment, it is necessary that the primer nucleic acid is designed to base pair with the chosen primer site.
  • a primer is used that hybridizes both to a portion of the target sequence and to a priming site on the RCP. This may be done to increase the specificity of the system, and allows the use of higher hybridization temperatures.
  • the primer may comprise the covalently attached ETMs.
  • the RCPs comprise a capture sequence.
  • a capture sequence as is outlined herein, is substantially complementary to a capture probe, as outlined herein.
  • the RCPs comprise a label sequence; i.e. a sequence that can be used to bind label probes and is substantially complementary to a label probe.
  • a label sequence i.e. a sequence that can be used to bind label probes and is substantially complementary to a label probe.
  • the RCPs comprise nucleotide analogs.
  • ETMs since it may be desirable to incorporate ETMs at specific locations within the amplicon (for example, at a cluster of 8- 10 ETMs in a 20-30 basepair stretch, to allow optimal signaling and configuration of the detection hybridization complex), unique bases may be incorporated into the RCP.
  • isocytosine is a nucleoside analog that will only basepair with isoguanine, as is generally described in U.S. Patent No. 5,681 ,702, hereby incorporated by reference in its entirety.
  • deoxy-isoC or deoxy-isoG labeled with an ETM can be added to the pool of nucleotides, resulting in the incorporation of ETMs at predetermined, specific locations.
  • the RCP/primer sets are designed to allow an additional level of amplification, sometimes referred to as "hyperbranching" or "cascade amplification".
  • a first concatamer can serve as the template for additional concatamers.
  • a polymerase that has high displacement activity is preferably used.
  • a first antisense primer is used, followed by the use of sense primers, to generate large numbers of concatamers and amplicons, when cleavage is used.
  • the invention provides for methods of detecting using RCPs as described herein.
  • the RCP primer is added, if necessary, along with a polymerase and dNTPs (or NTPs, if necessary).
  • the polymerase can be any polymerase as outlined herein, but is preferably one lacking 3' exonuclease activity (3' exo " ). Examples of suitable polymerase include but are not limited to exonuclease minus DNA Polymerase I large (Klenow) Fragment, Phi29 DNA polymerase, Taq DNA Polymerase and the like.
  • a polymerase that will replicate single- stranded DNA i.e. without a primer forming a double stranded section
  • the OLA/RCA is performed in solution followed by restriction endonuclease cleavage of the RCA product.
  • the cleaved product is then applied to an array as described herein.
  • the incorporation of an endonuclease site allows the generation of short, easily hybridizable sequences.
  • the unique capture sequence in each rolling circle padlock probe sequence allows diverse sets of nucleic acid sequences to be analyzed in parallel on an array, since each sequence is resolved on the basis of hybridization specificity.
  • these copies are subsequently detected by one of two methods; either hybridizing a label probe comprising ETMs which is complementary to the circular target or via the incorporation of ETM- labeled nucleotides in the amplification reaction.
  • the label is detected a described herein.
  • the polymerase creates more than 100 copies of the circular DNA. In more preferred embodiments the polymerase creates more than 1000 copies of the circular DNA; while in a most preferred embodiment the polymerase creates more than 10,000 copies or more than 50,000 copies of the template.
  • the amplified circular DNA sequence is then detected by methods known in the art and as described herein. Detection is accomplished by hybridizing with a labeled probe.
  • the probe is labeled directly or indirectly.
  • labeled nucleotides are incorporated into the amplified circular DNA product.
  • the nucleotides can be labeled directly, or indirectly as is further described herein.
  • the RCA as described herein finds use in allowing highly specific and highly sensitive detection of nucleic acid target sequences.
  • the method finds use in improving the multiplexing ability of DNA arrays and eliminating costly sample or target preparation.
  • a substantial savings in cost can be realized by directly analyzing genomic DNA on an array, rather than employing an intermediate PCR amplification step.
  • the method finds use in examining genomic DNA and other samples including mRNA.
  • the RCA finds use in allowing rolling circle amplification products to be easily detected by hybridization to probes in a solid-phase format.
  • An additional advantage of the RCA is that it provides the capability of multiplex analysis so that large numbers of sequences can be analyzed in parallel. By combining the sensitivity of RCA and parallel detection on arrays, many sequences can be analyzed directly from genomic DNA.
  • the signal amplification technique is CPT. CPT technology is described in a number of patents and patent applications, including U.S. Patent Nos.
  • CPT may be described as follows.
  • a CPT primer (also sometimes referred to herein as a "scissile primer”), comprises two probe sequences separated by a scissile linkage.
  • the CPT primer is substantially complementary to the target sequence and thus will hybridize to it to form a hybridization complex.
  • the scissile linkage is cleaved, without cleaving the target sequence, resulting in the two probe sequences being separated.
  • the two probe sequences can thus be more easily disassociated from the target, and the reaction can be repeated any number of times.
  • the cleaved primer is then detected as outlined herein.
  • scissile linkage herein is meant a linkage within the scissile probe that can be cleaved when the probe is part of a hybridization complex, that is, when a double-stranded complex is formed. It is important that the scissile linkage cleave only the scissile probe and not the sequence to which it is hybridized (i.e. either the target sequence or a probe sequence), such that the target sequence may be reused in the reaction for amplification of the signal.
  • the scissile linkage is any connecting chemical structure which joins two probe sequences and which is capable of being selectively cleaved without cleavage of either the probe sequences or the sequence to which the scissile probe is hybridized.
  • the scissile linkage may be a single bond, or a multiple unit sequence.
  • the scissile linkage comprises RNA.
  • RNA This system, previously described in as outlined above, is based on the fact that certain double-stranded nucleases, particularly ribonucleases, will nick or excise RNA nucleosides from a RNA:DNA hybridization complex. Of particular use in this embodiment is RNAseH, Exo III, and reverse transcriptase.
  • the entire scissile probe is made of RNA
  • the nicking is facilitated especially when carried out with a double-stranded ribonuclease, such as RNAseH or Exo III.
  • RNA probes made entirely of RNA sequences are particularly useful because first, they can be more easily produced enzymatically, and second, they have more cleavage sites which are accessible to nicking or cleaving by a nicking agent, such as the ribonucleases.
  • scissile probes made entirely of RNA do not rely on a scissile linkage since the scissile linkage is inherent in the probe.
  • the methods of the invention may be used to detect mismatches, as is generally described in U.S. Patent Nos. 5,660,988, and WO 95/14106, hereby expressly incorporated by reference. These mismatch detection methods are based on the fact that RNAseH may not bind to and/or cleave an RNA:DNA duplex if there are mismatches present in the sequence.
  • NA, and NA 2 are non-RNA nucleic acids, preferably DNA.
  • the mismatch is within the RNA:DNA duplex, but in some embodiments the mismatch is present in an adjacent sequence very close to the desired sequence, close enough to affect the RNAseH (generally within one or two bases).
  • the nucleic acid scissile linkage is designed such that the sequence of the scissile linkage reflects the particular sequence to be detected, i.e. the area of the putative mismatch.
  • the rate of generation of the released fragments is such that the methods provide, essentially, a yes/no result, whereby the detection of the virtually any released fragment indicates the presence of the desired target sequence.
  • a minimal mismatch for example, a 1-, 2- or 3-base mismatch, or a 3-base delection
  • the rate of generation of cleaved fragments, and/or the final amount of cleaved fragments is quantified to indicate the presence or absence of the target.
  • secondary and tertiary scissile probes may be particularly useful in this embodiment, as this can amplify the differences between a perfect match and a mismatch. These methods may be particularly useful in the determination of homozygotic or heterozygotic states of a patient.
  • the scissile linkage it is an important feature of the scissile linkage that its length is determined by the suspected difference between the target and the probe.
  • the scissile linkage is between 3 to 5 nucleotides in length, such that a suspected nucleotide difference from 1 nucleotide to 3 nucleotides is encompassed by the scissile linkage, and 0, 1 or 2 nucleotides are on either side of the difference.
  • preferred embodiments utilize from 1 to about 100 nucleotides, with from about 2 to about 20 being preferred and from about 5 to about 10 being particularly preferred.
  • CPT may be done enzymatically or chemically. That is, in addition to RNAseH, there are several other cleaving agents which may be useful in cleaving RNA (or other nucleic acid) scissile bonds. For example, several chemical nucleases have been reported; see for example Sigman et al., Annu. Rev. Biochem. 1990, 59, 207-236; Sigman et al., Chem. Rev. 1993, 93, 2295-2316; Bashkin et al., J. Org. Chem. 1990, 55, 5125-5132; and Sigman et al., Nucleic Acids and Molecular Biology, vol. 3, F. Eckstein and D.M.J. Lilley (Eds), Springer-Verlag, Heidelberg 1989, pp. 13-27; all of which are hereby expressly incorporated by reference.
  • RNA hydrolysis is also an active area; see for example Chin, Ace. Chem. Res. 1991 , 24, 145- 152; Breslow et al., Tetrahedron, 1991 , 47, 2365-2376; Anslyn et al., Angew. Chem. Int. Ed. Engl.,
  • RNA hydrolysis Current approaches to site-directed RNA hydrolysis include the conjugation of a reactive moiety capable of cleaving phosphodiester bonds to a recognition element capable of sequence-specifically hybridizing to RNA. In most cases, a metal complex is covalently attached to a DNA strand which forms a stable heteroduplex. Upon hybridization, a Lewis acid is placed in close proximity to the RNA backbone to effect hydrolysis; see Magda et al., J. Am. Chem. Soc. 1994, 116, 7439; Hall et al., Chem. Biology 1994, 1 , 185-190; Bashkin et al., J. Am. Chem. Soc.
  • the scissile linkage is not necessarily RNA.
  • chemical cleavage moieties may be used to cleave basic sites in nucleic acids; see Belmont, et al.,New J. Chem. 1997, 21 , 47-54; and references therein, all of which are expressly incorporated herein by reference.
  • photocleavable moieties for example, using transition metals, may be used; see Moucheron, et al., Inorg. Chem. 1997, 36, 584-592, hereby expressly by reference.
  • the first step of the CPT method requires hybridizing a primary scissile primer (also called a primary scissile probe) obe to the target. This is preferably done at a temperature that allows both the binding of the longer primary probe and disassociation of the shorter cleaved portions of the primary probe, as will be appreciated by those in the art. As outlined herein, this may be done in solution, or either the target or one or more of the scissile probes may be attached to a solid support. For example, it is possible to utilize "anchor probes" on a solid support or the electrode which are substantially complementary to a portion of the target sequence, preferably a sequence that is not the same sequence to which a scissile probe will bind.
  • a primary scissile primer also called a primary scissile probe
  • a preferred embodiment has one or more of the scissile probes attached to a solid support such as a bead.
  • the soluble target diffuses to allow the formation of the hybridization complex between the soluble target sequence and the support-bound scissile probe.
  • Such embodiments are generally depicted in Figures 34 and 35.
  • preferred methods utilize cutting or shearing techniques to cut the nucleic acid sample containing the target sequence into a size that will allow sufficient diffusion of the target sequence to the surface of a bead. This may be accomplished by shearing the nucleic acid through mechanical forces (e.g. sonication) or by cleaving the nucleic acid using restriction endonucleases. Alternatively, a fragment containing the target may be generated using polymerase, primers and the sample as a template, as in polymerase chain reaction (PCR). In addition, amplification of the target using PCR or LCR or related methods may also be done; this may be particularly useful when the target sequence is present in the sample at extremely low copy numbers. Similarly, numerous techniques are known in the art to increase the rate of mixing and hybridization including agitation, heating, techniques that increase the overall concentration such as precipitation, drying, dialysis, centrifugation, electrophoresis, magnetic bead concentration, etc.
  • the scissile probes are introduced in a molar excess to their targets (including both the target sequence or other scissile probes, for example when secondary or tertiary scissile probes are used), with ratios of scissile probe:target of at least about 100:1 being preferred, at least about 1000:1 being particularly preferred, and at least about 10,000:1 being especially preferred. In some embodiments the excess of probe:target will be much greater. In addition, ratios such as these may be used for all the amplification techniques outlined herein.
  • the complex is subjected to cleavage conditions. As will be appreciated, this depends on the composition of the scissile probe; if it is RNA, RNAseH is introduced. It should be noted that under certain circumstances, such as is generally outlined in WO 95/00666 and WO 95/00667, hereby incorporated by reference, the use of a double-stranded binding agent such as RNAseH may allow the reaction to proceed even at temperatures above the Tm of the primary probe:target hybridization complex. Accordingly, the addition of scissile probe to the target can be done either first, and then the cleavage agent or cleavage conditions introduced, or the probes may be added in the presence of the cleavage agent or conditions.
  • the cleavage conditions result in the separation of the two (or more) probe sequences of the primary scissile probe. As a result, the shorter probe sequences will no longer remain hybridized to the target sequence, and thus the hybridization complex will disassociate, leaving the target sequence intact.
  • the optimal temperature for carrying out the CPT reactions is generally from about 5°C to about 25°C below the melting temperatures of the probe:target hybridization complex. This provides for a rapid rate of hybridization and high degree of specificity for the target sequence.
  • the Tm of any particular hybridization complex depends on salt concentration, G-C content, and length of the complex, as is known in the art.
  • nucleases are generally removed from the sample during the isolation of the DNA by heating or extraction procedures.
  • a number of inhibitors of single-stranded nucleases such as vanadate, inhibitors it-ACE and RNAsin, a placental protein, do not affect the activity of RNAseH. This may not be necessary depending on the purity of the RNAseH and/or the target sample.
  • each molecule of the target sequence will turnover between 100 and 1000 times in this period, depending on the length and sequence of the probe, the specific reaction conditions, and the cleavage method. For example, for each copy of the target sequence present in the test sample 100 to 1000 molecules will be cleaved by RNAseH. Higher levels of amplification can be obtained by allowing the reaction to proceed longer, or using secondary, tertiary, or quaternary probes, as is outlined herein.
  • the uncleaved scissile probes Upon completion of the reaction, generally determined by time or amount of cleavage, the uncleaved scissile probes must be removed or neutralized prior to detection, such that the uncleaved probe does not bind to a detection probe, causing false positive signals. This may be done in a variety of ways, as is generally described below.
  • the separation is facilitated by the use of beads containing the primary probe.
  • beads containing the primary probe When the scissile probes are attached to beads, removal of the beads by filtration, centrifugation, the application of a magnetic field, electrostatic interactions for charged beads, adhesion, etc., results in the removal of the uncleaved probes.
  • the separation is based on gel electrophoresis of the reaction products to separate the longer uncleaved probe from the shorter cleaved probe sequences as is known in the art.
  • the separation is based on strong acid precipitation. This is useful to separate long (generally greater than 50 nucleotides) from smaller fragments (generally about 10 nucleotides).
  • a strong acid such as trichloroacetic acid
  • the solution can be centrifuged or filtered to remove the precipitate, and the cleaved probe sequences can be quantitated.
  • the scissile probe contains both an ETM and an affinity binding ligand or moiety, such that an affinity support is used to carry out the separation.
  • ETM used for detection is not on the same probe sequence that contains the affinity moiety, such that removal of the uncleaved probe, and the cleaved probe containing the affinity moiety, does not remove all the detectable ETMs.
  • the scissile probe may not contain a covalently attached ETM, but just an affinity label.
  • Suitable affinity moieties include, but are not limited to, biotin, avidin, streptavidin, lectins, haptens, antibodies, etc.
  • the binding partner of the affinity moiety is attached to a solid support such as glass beads, latex beads, dextrans, etc. and used to pull out the uncleaved probes, as is known in the art.
  • the cleaved probe sequences, which do not contain the affinity moiety, remain in solution and then can be detected as outlined below.
  • a separation sequence of nucleic acid is included in the scissile probe, which is not cleaved during the reaction.
  • a nucleic acid complementary to the separation sequence is attached to a solid support such as a bead and serves as a catcher sequence.
  • the separation sequence is added to the scissile probes, and is not recognized by the target sequence, such that a generalized catcher sequence may be utilized in a variety of assays.
  • the uncleaved probe is neutralized by the addition of a substantially complementary neutralization nucleic acid, as is generally depicted in Figure 32.
  • a substantially complementary neutralization nucleic acid as is generally depicted in Figure 32.
  • This is particularly useful in embodiments utilizing capture sequences, separation sequences, and one-step systems, as the complement to a probe containing capture sequences forms hybridization complexes that are more stable due to its length than the cleaved probe sequence:detection probe complex.
  • complete removal of the uncleaved probe is not required, since detection is based on electron transfer through nucleic acid; rather, what is important is that the uncleaved probe is not available for binding to a detection electrode probe specific for cleaved sequences.
  • the neutralization nucleic acid is a detection probe on the surface of the electrode, at a separate "address", such that the signal from the neutralization hybridization complex does not contribute to the signal of the cleaved fragments.
  • the neutralization nucleic acid may be attached to a bead; the neutralization beads are added to quench the reaction, and then removed prior to detection.
  • a mechanism-1 system can be described as follows; the cleaved probe sequences hybridize to a first detection single-stranded probe covalently attached via a conductive oligomer to an electrode, that thus forms a second hybridization complex.
  • the second hybridization complex comprising detection probe:probe sequence, contains at least a first ETM.
  • this ETM may be covalently attached to either the probe (primer) sequence or the detection probe, or it may be added non-covalently as a hybridization indicator, or both.
  • preferred embodiments utilize more than one ETM per hybridization complex for detection.
  • the electrode comprises at least a first detection probe which is substantially complementary to all or part of a cleaved portion of the primary scissile probe.
  • only one type of detection probe is utilized, which can be substantially complementary to all or part of any probe sequence of the primary probe.
  • more than one type of detection probe is utilized, with each detection probe being substantially complementary to all or part of each probe sequence of the primary probe.
  • hybridization complexes which either contain covalently bound ETMs or ETMs in the form of hybridization indicators are added to the system (or both), which are then detected as is outlined herein.
  • hybridization indicators when hybridization indicators are used, they are only added to the system after the reaction is complete, to avoid the association of hybridization indicators to the probe:target complexes, although in some embodiments it may be possible to have the hybridization indicators present during the reaction.
  • the detection probes are mixed on the surface of the electrode, such that the signal from each is combined. This may be particularly preferred when the target sequence is present in low copy number.
  • the detection probes may each be at a different "address" on the surface. While this reduces the possible signal from the system, it serves as an internal control in that the signal from each should be equal, all other parameters being equal.
  • different addresses can have different densities of probes creating addresses with various sensitivities to target sequences. Systems utilizing two detection probes, each to a probe sequence of the primary probe, that is bound to a bead, are generally depicted in Figure 30, utilizing bound ETMs.
  • At least one, and preferably more, secondary probes are used.
  • the secondary scissile probes may be added to the reaction in several ways. It is important that the secondary scissile probes be prevented from hybridizing to the uncleaved primary probes, as this results in the generation of false positive signal. These methods may be described in several ways, depending on whether bead-bound probes are used.
  • the primary and secondary probes are bound to solid supports.
  • the primary and secondary probes are added together, since generally the support-bound secondary probes will be unable to bind to the uncleaved primary probes on the surface of a bead. It is only upon hybridization of the primary probes with the target, resulting in cleavage and release of primary probe sequences from the bead, that the now diffusible primary probe sequences may bind to the secondary probes.
  • the primary probe sequences serve as targets for the secondary scissile probes, resulting in cleavage and release of secondary probe sequences.
  • the beads containing the primary probes are added, the reaction is allowed to proceed for some period of time, and then the beads containing the secondary probes are added, either with removal of the primary beads or not.
  • the beads containing the primary probes are removed and soluble secondary scissile probes are added.
  • the complete reaction is done in solution.
  • the primary probes are added, the reaction is allowed to proceed for some period of time, and the uncleaved primary scissile probes are removed, as outlined above.
  • the secondary probes are then added, and the reaction proceeds.
  • the secondary uncleaved probes are then removed, and the cleaved sequences are detected as is generally outlined herein.
  • preferred embodiments utilize detection probes that are substantially complementary to all or part of each probe sequence of a scissile probe. Alternatively, only detection probes for "higher order" probe sequences are used.
  • detection probes for only one of the probe sequences of any scissile probe may be used. Furthermore, as outlined above, in these embodiments, as for the others, the detection probes may be separated by sequence, or mixed, depending on the desired results.
  • tertiary probes are used.
  • the tertiary scissile probes may be added to the reaction in several ways. It is important that the tertiary scissile probes be prevented from hybridizing to the uncleaved secondary probes, as this results in the generation of false positive signal. These methods may be described in several ways, depending on whether bead-bound probes are used.
  • the primary, secondary and tertiary probes are bound to solid supports.
  • the primary, secondary and tertiary probes are added together, since generally the support-bound secondary probes will be unable to bind to the uncleaved primary probes on the surface of a bead and the support-bound tertiary probes will be unable to bind to the uncleaved secondary probes on the surface of a bead. It is only upon hybridization of the scissile probe with its target (i.e. either the target sequences of the sample for the primary probes, or probe sequences for each of the secondary and tertiary probes), that results in cleavage and release of probe sequences from the bead, that the now diffusable probe sequences may bind to the higher order probes.
  • target i.e. either the target sequences of the sample for the primary probes, or probe sequences for each of the secondary and tertiary probes
  • combinations of beads and solution probes are used, with any combination being possible: primary probe beads, soluble secondary probes, tertiary beads; soluble primary probes, soluble secondary probes, tertiary beads; primary probe beads, secondary probe beads, soluble tertiary probes; etc. What is important is that if soluble probes are used, they must be removed prior to the addition of the next higher order probe.
  • the complete reaction is done in solution.
  • the primary probes are added, the reaction is allowed to proceed for some period of time, and the uncleaved primary scissile probes are removed, as outlined above.
  • the secondary probes are then added, and the reaction proceeds.
  • the secondary uncleaved probes are then removed, and the tertiary probes are added, and the reaction proceeds.
  • the uncleaved tertiary probes are then removed and the cleaved sequences are detected as is generally outlined herein.
  • preferred embodiments utilize detection probes that are substantially complementary to all or part of each probe sequence of a scissile probe. Again, the detection sequences may be separated on the electrode, or mixed, to allow the greatest signal amplification.
  • At least one, and preferably more, quaternary probes are used. This proceeds as outlined above for tertiary probes.
  • CPT requires, again in no particular order, a first CPT primer comprising a first probe sequence, a scissile linkage and a second probe sequence; and a cleavage agent.
  • InvaderTM technology is used. InvaderTM technology is based on structure-specific polymerases that cleave nucleic acids in a site-specific manner. Two probes are used: an "invader” probe and a “signalling" probe, that adjacently hybridize to a target sequence with a non-complementary overlap. The enzyme cleaves at the overlap due to its recognition of the "tail", and releases the "tail” . This can then be detected.
  • the InvaderTM technology is described in U.S.
  • the invention provides a first primer, sometimes referred to herein as an "invader primer", that hybridizes to a first domain of a target sequence, and a second primer, sometimes referred to herein as the signalling primer, that hybridizes to a second domain of the target sequence.
  • the first and second target domains are adjacent.
  • the signalling primer further comprises an overlap sequence, comprising at least one nucleotide, that is perfectly complementary to at least one nucleotide of the first target domain, and a non-complementary "tail" region.
  • the cleavage enzyme recognizes the overlap structure and the noncomplementary tail, and cleaves the tail from the second primer.
  • Suitable cleavage enzymes are described in the Patents outlined above, and include, but are not limited to, 5' thermostable nucleases from Thermus species, including Thermus aquaticus, Thermus flavus and Thermus thermophilus.
  • the entire reaction is done isothermally at a temperature such that upon cleavage, the invader probe and the cleaved signalling probe come off the target stand, and new primers can bind. In this way large amounts of cleaved signalling probe (i.e. "tails”) are made.
  • the uncleaved signalling probes are removed (for example by binding to a solid support such as a bead, either on the basis of the sequence or through the use of a binding ligand attached to the portion of the signalling probe that hybridizes to the target).
  • the cleaved signalling probes are then detected by forming an assay complex on an electrode comprising the cleaved probe ("tail") as the target, a capture probe and at least one ETM.
  • the ETM may be covalently attached to the tail, or to a label probe which hybridizes either directly or indirectly (e.g. through the use of an amplifier probe) to the assay complex.
  • the signal amplification technique is a "sandwich" assay, as is generally described in U.S.S.N. 60/073,011 and in U.S. Patent Nos. 5,681 ,702, 5,597,909, 5,545,730, 5,594,117, 5,591 ,584, 5,571 ,670, 5,580,731 , 5,571 ,670, 5,591 ,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681 ,697, all of which are hereby incorporated by reference.
  • sandwich assays do not result in the alteration of primers, sandwich assays can be considered signal amplification techniques since multiple signals (i.e. label probes) are bound to a single target, resulting in the amplification of the signal.
  • Sandwich assays are used when the target sequence comprises little or no ETM labels; that is, when a secondary probe, comprising the ETM labels, is used to generate the signal.
  • the sandwich assays can be used for the detection of primary target sequences (e.g.
  • any of the newly synthesized strands outlined above, for example using PCR, LCR, NASBA, SDA, etc., may be used as the "target sequence" in a sandwich assay.
  • sandwich signal amplification techniques may be described as follows.
  • the target sequences are immobilized on the electrode surface. This is preferably done using capture probes and optionally one or more capture extender probes; see Figure 15.
  • capture extender probes may be used, that allow a "universal" surface, i.e. a surface containing a single type of capture probe that can be used to detect any target sequence.
  • Capture extender probes are generally depicted in Figure 15, and other figures, and have a first portion that will hybridize to all or part of the capture probe, and a second portion that will hybridize to a first portion of the target sequence. This then allows the generation of customized soluble probes, which as will be appreciated by those in the art is generally simpler and less costly. As shown herein, two capture extender probes may be used. This has generally been done to stabilize assay complexes (for example when the target sequence is large, or when large amplifier probes (particularly branched or dendrimer amplifier probes) are used.
  • the nucleic acids are added after the formation of the SAM, discussed below. This may be done in a variety of ways, as will be appreciated by those in the art.
  • conductive oligomers with terminal functional groups are made, with preferred embodiments utilizing activated carboxylates and isothiocyanates, that will react with primary amines that are put onto the nucleic acid, as is generally depicted in Figure 6 using an activated carboxylate.
  • activated carboxylates and isothiocyanates that will react with primary amines that are put onto the nucleic acid, as is generally depicted in Figure 6 using an activated carboxylate.
  • These two reagents have the advantage of being stable in aqueous solution, yet react with primary alkylamines.
  • the primary aromatic amines and secondary and tertiary amines of the bases should not react, thus allowing site specific addition of nucleic acids to the surface. This allows the spotting of probes (either capture or detection probes, or both) using known
  • binding partner pairs can be utilized; i.e. one binding partner is attached to the terminus of an attachment linker, described below, and the other to the end of the nucleic acid.
  • This may also be done without using a nucleic acid capture probe; that is, one binding partner serves as the capture probe and the other is attached to either the target sequence or a capture extender probe. That is, either the target sequence comprises the binding partner, or a capture extender probe that will hybridize to the target sequence comprises the binding partner.
  • Suitable binding partner pairs include, but are not limited to, hapten pairs such as biotin/streptavidin; antigens/antibodies; NTA/histidine tags; etc. In general, smaller binding partners are preferred, such that the electrons can pass from the nucleic acid into the conductive oligomer to allow detection.
  • the binding partner when the target sequence itself is modified to contain a binding partner, is attached via a modified nucleotide that can be enzymatically attached to the target sequence, for example during a PCR target amplification step.
  • the binding partner should be easily attached to the target sequence.
  • a capture extender probe may be utilized that has a nucleic acid portion for hybridization to the target as well as a binding partner (for example, the capture extender probe may comprise a non-nucleic acid portion such as an alkyl linker that is used to attach a binding partner).
  • the capture extender probe may comprise a non-nucleic acid portion such as an alkyl linker that is used to attach a binding partner.
  • it may be desirable to cross-link the double-stranded nucleic acid of the target and capture extender probe for stability, for example using psoralen as is known in the art.
  • the target is not bound to the electrode surface using capture probes.
  • excess label probes be removed prior to detection and that the assay complex be in proximity to the surface.
  • the assay complex comprising the ETMs may be present on beads that are added to the electrode comprising the monolayer, and then the beads brought into proximity of the electrode surface using techniques well known in the art, including gravity settling of the beads on the surface, electrostatic or magnetic interactions between bead components and the surface, using binding partner attachment as outlined above.
  • the assay complex may be driven down to the surface, for example by pulsing the system with a voltage sufficient to drive the assay complex to the surface.
  • preferred embodiments utilize assay complexes attached via nucleic acid capture probes.
  • an amplifier probe is hybridized to the target sequence, either directly, or through the use of one or more label extender probes, which serves to allow "generic" amplifier probes to be made.
  • the amplifier probe contains a multiplicity of amplification sequences, although in some embodiments, as described below, the amplifier probe may contain only a single amplification sequence, or at least two amplification sequences.
  • the amplifier probe may take on a number of different forms; either a branched conformation, a dendrimer conformation, or a linear "string" of amplification sequences.
  • Label probes comprising ETMs then hybridize to the amplification sequences (or in some cases the label probes hybridize directly to the target sequence), and the ETMs are detected using the electrode, as is more fully outlined below.
  • the systems of the invention may take on a large number of different configurations, as is generally depicted in Figures 15, 16, 27, etc.
  • non-sandwich systems also referred to herein as “direct” detection
  • the target sequence itself is labeled with ETMs (again, either because the primers comprise ETMs or due to the incorporation of ETMs into the newly synthesized strand)
  • systems in which label probes directly bind to the target analytes and (3) systems in which label probes are indirectly bound to the target sequences, for example through the use of amplifier probes.
  • compositions comprising an amplifier probe.
  • amplifier probe or “nucleic acid multimer” or “amplification multimer” or grammatical equivalents herein is meant a nucleic acid probe that is used to facilitate signal amplification.
  • Amplifier probes comprise at least a first single-stranded nucleic acid probe sequence, as defined below, and at least one single- stranded nucleic acid amplification sequence, with a multiplicity of amplification sequences being preferred.
  • Amplifier probes comprise a first probe sequence that is used, either directly or indirectly, to hybridize to the target sequence. That is, the amplifier probe itself may have a first probe sequence that is substantially complementary to the target sequence, or it has a first probe sequence that is substantially complementary to a portion of an additional probe, in this case called a label extender probe, that has a first portion that is substantially complementary to the target sequence. In a preferred embodiment, the first probe sequence of the amplifier probe is substantially complementary to the target sequence.
  • the first probe sequence is of a length sufficient to give specificity and stability.
  • the probe sequences of the invention that are designed to hybridize to another nucleic acid are at least about 5 nucleosides long, with at least about 10 being preferred and at least about 15 being especially preferred.
  • the amplifier probes may form hairpin stem-loop structures in the absence of their target.
  • the length of the stem double-stranded sequence will be selected such that the hairpin structure is not favored in the presence of target.
  • these hairpin structures comprise four components.
  • the first component is a target binding sequence, i.e. a region complementary to the target (which may be the sample target sequence or another probe sequence to which binding is desired), that is about 10 nucleosides long, with about 15 being preferred.
  • the second component is a loop sequence, that can facilitate the formation of nucleic acid loops.
  • Particularly preferred in this regard are repeats of GTC, which has been identified in Fragile X Syndrome as forming turns. (When PNA analogs are used, turns comprising proline residues may be preferred). Generally, from three to five repeats are used, with four to five being preferred.
  • the third component is a self-complementary region, which has a first portion that is complementary to a portion of the target sequence region and a second portion that comprises a first portion of the label probe binding sequence.
  • the fourth component is substantially complementary to a label probe (or other probe, as the case may be).
  • the fourth component further comprises a "sticky end", that is, a portion that does not hybridize to any other portion of the probe, and preferably contains most, if not all, of the ETMs.
  • the general structure is depicted in Figure 18.
  • the any or all of the probes described herein may be configured to form hairpins in the absence of their targets, including the amplifier, capture, capture extender, label and label extender probes.
  • the amplifier probe provides an amplification of signal due to a multiplicity of labelling events, and several different amplifier probes, each with this multiplicity of labels, for each target sequence is used.
  • preferred embodiments utilize at least two different pools of amplifier probes, each pool having a different probe sequence for hybridization to different portions of the target sequence; the only real limitation on the number of different amplifier probes will be the length of the original target sequence.
  • the different amplifier probes contain different amplification sequences, although this is generally not preferred.
  • the amplifier probe does not hybridize to the sample target sequence directly, but instead hybridizes to a first portion of a label extender probe.
  • This is particularly useful to allow the use of "generic" amplifier probes, that is, amplifier probes that can be used with a variety of different targets. This may be desirable since several of the amplifier probes require special synthesis techniques.
  • the addition of a relatively short probe as a label extender probe is preferred.
  • the first probe sequence of the amplifier probe is substantially complementary to a first portion or domain of a first label extender single-stranded nucleic acid probe.
  • the label extender probe also contains a second portion or domain that is substantially complementary to a portion of the target sequence.
  • Both of these portions are preferably at least about 10 to about 50 nucleotides in length, with a range of about 15 to about 30 being preferred.
  • the terms "first” and “second” are not meant to confer an orientation of the sequences with respect to the 5'-3' orientation of the target or probe sequences.
  • the first portion may be located either 5' to the second portion, or 3' to the second portion.
  • the order of probe sequences are generally shown from left to right.
  • more than one label extender probe-amplifier probe pair may be used, that is, n is more than 1.
  • a plurality of label extender probes may be used, each with a portion that is substantially complementary to a different portion of the target sequence; this can serve as another level of amplification.
  • a preferred embodiment utilizes pools of at least two label extender probes, with the upper limit being set by the length of the target sequence.
  • more than one label extender probe is used with a single amplifier probe to reduce non-specific binding, as is generally outlined in U.S. Patent No. 5,681 ,697, incorporated by reference herein.
  • a first portion of the first label extender probe hybridizes to a first portion of the target sequence
  • the second portion of the first label extender probe hybridizes to a first probe sequence of the amplifier probe.
  • a first portion of the second label extender probe hybridizes to a second portion of the target sequence
  • the second portion of the second label extender probe hybridizes to a second probe sequence of the amplifier probe.
  • the label extender probes may interact with a preamplifier probe, described below, rather than the amplifier probe directly.
  • a preferred embodiment utilizes several different amplifier probes, each with first probe sequences that will hybridize to a different portion of the label extender probe.
  • the different amplifier probes contain different amplification sequences, although this is generally not preferred.
  • the amplifier probe also comprises at least one amplification sequence.
  • An "amplification sequence” or “amplification segment” or grammatical equivalents herein is meant a sequence that is used, either directly or indirectly, to bind to a first portion of a label probe as is more fully described below (although in some cases the amplification sequence may bind to a detection probe; see Figure 27C).
  • the amplifier probe comprises a multiplicity of amplification sequences, with from about 3 to about 1000 being preferred, from about 10 to about 100 being particularly preferred, and about 50 being especially preferred. In some cases, for example when linear amplifier probes are used, from 1 to about 20 is preferred with from about 5 to about 10 being particularly preferred.
  • the amplification sequences may be linked to each other in a variety of ways, as will be appreciated by those in the art. They may be covalently linked directly to each other, or to intervening sequences or chemical moieties, through nucleic acid linkages such as phosphodiester bonds, PNA bonds, etc., or through interposed linking agents such amino acid, carbohydrate or polyol bridges, or through other cross-linking agents or binding partners.
  • the site(s) of linkage may be at the ends of a segment, and/or at one or more internal nucleotides in the strand.
  • the amplification sequences are attached via nucleic acid linkages.
  • branched amplifier probes are used, as are generally described in U.S. Patent No. 5,124,246, hereby incorporated by reference.
  • Branched amplifier probes may take on “fork-like” or “comb-like” conformations.
  • "Fork-like” branched amplifier probes generally have three or more oligonucleotide segments emanating from a point of origin to form a branched structure. The point of origin may be another nucleotide segment or a multifunctional molecule to whcih at least three segments can be covalently or tightly bound.
  • "Comb-like" branched amplifier probes have a linear backbone with a multiplicity of sidechain oligonucleotides extending from the backbone.
  • the pendant segments will normally depend from a modified nucleotide or other organic moiety having the appropriate functional groups for attachment of oligonucleotides.
  • a large number of amplification sequences are available for binding, either directly or indirectly, to detection probes.
  • these structures are made as is known in the art, using modified multifunctional nucleotides, as is described in U.S. Patent Nos. 5,635,352 and 5,124,246, among others.
  • dendrimer amplifier probes are used, as are generally described in U.S. Patent No. 5,175,270, hereby expressly incorporated by reference.
  • Dendrimeric amplifier probes have amplification sequences that are attached via hybridization, and thus have portions of double-stranded nucleic acid as a component of their structure.
  • the outer surface of the dendrimer amplifier probe has a multiplicity of amplification sequences.
  • linear amplifier probes are used, that have individual amplification sequences linked end-to-end either directly or with short intervening sequences to form a polymer. As with the other amplifier configurations, there may be additional sequences or moieties between the amplification sequences. In addition, as outlined herein, linear amplification probes may form hairpin stem-loop structures, as is depicted in Figure 18.
  • the linear amplifier probe has a single amplification sequence. This may be useful when cycles of hybridization/disassociation occurs, forming a pool of amplifier probe that was hybridized to the target and then removed to allow more probes to bind, or when large numbers of ETMs are used for each label probe.
  • linear amplifier probes comprise a multiplicity of amplification sequences.
  • the amplifier probe may be totally linear, totally branched, totally dendrimeric, or any combination thereof.
  • the amplification sequences of the amplifier probe are used, either directly or indirectly, to bind to a label probe to allow detection.
  • the amplification sequences of the amplifier probe are substantially complementary to a first portion of a label probe.
  • amplifier extender probes are used, that have a first portion that binds to the amplification sequence and a second portion that binds to the first portion of the label probe.
  • compositions of the invention may include "preamplifier” molecules, which serves a bridging moiety between the label extender molecules and the amplifier probes. In this way, more amplifier and thus more ETMs are ultimately bound to the detection probes.
  • Preamplifier molecules may be either linear or branched, and typically contain in the range of about 30-3000 nucleotides.
  • label probes are either substantially complementary to an amplification sequence or to a portion of the target sequence. Accordingly, the label probes can be configured in a variety of ways, as is generally described herein, depending on whether a "mechanism-1" or “mechanism-2" detection system is utilized, as described below.
  • Detection of the amplification reactions of the invention is done by detecting assay complexes comprising ETMs, which can be attached to the assay complex in a variety of ways, as is more fully described below.
  • ETMs assay complexes comprising ETMs
  • detection of an ETM is based on electron transfer through the stacked ⁇ -orbitals of double stranded nucleic acid. This basic mechanism is described in U.S. Patent Nos.
  • ETMs either covalently to one of the strands or non-covalently to the hybridization complex through the use of hybridization indicators, described below
  • a nucleic acid that is attached to a detection electrode via a conductive oligomer electron transfer between the ETM and the electrode, through the nucleic acid and conductive oligomer, may be detected.
  • This general idea is depicted in Figure 27.
  • the ETM can be detected, not necessarily via electron transfer through nucleic acid, but rather can be directly detected on the surface of the electrode.
  • the detection electrode preferably comprises a self-assembled monolayer (SAM) that serves to shield the electrode from redox-active species in the sample.
  • SAM self-assembled monolayer
  • the presence of ETMs on the surface of a SAM, that has been formulated to comprise slight "defects” can be directly detected.
  • This basic idea is termed "mechanism-2" herein.
  • the electroconduits allow particular ETMs access to the surface.
  • the configuration of the electroconduit depends in part on the ETM chosen.
  • the use of relatively hydrophobic ETMs allows the use of hydrophobic electroconduit forming species, which effectively exclude hydrophilic or charged ETMs.
  • the use of more hydrophilic or charged species in the SAM may serve to exclude hydrophobic ETMs.
  • the electroconduits can be generated in several general ways, including but not limited to the use of rough electrode surfaces, such as gold electrodes formulated on PC circuit boards; or the inclusion of at least two different species in the monolayer, i.e. using a "mixed monolayer", at least one of which is a electroconduit-forming species (EFS).
  • ETM electroconduit-forming species
  • the role of the SAM comprising the defects is to allow contact of the ETM with the electronic surface of the electrode, while still providing the benefits of shielding the electrode from solution components and reducing the amount of non-specific binding to the electrodes.
  • the role of the binding ligand is to provide specificity for a recruitment of ETMs to the surface, where they can be directly detected.
  • the present invention is directed to the formation of assay complexes on electrodes, generally comprising SAMs.
  • the presence or absence of the ETMs are detected as is described below and in U.S. Patent Nos. 5,591 ,578; 5,824,473; 5,770,369; 5,705,348 and 5,780,234; U.S.S.N.s 08/911 ,589; 09/135,183; 09/306,653; 09/134,058; 09/295,691 ; 09/238,351 ; 09/245,105 and 09/338,726; and PCT applications WO98/20162; PCT US99/01705; PCT US99/01703; PCT US99/10104, all of which are expressly incorporated herein by reference in their entirety.
  • an assay complex is formed that contains an ETM, which is then detected using the detection electrode.
  • the invention thus provides assay complexes that minimally comprise a target sequence.
  • "Assay complex” herein is meant the collection of hybridization complexes comprising nucleic acids, including probes and targets, that contains at least one ETM and thus allows detection.
  • the composition of the assay complex depends on the use of the different probe components outlined herein.
  • the assay complex comprises the capture probe and the target sequence.
  • the assay complexes may also include label probes, capture extender probes, label extender probes, and amplifier probes, as outlined herein, depending on the configuration used.
  • the assay complexes comprise at least one ETM, which can either be covalently attached to a component of the assay complex as described herein or a "hybridization indicator", described below.
  • ETM electrospray transfer indicator
  • ETMs electrospray transfer indicator
  • electron donor and acceptor capabilities are relative; that is, a molecule which can lose an electron under certain experimental conditions will be able to accept an electron under different experimental conditions.
  • ETMs include, but are not limited to, transition metal complexes, organic ETMs, and electrodes.
  • the ETMs are transition metal complexes.
  • Transition metals are those whose atoms have a partial or complete d shell of electrons. Suitable transition metals for use in the invention include, but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, the first series of transition metals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred.
  • ruthenium particularly preferred are ruthenium, rhenium, osmium, platinium, cobalt and iron.
  • the transition metals are complexed with a variety of ligands, to form suitable transition metal complexes, as is well known in the art.
  • L are the co-ligands, that provide the coordination atoms for the binding of the metal ion.
  • the number and nature of the co-ligands will depend on the coordination number of the metal ion.
  • Mono-, di- or polydentate co- ligands may be used at any position.
  • the co-ligands can be the same or different. Suitable ligands fall into two categories: ligands which use nitrogen, oxygen, sulfur, carbon or phosphorus atoms (depending on the metal ion) as the coordination atoms (generally referred to in the literature as sigma ( ⁇ ) donors) and organometallic ligands such as metallocene ligands (generally referred to in the literature as pi ( ⁇ ) donors, and depicted herein as L m ).
  • Suitable nitrogen donating ligands are well known in the art and include, but are not limited to, NH 2 ; NHR; NRR'; pyridine; pyrazine; isonicotinamide; imidazole; bipyridine and substituted derivatives of bipyridine; terpyridine and substituted derivatives; phenanthrolines, particularly 1 ,10-phenanthroline (abbreviated phen) and substituted derivatives of phenanthrolines such as 4,7-dimethylphenanthroline and dipyridol[3,2-a:2',3'-c]phenazine (abbreviated dppz); dipyridophenazine; 1 ,4,5,8,9,12-hexaazatriphenylene (abbreviated hat); 9,10- phenanthrenequinone diimine (abbreviated phi); 1 ,4,5,8-tetraazaphenanthrene (abbreviated tap); 1 ,4,8,11-tetra-azacyclo
  • Substituted derivatives including fused derivatives, may also be used.
  • porphyrins and substituted derivatives of the porphyrin family may be used. See for example, Comprehensive Coordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987, Chapters 13.2 (pp73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), all of which are hereby expressly incorporated by reference.
  • Suitable sigma donating ligands using carbon, oxygen, sulfur and phosphorus are known in the art.
  • suitable sigma carbon donors are found in Cotton and Wilkenson, Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference; see page 38, for example.
  • suitable oxygen ligands include crown ethers, water and others known in the art.
  • Phosphines and substituted phosphines are also suitable; see page 38 of Cotton and Wilkenson.
  • oxygen, sulfur, phosphorus and nitrogen-donating ligands are attached in such a manner as to allow the heteroatoms to serve as coordination atoms.
  • organometallic ligands are used.
  • transition metal organometallic compounds with ⁇ -bonded organic ligands see Advanced Inorganic Chemistry, 5th Ed., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26; Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed., 1992, VCH; and Comprehensive Organometallic
  • organometallic ligands include cyclic aromatic compounds such as the cyclopentadienide ion [C 5 H 5 (-1 )] and various ring substituted and ring fused derivatives, such as the indenylide (-1 ) ion, that yield a class of bis(cyclopentadieyl) metal compounds, (i.e. the metallocenes); see for example Robins et al., J. Am. Chem. Soc.
  • ferrocene [(C 5 H 5 ) 2 Fe] and its derivatives are prototypical examples which have been used in a wide variety of chemical (Connelly et al., Chem. Rev. 96:877- 910 (1996), incorporated by reference) and electrochemical (Geiger et al., Advances in Organometallic Chemistry 23:1-93; and Geiger et al., Advances in Organometallic Chemistry 24:87, incorporated by reference) electron transfer or "redox" reactions.
  • Metallocene derivatives of a variety of the first, second and third row transition metals are potential candidates as redox moieties that are covalently attached to the nucleic acid.
  • Other potentially suitable organometallic ligands include cyclic arenes such as benzene, to yield bis(arene)metal compounds and their ring substituted and ring fused derivatives, of which bis(benzene)chromium is a prototypical example,
  • Other acyclic ⁇ -bonded ligands such as the allyl(-1 ) ion, or butadiene yield potentially suitable organometallic compounds, and all such ligands, in conjuction with other ⁇ -bonded and ⁇ -bonded ligands constitute the general class of organometallic compounds in which there is a metal to carbon bond.
  • organometallic ligands include metallocene ligands, including substituted derivatives and the metalloceneophanes (see page 1174 of Cotton and Wilkenson, supra).
  • derivatives of metallocene ligands such as methylcyclopentadienyl, with multiple methyl groups being preferred, such as pentamethylcyclopentadienyl, can be used to increase the stability of the metallocene.
  • metallocene ligands such as methylcyclopentadienyl, with multiple methyl groups being preferred, such as pentamethylcyclopentadienyl, can be used to increase the stability of the metallocene.
  • only one of the two metallocene ligands of a metallocene are derivatized.
  • any combination of ligands may be used.
  • Preferred combinations include: a) all ligands are nitrogen donating ligands; b) all ligands are organometallic ligands; and c) one ligand is a metallocene ligand and another is a nitrogen donating ligand, with the other ligands, if needed, are either nitrogen donating ligands or metallocene ligands, or a mixture.
  • organic electron donors and acceptors may be covalently attached to the nucleic acid for use in the invention.
  • organic molecules include, but are not limited to, riboflavin, xanthene dyes, azine dyes, acridine orange, A/,/V-dinnet.hyl-2,7- diazapyrenium dichloride (DAP 2+ ), methylviologen, ethidium bromide, quinones such as N,N'- dimethylanthra(2,1 ,9-def:6,5,10-d'e'Odiisoquinoline dichloride (ADIQ 2+ ); porphyrins ([meso-tetrakis(N- methyl-x-pyridinium)porphyrin tetrachloride], varlamine blue B hydrochloride, Bindschedler's green; 2,6-dichloroindophenol, 2,6-dibromophenolindophenol;
  • the electron donors and acceptors are redox proteins as are known in the art. However, redox proteins in many embodiments are not preferred.
  • ETMs The choice of the specific ETMs will be influenced by the type of electron transfer detection used, as is generally outlined below.
  • Preferred ETMs are metallocenes, with ferrocene being particularly preferred.
  • hydrophobic ETMs are preferred in mechanism-2 systems; however, transition metal complexes, although charged, with one or more hydrophobic ligands, such as Ru and Os complexes, also give rise to good signals. Similarly, electron transfer between the ETM and the electrode is facilitated by the use of linkers or spacers that allow the
  • the N6 compositions of the invention have a four carbon linker attaching the ETM to the nucleic acid.
  • a plurality of ETMs are used.
  • the use of multiple ETMs provides signal amplification and thus allows more sensitive detection limits. While the use of multiple ETMs on nucleic acids that hybridize to complementary strands (i.e. mechanism-1 systems) can cause decreases in T m s of the hybridization complexes depending on the number, site of attachment and spacing between the multiple ETMs, this is not a factor when the ETMs are on the recruitment linker, since this does not hybridize to a complementary sequence. Accordingly, pluralities of ETMs are preferred, with at least about 2 ETMs per assay complex being preferred, and at least about 10 being particularly preferred, and at least about 20 to 50 being especially preferred. In some instances, very large numbers of ETMs (100 to 10000 or greater) can be used.
  • Attachment of the ETM to the assay complex can be done in a wide variety of ways, and depends on the mechanism of detection and whether direct or indirect detection is done. In general, methods and compositions outlined in WO98/20162, expressly incorporated by reference in its entirety, can be used.
  • the label probe is a label probe that comprises the ETMs.
  • the label probe is used in a mechanism-2 detection system.
  • the portion of the label probe (or target, in some embodiments) that comprises the ETMs can be nucleic acid, or it can be a non-nucleic acid linker that links the first hybridizable portion of the label probe to the ETMs. That is, since this portion of the label probe is not required for hybridization if a mechanism-2 system is used, it need not be nucleic acid, although this may be done for ease of synthesis.
  • the recruitment linker may comprise double-stranded portions.
  • the recruitment linker is nucleic acid (including analogs), and attachment of the ETMs can be via (1 ) a base; (2) the backbone, including the ribose, the phosphate, or comparable structures in nucleic acid analogs; (3) nucleoside replacement, described below; or (4) metallocene polymers, as described below.
  • the linker is non-nucleic acid, and can be either a metallocene polymer or an alkyl-type polymer (including heteroalkyl, as is more fully described below) containing ETM substitution groups.
  • the ETMs are generally attached via either (1 ) or (2) above, since hybridization of the label probe to a detection probe, as outlined herein, requires the formation of a hybridization complex.
  • the recruitment linker is a nucleic acid, and comprises covalently attached ETMs.
  • the ETMs may be attached to nucleosides within the nucleic acid in a variety of positions. Preferred embodiments include, but are not limited to, (1 ) attachment to the base of the nucleoside, (2) attachment of the ETM as a base replacement, (3) attachment to the backbone of the nucleic acid, including either to a ribose of the ribose-phosphate backbone or to a phosphate moiety, or to analogous structures in nucleic acid analogs, and (4) attachment via metallocene polymers, with the latter being preferred.
  • the recruitment linker is nucleic acid
  • secondary label probes that have a first portion that will hybridize to a portion of the primary label probes and a second portion comprising a recruitment linker as is defined herein. This is generally depicted in Figure 16H; this is similar to the use of an amplifier probe, except that both the primary and the secondary label probes comprise ETMs.
  • the ETM is attached to the base of a nucleoside as is generally outlined in WO 98/20162, incorporated by reference, for attachment of conductive oligomers. Attachment can be to an internal nucleoside or a terminal nucleoside.
  • the covalent attachment to the base will depend in part on the ETM chosen, but in general is similar to the attachment of conductive oligomers to bases, as outlined in WO 98/20162. Attachment may generally be done to any position of the base.
  • the ETM is a transition metal complex, and thus attachment of a suitable metal ligand to the base leads to the covalent attachment of the ETM.
  • similar types of linkages may be used for the attachment of organic ETMs, as will be appreciated by those in the art.
  • the C4 attached amino group of cytosine, the C6 attached amino group of adenine, or the C2 attached amino group of guanine may be used as a transition metal ligand.
  • Ligands containing aromatic groups can be attached via acetylene linkages as is known in the art (see Comprehensive Organic Synthesis, Trost et al., Ed., Pergamon Press, Chapter 2.4: Coupling
  • Structure 1 depicts a representative structure in the presence of the metal ion and any other necessary ligands; Structure 1 depicts uridine, although as for all the structures herein, any other base may also be used.
  • L a is a ligand, which may include nitrogen, oxygen, sulfur or phosphorus donating ligands or organometallic ligands such as metallocene ligands.
  • Suitable L a ligands include, but not limited to, phenanthroline, imidazole, bpy and terpy.
  • L r and M are as defined above.
  • a linker (“Z") may be included between the nucleoside and the ETM.
  • the linkage may be done using a linker, which may utilize an amide linkage (see generally Telser et al., J. Am. Chem. Soc. 111 :7221-7226 (1989); Telser et al., J. Am. Chem. Soc. 111 :7226-7232 (1989), both of which are expressly incorporated by reference).
  • a linker which may utilize an amide linkage (see generally Telser et al., J. Am. Chem. Soc. 111 :7221-7226 (1989); Telser et al., J. Am. Chem. Soc. 111 :7226-7232 (1989), both of which are expressly incorporated by reference).
  • amide linkage see generally Telser et al., J. Am. Chem. Soc. 111 :7221-7226 (1989); Telser et al., J. Am. Chem. Soc. 111 :7226-7232 (1989), both
  • L is a ligand as defined above, with L r and M as defined above as well.
  • L is amino, phen, byp and terpy.
  • the ETM attached to a nucleoside is a metallocene; i.e. the L and L r of Structure 2 are both metallocene ligands, L m , as described above.
  • Structure 3 depicts a preferred embodiment wherein the metallocene is ferrocene, and the base is uridine, although other bases may be used: Structure 3
  • Preferred metallocenes include ferrocene, cobaltocene and osmiumocene.
  • the ETM is attached to a ribose at any position of the ribose-phosphate backbone of the nucleic acid, i.e. either the 5' or 3' terminus or any internal nucleoside.
  • Ribose in this case can include ribose analogs.
  • nucleosides that are modified at either the 2' or 3' position of the ribose can be made, with nitrogen, oxygen, sulfur and phosphorus-containing modifications possible. Amino-modified and oxygen-modified ribose is preferred. See generally WO 95/15971 and WO 98/20162, incorporated herein by reference.
  • modification groups may be used as a transition metal ligand, or as a chemically functional moiety for attachment of other transition metal ligands and organometallic ligands, or organic electron donor moieties as will be appreciated by those in the art.
  • a linker such as depicted herein for "Z” may be used as well, or a conductive oligomer between the ribose and the ETM.
  • Preferred embodiments utilize attachment at the 2' or 3' position of the ribose, with the 2' position being preferred.
  • the conductive oligomers depicted in Structure 13, 14 and 15 of WO98/20162 may be replaced by ETMs; alternatively, the ETMs may be added to the free terminus of the conductive oligomer.
  • a metallocene serves as the ETM, and is attached via an amide bond as depicted below in Structure 4.
  • the examples outline the synthesis of a preferred compound when the metallocene is ferrocene.
  • amine linkages are used, as is generally depicted in Structure 5.
  • Structure 5
  • Z is a linker, as defined herein, with 1-16 atoms being preferred, and 2-4 atoms being particularly preferred, and t is either one or zero.
  • oxo linkages are used, as is generally depicted in Structure 6.
  • Z is a linker, as defined herein, and t is either one or zero.
  • an ETM is attached to a phosphate at any position of the ribose- phosphate backbone of the nucleic acid. This may be done in a variety of ways.
  • phosphodiester bond analogs such as phosphoramide or phosphoramidite linkages may be incorporated into a nucleic acid, where the heteroatom (i.e. nitrogen) serves as a transition metal ligand (see PCT publication WO 95/15971 and WO 98/20162, incorporated by reference).
  • the conductive oligomers depicted in Structures 23 and 24 of WO98/20162 may be replaced by ETMs.
  • the composition has the structure shown in Structure 7.
  • the ETM is attached via a phosphate linkage, generally through the use of a linker, Z.
  • alkyl-based linkers can be used to create multiple branching structures comprising one or more ETMs at the terminus of each branch. Generally, this is done by creating branch points containing multiple hydroxy groups, which optionally can then be used to add additional branch points. The terminal hydroxy groups can then be used in phosphoramidite reactions to add ETMs, as is generally done below for the nucleoside replacement and metallocene polymer reactions.
  • an ETM such as a metallocene is used as a "nucleoside replacement", serving as an ETM.
  • the distance between the two cyclopentadiene rings of ferrocene is similar to the orthongonal distance between two bases in a double stranded nucleic acid.
  • Other metallocenes in addition to ferrocene may be used, for example, air stable metallocenes such as those containing cobalt or ruthenium.
  • metallocene moieties may be incorporated into the backbone of a nucleic acid, as is generally depicted in Structure 8 (nucleic acid with a ribose- phosphate backbone) and Structure 9 (peptide nucleic acid backbone).
  • Structures 8 and 9 depict ferrocene, although as will be appreciated by those in the art, other metallocenes may be used as well. In general, air stable metallocenes are preferred, including metallocenes utilizing ruthenium and cobalt as the metal.
  • Z is a linker as defined above, with generally short, alkyl groups, including heteroatoms such as oxygen being preferred. Generally, what is important is the length of the linker, such that minimal perturbations of a double stranded nucleic acid is effected, as is more fully described below. Thus, methylene, ethylene, ethylene glycols, propylene and butylene are all preferred, with ethylene and ethylene glycol being particularly preferred.
  • each Z linker may be the same or different.
  • Structure 8 depicts a ribose-phosphate backbone, although as will be appreciated by those in the art, nucleic acid analogs may also be used, including ribose analogs and phosphate bond analogs.
  • each Z linker can be the same or different.
  • other nucleic acid analogs may be used as well.
  • ETMs in addition to metallocenes, as nucleoside replacements or in polymer embodiments, described below.
  • the ETM is a transition metal complex other than a metallocene, comprising one, two or three (or more) ligands
  • the ligands can be functionalized as depicted for the ferrocene to allow the addition of phosphoramidite groups.
  • complexes comprising at least two ring (for example, aryl and substituted aryl) ligands, where each of the ligands comprises functional groups for attachment via phosphoramidite chemistry.
  • this type of reaction creating polymers of ETMs either as a portion of the backbone of the nucleic acid or as "side groups" of the nucleic acids, to allow amplification of the signals generated herein, can be done with virtually any ETM that can be functionalized to contain the correct chemical groups.
  • nucleic acid analogs are made; that is, the invention provides nucleic acids having a backbone comprising at least one metallocene.
  • a metallocene such as ferrocene (or other ETM)
  • the invention provides nucleic acids having a backbone comprising at least one metallocene.
  • nucleic acids having metallocenes attached to the backbone i.e. via a ribose, a phosphate, etc. That is, two nucleic acids each made up of a traditional nucleic acid or analog (nucleic acids in this case including a single nucleoside), may be covalently attached to each other via a metallocene.
  • a metallocene derivative or substituted metallocene is provided, wherein each of the two aromatic rings of the metallocene has a nucleic acid substitutent group.
  • metallocene polymers that is, there are areas of metallocene polymers within the backbone.
  • nucleic acid substitutent groups it is also desirable in some instances to add additional substituent groups to one or both of the aromatic rings of the metallocene (or ETM).
  • ETM metallocene
  • these nucleoside replacements are generally part of probe sequences to be hybridized with a substantially complementary nucleic acid, for example a target sequence or another probe sequence
  • substitutent groups to the metallocene rings to facilitate hydrogen bonding to the base or bases on the opposite strand. These may be added to any position on the metallocene rings.
  • Suitable substitutent groups include, but are not limited to, amide groups, amine groups, carboxylic acids, and alcohols, including substituted alcohols.
  • these substitutent groups can be attached via linkers as well, although in general this is not preferred.
  • substituent groups on an ETM may be added to alter the redox properties of the ETM.
  • substituent groups on an ETM may be added in different ways (i.e. base or ribose attachment), on different probes, or for different purposes (for example, calibration or as an internal standard).
  • the addition of substituent groups on the metallocene may allow two different ETMs to be distinguished.
  • the intermediate components are also provided.
  • the invention provides phosphoramidite metallocenes, as generally depicted in Structure 10:
  • PG is a protecting group, generally suitable for use in nucleic acid synthesis, with DMT, MMT and TMT all being preferred.
  • the aromatic rings can either be the rings of the metallocene, or aromatic rings of ligands for transition metal complexes or other organic ETMs.
  • the aromatic rings may be the same or different, and may be substituted as discussed herein.
  • Structure 11 depicts the ferrocene derivative:
  • Structure 12 depicts the ferrocene peptide nucleic acid (PNA) monomer, that can be added to PNA synthesis as is known in the art and depicted within the Figures and Examples: Structure 12
  • PNA ferrocene peptide nucleic acid
  • the PG protecting group is suitable for use in peptide nucleic acid synthesis, with MMT, boc and Fmoc being preferred.
  • ETM or metallocene polymers which are added to the nucleic acids, rather than as backbone replacements, as is more fully described below.
  • the ETMs are attached as polymers, for example as metallocene polymers, in a "branched" configuration similar to the "branched DNA” embodiments herein and as outlined in U.S. Patent No. 5,124,246, using modified functionalized nucleotides.
  • the general idea is as follows. A modified phosphoramidite nucleotide is generated that can ultimately contain a free hydroxy group that can be used in the attachment of phosphoramidite ETMs such as metallocenes.
  • This free hydroxy group could be on the base or the backbone, such as the ribose or the phosphate (although as will be appreciated by those in the art, nucleic acid analogs containing other structures can also be used).
  • the modified nucleotide is incorporated into a nucleic acid, and any hydroxy protecting groups are removed, thus leaving the free hydroxyl.
  • ETMs such as metallocene ETMs, are added.
  • ETM polymers including "metallocene polymers” as depicted herein, particularly for ferrocene.
  • a "capping” group to the terminal ETM in the polymer, for example a final phosphate group to the metallocene as is generally depicted in Figure 12.
  • Other suitable solubility enhancing "capping” groups will be appreciated by those in the art. It should be noted that these solubility enhancing groups can be added to the polymers in other places, including to the ligand rings, for example on the metallocenes as discussed herein
  • the 2' position of a ribose of a phosphoramidite nucleotide is first functionalized to contain a protected hydroxy group, in this case via an oxo-linkage, although any number of linkers can be used, as is generally described herein for Z linkers.
  • the protected modified nucleotide is then incorporated via standard phosphoramidite chemistry into a growing nucleic acid.
  • the protecting group is removed, and the free hydroxy group is used, again using standard phosphoramidite chemistry to add a phosphoramidite metallocene such as ferrocene.
  • a similar reaction is possible for nucleic acid analogs. For example, using peptide nucleic acids and the metallocene monomer shown in Structure 12, peptide nucleic acid structures containing metallocene polymers could be generated.
  • the present invention provides recruitment linkers of nucleic acids comprising "branches" of metallocene polymers as is generally depicted in Figures 12 and 13.
  • Preferred embodiments also utilize metallocene polymers from one to about 50 metallocenes in length, with from about 5 to about 20 being preferred and from about 5 to about 10 being especially preferred.
  • the recruitment linker is nucleic acid
  • any combination of ETM attachments may be done.
  • the recruitment linker is not nucleic acid, and instead may be any sort of linker or polymer.
  • linker or polymer that can be modified to contain ETMs can be used.
  • the polymers or linkers should be reasonably soluble and contain suitable functional groups for the addition of ETMs.
  • a "recruitment polymer” comprises at least two or three subunits, which are covalently attached. At least some portion of the monomeric subunits contain functional groups for the covalent attachment of ETMs. In some embodiments coupling moieties are used to covalently link the subunits with the ETMs. Preferred functional groups for attachment are amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred. As will be appreciated by those in the art, a wide variety of recruitment polymers are possible.
  • Suitable linkers include, but are not limited to, alkyl linkers (including heteroalkyl (including
  • linkers will comprise one or more functional groups for the attachment of ETMs, which will be done as will be appreciated by those in the art, for example through the use homo-or hetero- bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • Suitable recruitment polymers include, but are not limited to, functionalized styrenes, such as amino styrene, functionalized dextrans, and polyamino acids.
  • Preferred polymers are polyamino acids (both poly-D-amino acids and poly-L-amino acids), such as polylysine, and polymers containing lysine and other amino acids being particularly preferred.
  • Other suitable polyamino acids are polyglutamic acid, polyaspartic acid, co-polymers of lysine and glutamic or aspartic acid, co-polymers of lysine with alanine, tyrosine, phenylalanine, serine, tryptophan, and/or proline.
  • the recruitment linker comprises a metallocene polymer, as is described above.
  • the attachment of the recruitment linkers to the first portion of the label probe will depend on the composition of the recruitment linker, as will be appreciated by those in the art.
  • the recruitment linker is nucleic acid, it is generally formed during the synthesis of the first portion of the label probe, with incorporation of nucleosides containing ETMs as required.
  • the first portion of the label probe and the recruitment linker may be made separately, and then attached. For example, there may be an overlapping section of complementarity, forming a section of double stranded nucleic acid that can then be chemically crosslinked, for example by using psoralen as is known in the art.
  • attachment of the linker/polymer of the recruitment linker will be done generally using standard chemical techniques, such as will be appreciated by those in the art.
  • attachment can be similar to the attachment of insulators to nucleic acids.
  • recruitment linkers that are mixtures of nucleic acids and non-nucleic acids, either in a linear form (i.e. nucleic acid segments linked together with alkyl linkers) or in branched forms (nucleic acids with alkyl "branches" that may contain ETMs and may be additionally branched).
  • the ETM is attached to the target sequence, either by incorporation into a primer, or by incorporation into the newly synthesized portion of the target sequence.
  • Target sequences comprising covalently attached ETMs can be directly detected using either a mechanism-1 or mechanism-2 system, as is shown in the figures.
  • the target sequence itself that carries the ETMs, rather than the recruitment linker of a label probe.
  • this may be done using target sequences that have ETMs incorporated at any number of positions, including either within a primer or within the newly synthesized strand, and can be attached to the nucleic acid in a variety of positions, as outlined herein.
  • the 3'-5' orientation of the probes and targets is chosen to get the ETM-containing structures (i.e. label probes, recruitment linkers or target sequences) as close to the surface of the monolayer as possible, and in the correct orientation. This may be done using attachment via insulators or conductive oligomers as is generally shown in the Figures, and may depend on which mechanism is used.
  • multiple capture probes can be utilized, either in a configuration wherein the 5'-3' orientation of the capture probes is different, or where "loops" of target form when multiples of capture probes are used. Labelling of the target sequence with ETMs can also occur during synthesis of the new target strand.
  • triphosphate nucleotides comprising the ETMs of the invention to a growing nucleic acid, for example during the previously described amplification techniques.
  • modified nucleotides of the invention for example the "nucleoside replacement" embodiments and putatively some of the phosphate attachments, may or may not be recognized by the enzymes to allow incorporation into a growing nucleic acid. Therefore, preferred attachments in this embodiment are to the base or ribose of the nucleotide.
  • compositions utilizing electrodes comprising monolayers of conductive oligomers and capture probes, and target sequences that comprises a first portion that is capable of hybridizing to a component of an assay complex, and a second portion that does not hybridize to a component of an assay complex and comprises at least one covalently attached electron transfer moiety.
  • methods utilizing these compositions are also provided.
  • ETMs may be connected to probe sequences, i.e. sequences designed to hybridize to complementary sequences.
  • ETMs may be added to non-recruitment linkers as well.
  • ETMs may be added to sections of label probes that do hybridize to components of the assay complex, for example the first portion, or to the target sequence as outlined above.
  • These ETMs may be used for electron transfer detection in some embodiments, or they may not, depending on the location and system.
  • the target sequence containing randomly incorporated ETMs is hybridized directly to the capture probe, as is depicted in Figure 16A, there may be ETMs in the portion hybridizing to the capture probe. If the capture probe is attached to the electrode using a conductive oligomer, these ETMs can be used to detect electron transfer as has been previously described. Alternatively, these ETMs may not be specifically detected.
  • the recruitment linker when the recruitment linker is nucleic acid, it may be desirable in some instances to have some or all of the recruitment linker be double stranded.
  • the first recruitment linker comprises the covalently attached ETMs.
  • the second recruitment linker contains the ETMs, and the first recruitment linker does not, and the ETMs are recruited to the surface by hybridization of the second recruitment linker to the first.
  • both the first and second recruitment linkers comprise ETMs.
  • nucleic acids comprising a large number of ETMs may not hybridize as well, i.e. the T m may be decreased, depending on the site of attachment and the characteristics of the ETM.
  • the T m may be decreased, depending on the site of attachment and the characteristics of the ETM.
  • the ETMs when multiple ETMs are used on hybridizing strands, generally there are less than about 5, with less than about 3 being preferred, or alternatively the ETMs should be spaced sufficiently far apart that the intervening nucleotides can sufficiently hybridize to allow good kinetics.
  • non-covalently attached ETMs may be used.
  • the ETM is a hybridization indicator.
  • Hybridization indicators serve as an ETM that will preferentially associate with double stranded nucleic acid is added, usually reversibly, similar to the method of Millan et al., Anal. Chem. 65:2317-2323 (1993); Millan et al., Anal. Chem. 662943-2948 (1994), both of which are hereby expressly incorporated by reference.
  • increases in the local concentration of ETMs, due to the association of the ETM hybridization indicator with double stranded nucleic acid at the surface can be monitored using the monolayers comprising the conductive oligomers.
  • Hybridization indicators include intercalators and minor and/or major groove binding moieties.
  • intercalators may be used; since intercalation generally only occurs in the presence of double stranded nucleic acid, only in the presence of double stranded nucleic acid will the ETMs concentrate. Intercalating transition metal complex ETMs are known in the art. Similarly, major or minor groove binding moieties, such as methylene blue, may also be used in this embodiment.
  • the systems of the invention may utilize non-covalently attached ETMs, as is generally described in Napier et al., Bioconj. Chem. 8:906 (1997), hereby expressly incorporated by reference.
  • changes in the redox state of certain molecules as a result of the presence of DNA can be detected using the SAMs comprising conductive oligomers as well.
  • Figure 27 shows direct detection (i.e. target sequence comprising the ETM), and indirect detection (using label probes).
  • mechanism-2 systems are depicted in Figure 16, again showing direct detection (i.e. target sequence comprising the ETM), and indirect detection (using label probes).
  • the label probes directly hybridize to the target sequences, as is generally depicted in the figures.
  • the target sequence is preferably, but not required to be, immobilized on the surface using capture probes, including capture extender probes.
  • Label probes are then used to bring the ETMs into proximity of the surface of the monolayer comprising conductive oligomers.
  • multiple label probes are used; that is, label probes are designed such that the portion that hybridizes to the target sequence can be different for a number of different label probes, such that amplification of the signal occurs, since multiple label probes can bind for every target sequence.
  • n is an integer of at least one.
  • n is from 1 to 50, with from about 1 to about 20 being particularly preferred, and from about 2 to about 5 being especially preferred.
  • label extender probes can be used as generally described below for use with amplifier probes.
  • the configuration of the system and the label probes are designed to recruit the ETMs as close as possible to the monolayer surface.
  • the label probes are hybridized to the target sequence indirectly. That is, the present invention finds use in novel combinations of signal amplification technologies and electron transfer detection on electrodes, which may be particularly useful in sandwich hybridization assays, as generally depicted in the Figures.
  • the amplifier probes of the invention are bound to the target sequence in a sample either directly or indirectly. Since the amplifier probes preferably contain a relatively large number of amplification sequences that are available for binding of label probes, the detectable signal is significantly increased, and allows the detection limits of the target to be significantly improved.
  • label and amplifier probes and the detection methods described herein, may be used in essentially any known nucleic acid hybridization formats, such as those in which the target is bound directly to a solid phase or in sandwich hybridization assays in which the target is bound to one or more nucleic acids that are in turn bound to the solid phase.
  • the assay complexes of the invention are detected using electrodes.
  • electrode herein is meant a composition, which, when connected to an electronic device, is able to sense a current or charge and convert it to a signal.
  • an electrode can be defined as a composition which can apply a potential to and/or pass electrons to or from species in the solution.
  • an electrode is an ETM as described herein.
  • Preferred electodes include, but are not limited to, certain metals and their oxides, including gold; platinum; palladium; silicon; aluminum; metal oxide electrodes including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo 2 0 6 ), tungsten oxide (W0 3 ) and ruthenium oxides; and carbon (including glassy carbon electrodes, graphite and carbon paste).
  • Preferred electrodes include gold, silicon, platinum, carbon and metal oxide electrodes, with gold being particularly preferred.
  • the electrodes described herein are depicted as a flat surface, which is only one of the possible conformations of the electrode and is for schematic purposes only.
  • the conformation of the electrode will vary with the detection method used.
  • flat planar electrodes may be preferred for optical detection methods, or when arrays of nucleic acids are made, thus requiring addressable locations for both synthesis and detection.
  • the electrode may be in the form of a tube, with the SAMs comprising conductive oligomers and nucleic acids bound to the inner surface.
  • Electrode coils may be preferred in some embodiments as well. This allows a maximum of surface area containing the nucleic acids to be exposed to a small volume of sample.
  • the detection electrodes are formed on a substrate.
  • the discussion herein is generally directed to the formation of gold electrodes, but as will be appreciated by those in the art, other electrodes can be used as well.
  • the substrate can comprise a wide variety of materials, as will be appreciated by those in the art, with printed circuit board (PCB) materials being particularly preferred.
  • the suitable substrates include, but are not limited to, fiberglass, teflon, ceramics, glass, silicon, mica, plastic (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, TeflonTM, and derivatives thereof, etc.), GETEK (a blend of polypropylene oxide and fiberglass), etc.
  • circuit board materials are those that comprise an insulating substrate that is coated with a conducting layer and processed using lithography techniques, particularly photolithography techniques, to form the patterns of electrodes and interconnects (sometimes referred to in the art as interconnections or leads).
  • the insulating substrate is generally, but not always, a polymer.
  • one or a plurality of layers may be used, to make either "two dimensional" (e.g. all electrodes and interconnections in a plane) or "three dimensional” (wherein the electrodes are on one surface and the interconnects may go through the board to the other side) boards.
  • Circuit board materials are often provided with a foil already attached to the substrate, such as a copper foil, with additional copper added as needed (for example for interconnections), for example by electroplating.
  • the copper surface may then need to be roughened, for example through etching, to allow attachment of the adhesion layer.
  • glass may not be preferred as a substrate.
  • the present invention provides biochips (sometimes referred to herein "chips") that comprise substrates comprising a plurality of electrodes, preferably gold electrodes.
  • the number of electrodes is as outlined for arrays.
  • Each electrode preferably comprises a self-assembled monolayer as outlined herein.
  • one of the monolayer- forming species comprises a capture ligand as outlined herein.
  • each electrode has an interconnection, that is attached to the electrode at one end and is ultimately attached to a device that can control the electrode. That is, each electrode is independently addressable.
  • the substrates can be part of a larger device comprising a detection chamber that exposes a given volume of sample to the detection electrode. Generally, the detection chamber ranges from about 1 nL to 1 ml, with about 10 ⁇ L to 500 ⁇ L being preferred. As will be appreciated by those in the art, depending on the experimental conditions and assay, smaller or larger volumes may be used.
  • the detection chamber and electrode are part of a cartridge that can be placed into a device comprising electronic components (an AC/DC voltage source, an ammeter, a processor, a read-out display, temperature controller, light source, etc.).
  • electronic components an AC/DC voltage source, an ammeter, a processor, a read-out display, temperature controller, light source, etc.
  • the interconnections from each electrode are positioned such that upon insertion of the cartridge into the device, connections between the electrodes and the electronic components are established.
  • Detection electrodes on circuit board material are generally prepared in a wide variety of ways.
  • high purity gold is used, and it may be deposited on a surface via vacuum deposition processes (sputtering and evaporation) or solution deposition (electroplating or electroless processes).
  • the substrate When electroplating is done, the substrate must initially comprise a conductive material; fiberglass circuit boards are frequently provided with copper foil.
  • an adhesion layer between the substrate and the gold in order to insure good mechanical stability is used.
  • preferred embodiments utilize a deposition layer of an adhesion metal such as chromium, titanium, titanium/tungsten, tantalum, nickel or palladium, which can be deposited as above for the gold.
  • grain refining additives When electroplated metal (either the adhesion metal or the electrode metal) is used, grain refining additives, frequently referred to in the trade as brighteners, can optionally be added to alter surface deposition properties.
  • Preferred brighteners are mixtures of organic and inorganic species, with cobalt and nickel being preferred.
  • the adhesion layer is from about 100 A thick to about 25 microns (1000 microinches).
  • the electrode metal preferably gold
  • the electrode metal is deposited at thicknesses ranging from about 500 A to about 5 microns (200 microinches), with from about 30 microinches to about 50 microinches being preferred.
  • the gold is deposited to make electrodes ranging in size from about 5 microns to about 5 mm in diameter, with about 100 to 250 microns being preferred.
  • the detection electrodes thus formed are then preferably cleaned and SAMs added, as is discussed below.
  • the present invention provides methods of making a substrate comprising a plurality of gold electrodes.
  • the methods first comprise coating an adhesion metal, such as nickel or palladium (optionally with brightener), onto the substrate. Electroplating is preferred.
  • the electrode metal preferably gold, is then coated (again, with electroplating preferred) onto the adhesion metal.
  • the patterns of the device, comprising the electrodes and their associated interconnections are made using lithographic techniques, particularly photolithographic techniques as are known in the art, and wet chemical etching.
  • a non-conductive chemically resistive insulating material such as solder mask or plastic is laid down using these photolithographic techniques, leaving only the electrodes and a connection point to the leads exposed; the leads themselves are generally coated.
  • drop deposition techniques are used to add the required chemistry, i.e. the monolayer forming species, one of which is preferably a capture ligand comprising species.
  • Drop deposition techniques are well known for making "spot" arrays. This is done to add a different composition to each electrode, i.e. to make an array comprising different capture ligands.
  • the SAM species may be identical for each electrode, and this may be accomplished using a drop deposition technique or the immersion of the entire substrate or a surface of the substrate into the solution.
  • the electrode comprises a monolayer, comprising electroconduit forming species (EFS).
  • EFS electroconduit forming species
  • the efficiency of target analyte binding may increase when the analyte is at a distance from the electrode.
  • non-specific binding of biomolecules, including the target analytes, to an electrode is generally reduced when a monolayer is present.
  • a monolayer facilitates the maintenance of the analyte away from the electrode surface.
  • a monolayer serves to keep charged species away from the surface of the electrode. Thus, this layer helps to prevent electrical contact between the electrodes and the ETMs, or between the electrode and charged species within the solvent.
  • the monolayer is preferably tightly packed in a uniform layer on the electrode surface, such that a minimum of "holes" exist.
  • the monolayer thus serves as a physical barrier to block solvent community to the electrode.
  • the detection electrode comprises a self-assembled monolayer (SAM) comprising conductive oligomers.
  • SAM self-assembled monolayer
  • a relatively ordered assembly of molecules spontaneously chemisorbed on a surface in which the molecules are oriented approximately parallel to each other and roughly perpendicular to the surface.
  • Each of the molecules includes a functional group that adheres to the surface, and a portion that interacts with neighboring molecules in the monolayer to form the relatively ordered array.
  • a “mixed” monolayer comprises a heterogeneous monolayer, that is, where at least two different molecules make up the monolayer.
  • the SAM may comprise conductive oligomers alone, or a mixture of conductive oligomers and insulators.
  • the efficiency of oligonucleotide hybridization may increase when the analyte is at a distance from the electrode.
  • non-specific binding of biomolecules, including the target analytes, to an electrode is generally reduced when a monolayer is present.
  • a monolayer facilitates the maintenance of the nucleic acid away from the electrode surface.
  • a monolayer serves to keep charged species away from the surface of the electrode.
  • this layer helps to prevent electrical contact between the electrodes and the ETMs, or between the electrode and charged species within the solvent. Such contact can result in a direct "short circuit" or an indirect short circuit via charged species which may be present in the sample.
  • the monolayer is preferably tightly packed in a uniform layer on the electrode surface, such that a minimum of "holes" exist. The monolayer thus serves as a physical barrier to block solvent community to the electrode.
  • the SAMs of the invention can be generated in a number of ways and comprise a number of different components, depending on the electrode surface and the system used.
  • preferred embodiments utilize two monolayer forming species: a monolayer forming species (including insulators or conductive oligomers) and a conductive oligomer species comprising the capture binding ligand, although as will be appreciated by those in the art, additional monolayer forming species can be included as well.
  • the composition of the SAM depends on the detection electrode surface.
  • detection electrodes comprising "smooth” surfaces, such as gold ball electrodes
  • those comprising "rough” surfaces such as those that are made using commercial processes on PC circuit boards.
  • monolayers made on imperfect surfaces i.e. "rough” surfaces
  • spontaneously form monolayers containing enough electroconduits even in the absence of EFS probably due to the fact that the formation of a uniform monolayer on a rough surface is difficult.
  • “Smoother” surfaces may require the inclusion of sufficient numbers of EFS to generate the electroconduits, as the uniform surfaces allow a more uniform monolayer to form.
  • “smooth" surfaces comprise monolayers comprising three components: an insulator species, a EFS, and a species comprising the capture ligand, although in some circumstances, for example when the capture ligand species is included at high density, the capture ligand species can serve as the EFS.
  • “Smoothness" in this context is not measured physically but rather as a function of an increase in the measured signal when EFS are included. That is, the signal from a detection electrode coated with monolayer forming species is compared to a signal from a detection electrode coated with monolayer forming species including a EFS. An increase indicates that the surface is relatively smooth, since the inclusion of a EFS served to facilitate the access of the
  • ETM to the electrode. It should also be noted that while the discussion herein is mainly directed to gold electrodes and thiol-containing monolayer forming species, other types of electrodes and monolayer-forming species can be used.
  • electroconduits do not result in direct contact of sample components with the electrode surface; that is, the electroconduits are not large pores or holes that allow physical access to the electrode. Rather, without being bound by theory, it appears that the electroconduits allow certain types of ETMs, particularly hydrophobic ETMs, to penetrate sufficiently into the monolayer to allow detection. However, other types of redox active species, including some hydrophilic species, do not penentrate into the monolayer, even with electroconduits present. Thus, in general, redox active species that may be present in the sample do not give substantial signals as a result of the electroconduits.
  • the test for a suitable SAM to reduce non-specific binding that also has sufficient electroconduits for ETM detection is to add either ferrocene or ferrocyanide to the SAM; the former should give a signal and the latter should not.
  • the monolayer comprises a first species comprising a conductive oligomer comprising the capture binding ligand, as is more fully outlined below, and a second species comprising a monolayer forming species, including either or both insulators or conductive oligomers.
  • the monolayer comprises electroconduit-forming species.
  • electroconduit-forming species or “EFS” herein is meant a molecule that is capable of generating sufficient electroconduits in a monolayer, generally of insulators such as alkyl groups, to allow detection of ETMs at the surface.
  • EFS have one or more of the following qualities: they may be relatively rigid molecules, for example as compared to an alkyl chain; they may attach to the electrode surface with a geometry different from the other monolayer forming species (for example, alkyl chains attached to gold surfaces with thiol groups are thought to attach at roughly 45° angles, and phenyl-acetylene chains attached to gold via thiols are thought to go down at 90° angles); they may have a structure that sterically interferes or interrupts the formation of a tightly packed monolayer, for example through the inclusion of branching groups such as alkyl groups, or the inclusion of highly flexible species, such as polyethylene glycol units; or they may be capable of being activated to form electroconduits; for example, photoactivatible species that can be selectively removed from the surface upon photoactivation, leaving electroconduits.
  • Preferred EFS include conductive oligomers, as defined below, and phenyl-acetylene-polyethylene glycol species. However, in some embodiments, the EFS is not a conductive oligomer.
  • the monolayer comprises conductive oligomers.
  • conductive oligomer herein is meant a substantially conducting oligomer, preferably linear, some embodiments of which are referred to in the literature as “molecular wires”.
  • substantially conducting herein is meant that the oligomer is capable of transfering electrons at 100 Hz.
  • the conductive oligomer has substantially overlapping ⁇ -orbitals, i.e. conjugated ⁇ -orbitals, as between the monomeric units of the conductive oligomer, although the conductive oligomer may also contain one or more sigma ( ⁇ ) bonds.
  • a conductive oligomer may be defined functionally by its ability to inject or receive electrons into or from an associated ETM. Furthermore, the conductive oligomer is more conductive than the insulators as defined herein. Additionally, the conductive oligomers of the invention are to be distinguished from electroactive polymers, that themselves may donate or accept electrons.
  • the conductive oligomers have a conductivity, S, of from between about 10 '6 to about 10 4 ⁇ '1 cm “1 , with from about 10 '5 to about 10 3 ⁇ "1 cm '1 being preferred, with these S values being calculated for molecules ranging from about 2 ⁇ to about 200A.
  • insulators have a conductivity S of about 10 '7 ⁇ "1 cm '1 or lower, with less than about 10 '8 ⁇ '1 cm "1 being preferred. See generally Gardner et al., Sensors and Actuators A 51 (1995) 57-66, incorporated herein by reference.
  • Desired characteristics of a conductive oligomer include high conductivity, sufficient solubility in organic solvents and/or water for synthesis and use of the compositions of the invention, and preferably chemical resistance to reactions that occur i) during binding ligand synthesis (i.e. nucleic acid synthesis, such that nucleosides containing the conductive oligomers may be added to a nucleic acid synthesizer during the synthesis of the compositions of the invention, ii) during the attachment of the conductive oligomer to an electrode, or iii) during binding assays.
  • binding ligand synthesis i.e. nucleic acid synthesis, such that nucleosides containing the conductive oligomers may be added to a nucleic acid synthesizer during the synthesis of the compositions of the invention, ii) during the attachment of the conductive oligomer to an electrode, or iii) during binding assays.
  • oligomers of the invention comprise at least two monomeric subunits, as described herein. As is described more fully below, oligomers include homo- and hetero-oligomers, and include polymers.
  • the conductive oligomer has the structure depicted in Structure 13:
  • the conductive oligomer of Structure 1 may be attached to ETMs, such as electrodes, transition metal complexes, organic ETMs, and metallocenes, and to binding ligands such as nucleic acids, or to several of these.
  • ETMs such as electrodes, transition metal complexes, organic ETMs, and metallocenes
  • binding ligands such as nucleic acids, or to several of these.
  • the conductive oligomers depicted herein will be attached at the left side to an electrode; that is, as depicted in Structure 1 , the left "Y" is connected to the electrode as described herein.
  • the right "Y" is attached to the binding ligand such as a nucleic acid, either directly or through the use of a linker, as is described herein.
  • Y is an aromatic group
  • n is an integer from 1 to 50
  • g is either 1 or zero
  • e is an integer from zero to 10
  • m is zero or 1.
  • D is preferably carbonyl, or a heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen, silicon or phosphorus.
  • suitable heteroatom moieties include, but are not limited to, -NH and - NR, wherein R is as defined herein; substituted sulfur; sulfonyl (-S0 2 -) sulfoxide (-SO-); phosphine oxide (-PO- and -RPO-); and thiophosphine (-PS- and -RPS-).
  • sulfur derivatives are not preferred.
  • aromatic group or grammatical equivalents herein is meant an aromatic monocyclic or polycyclic hydrocarbon moiety generally containing 5 to 14 carbon atoms (although larger polycyclic rings structures may be made) and any carbocylic ketone or thioketone derivative thereof, wherein the carbon atom with the free valence is a member of an aromatic ring.
  • Aromatic groups include arylene groups and aromatic groups with more than two atoms removed. For the purposes of this application aromatic includes heterocycle.
  • Heterocycle or “heteroaryl” means an aromatic group wherein 1 to 5 of the indicated carbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen, sulfur, phosphorus, boron and silicon wherein the atom with the free valence is a member of an aromatic ring, and any heterocyclic ketone and thioketone derivative thereof.
  • heterocycle includes thienyl, furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl, isoquinolyl, thiazolyl, imidozyl, etc.
  • the Y aromatic groups of the conductive oligomer may be different, i.e. the conductive oligomer may be a heterooligomer. That is, a conductive oligomer may comprise a oligomer of a single type of Y groups, or of multiple types of Y groups.
  • the aromatic group may be substituted with a substitution group, generally depicted herein as R.
  • R groups may be added as necessary to affect the packing of the conductive oligomers, i.e. R groups may be used to alter the association of the oligomers in the monolayer. R groups may also be added to 1 ) alter the solubility of the oligomer or of compositions containing the oligomers; 2) alter the conjugation or electrochemical potential of the system; and 3) alter the charge or characteristics at the surface of the monolayer.
  • R groups are preferred to increase solubility when solution synthesis is done.
  • the R groups, and their positions, are chosen to minimally effect the packing of the conductive oligomers on a surface, particularly within a monolayer, as described below.
  • only small R groups are used within the monolayer, with larger R groups generally above the surface of the monolayer.
  • attachment of methyl groups to the portion of the conductive oligomer within the monolayer to increase solubility is preferred, with attachment of longer alkoxy groups, for example, C3 to C10, is preferably done above the monolayer surface.
  • R groups include, but are not limited to, hydrogen, alkyl, alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes, sulfonyl, silicon moieties, halogens, sulfur containing moieties, phosphorus containing moieties, and ethylene glycols.
  • R is hydrogen when the position is unsubstituted. It should be noted that some positions may allow two substitution groups, R and R', in which case the R and R' groups may be either the same or different.
  • alkyl group or grammatical equivalents herein is meant a straight or branched chain alkyl group, with straight chain alkyl groups being preferred. If branched, it may be branched at one or more positions, and unless specified, at any position.
  • the alkyl group may range from about 1 to about 30 carbon atoms (C1 -C30), with a preferred embodiment utilizing from about 1 to about 20 carbon atoms (C1 -C20), with about C1 through about C12 to about C15 being preferred, and C1 to C5 being particularly preferred, although in some embodiments the alkyl group may be much larger.
  • alkyl group also included within the definition of an alkyl group are cycloalkyl groups such as C5 and C6 rings, and heterocyclic rings with nitrogen, oxygen, sulfur or phosphorus.
  • Alkyl also includes heteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and silicone being preferred.
  • Alkyl includes substituted alkyl groups.
  • substituted alkyl group herein is meant an alkyl group further comprising one or more substitution moieties "R", as defined above.
  • amino groups or grammatical equivalents herein is meant -NH 2 , -NHR and -NR 2 groups, with R being as defined herein.
  • nitro group herein is meant an -N0 2 group.
  • sulfur containing moieties herein is meant compounds containing sulfur atoms, including but not limited to, thia-, thio- and sulfo- compounds, thiols (-SH and -SR), and sulfides (-RSR-).
  • phosphorus containing moieties herein is meant compounds containing phosphorus, including, but not limited to, phosphines and phosphates.
  • silicon containing moieties herein is meant compounds containing silicon.
  • ether herein is meant an -O-R group.
  • Preferred ethers include alkoxy groups, with -0-(CH 2 ) 2 CH 3 and -0-(CH 2 ) 4 CH 3 being preferred.
  • esters herein is meant a -COOR group.
  • halogen herein is meant bromine, iodine, chlorine, or fluorine.
  • Preferred substituted alkyls are partially or fully halogenated alkyls such as CF 3 , etc.
  • aldehyde herein is meant -RCHO groups.
  • alcohol herein is meant -OH groups, and alkyl alcohols -ROH.
  • ethylene glycol or "(poly)ethylene glycol” herein is meant a -(0-CH 2 -CH 2 ) n - group, although each carbon atom of the ethylene group may also be singly or doubly substituted, i.e. -(0-CR 2 -CR 2 ) n -, with R as described above.
  • Ethylene glycol derivatives with other heteroatoms in place of oxygen i.e. -(N-
  • substitution groups include, but are not limited to, methyl, ethyl, propyl, alkoxy groups such as -0-(CH 2 ) 2 CH 3 and -0-(CH 2 ) 4 CH 3 and ethylene glycol and derivatives thereof.
  • Preferred aromatic groups include, but are not limited to, phenyl, naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine, thiophene, porphyrins, and substituted derivatives of each of these, included fused ring derivatives.
  • B-D is a bond linking two atoms or chemical moieties.
  • B-D is a conjugated bond, containing overlapping or conjugated ⁇ -orbitals.
  • Preferred B-D bonds are selected from acetylene (-C ⁇ C-, also called alkyne or ethyne), alkene (-
  • B-D bonds are acetylene, alkene, amide, and substituted derivatives of these three, and azo.
  • Especially preferred B-D bonds are acetylene, alkene and amide.
  • the oligomer components attached to double bonds may be in the trans or cis conformation, or mixtures.
  • B or D may include carbon, nitrogen or silicon.
  • the substitution groups are as defined as above for R.
  • e is preferably 1 and the D moiety may be carbonyl or a heteroatom moiety as defined above.
  • the terminal B-D bond may be an amide bond
  • the rest of the B-D bonds may be acetylene bonds.
  • amide bonds when amide bonds are present, as few amide bonds as possible are preferable, but in some embodiments all the B-D bonds are amide bonds.
  • one type of B-D bond may be present in the conductive oligomer within a monolayer as described below, and another type above the monolayer level, for example to give greater flexibility for nucleic acid hybridization when the nucleic acid is attached via a conductive oligomer.
  • n is an integer from 1 to 50, although longer oligomers may also be used (see for example Schumm et al. , Angew. Chem. Int. Ed. Engl. 1994 33(13): 1360). Without being bound by theory, it appears that for efficient hybridization of nucleic acids on a surface, the hybridization should occur at a distance from the surface, i.e. the kinetics of hybridization increase as a function of the distance from the surface, particularly for long oligonucleotides of 200 to 300 basepairs.
  • the length of the conductive oligomer is such that the closest nucleotide of the nucleic acid is positioned from about 6A to about 100A (although distances of up to 500A may be used) from the electrode surface, with from about 15A to about 60A being preferred and from about 25A to about 60A also being preferred.
  • n will depend on the size of the aromatic group, but generally will be from about 1 to about 20, with from about 2 to about 15 being preferred and from about 3 to about 10 being especially preferred.
  • m is either 0 or 1. That is, when m is 0, the conductive oligomer may terminate in the B-D bond or D moiety, i.e. the D atom is attached to the nucleic acid either directly or via a linker. In some embodiments, for example when the conductive oligomer is attached to a phosphate of the ribose-phosphate backbone of a nucleic acid, there may be additional atoms, such as a linker, attached between the conductive oligomer and the nucleic acid. Additionally, as outlined below, the D atom may be the nitrogen atom of the amino-modified ribose. Alternatively, when m is 1 , the conductive oligomer may terminate in Y, an aromatic group, i.e. the aromatic group is attached to the nucleic acid or linker.
  • conductive oligomers may be utilized. These include conductive oligomers falling within the Structure 1 and Structure 8 formulas, as well as other conductive oligomers, as are generally known in the art, including for example, compounds comprising fused aromatic rings or Teflon®-like oligomers, such as -(CF 2 ) n -, -(CHF) n - and -(CFR) n -. See for example, Schumm et al., Angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev.
  • Structure 14 is Structure 13 when g is 1.
  • Preferred embodiments of Structure 14 include: e is zero, Y is pyrole or substituted pyrole; e is zero, Y is thiophene or substituted thiophene; e is zero, Y is furan or substituted furan; e is zero, Y is phenyl or substituted phenyl; e is zero, Y is pyridine or substituted pyridine; e is 1 , B-D is acetylene and Y is phenyl or substituted phenyl (see Structure 16 below).
  • a preferred embodiment of Structure 14 is also when e is one, depicted as Structure 15 below:
  • Preferred embodiments of Structure 15 are: Y is phenyl or substituted phenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D is acetylene; Y is phenyl or substituted phenyl and B-D is alkene; Y is pyridine or substituted pyridine and B-D is acetylene; Y is thiophene or substituted thiophene and B-D is acetylene; Y is furan or substituted furan and B-D is acetylene; Y is thiophene or furan (or substituted thiophene or furan) and B-D are alternating alkene and acetylene bonds.
  • Structure 15 oligomers may be substituted with any of the other structures depicted herein, i.e. Structure 13 or 20 oligomer, or other conducting oligomer, and the use of such Structure 15 depiction is not meant to limit the scope of the invention.
  • Structure 15 includes Structures 16, 17, 18 and 19, depicted below:
  • Particularly preferred embodiments of Structure 16 include: n is two, m is one, and R is hydrogen; n is three, m is zero, and R is hydrogen; and the use of R groups to increase solubility.
  • the conductive oligomers are pseudopeptide oligomers.
  • the amide bond in Structure 17 is depicted with the carbonyl to the left, i.e. - CONH-, the reverse may also be used, i.e. -NHCO-.
  • Particularly preferred embodiments of Structure 17 include: n is two, m is one, and R is hydrogen; n is three, m is zero, and R is hydrogen (in this embodiment, the terminal nitrogen (the D atom) may be the nitrogen of the amino-modified ribose); and the use of R groups to increase solubility.
  • Preferred embodiments of Structure 18 include the first n is two, second n is one, m is zero, and all R groups are hydrogen, or the use of R groups to increase solubility.
  • Preferred embodiments of Structure 19 include: the first n is three, the second n is from 1-3, with m being either 0 or 1 , and the use of R groups to increase solubility.
  • the conductive oligomer has the structure depicted in Structure 20:
  • C are carbon atoms
  • n is an integer from 1 to 50
  • m is 0 or 1
  • J is a heteroatom selected from the group consisting of oxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide
  • the G bond of each subunit may be the same or different than the G bonds of other subunits; that is, alternating oligomers of alkene and acetylene bonds could be used, etc.
  • G is an alkane bond
  • the number of alkane bonds in the oligomer should be kept to a minimum, with about six or less sigma bonds per conductive oligomer being preferred.
  • Alkene bonds are preferred, and are generally depicted herein, although alkane and acetylene bonds may be substituted in any structure or embodiment described herein as will be appreciated by those in the art.
  • the m of Structure 20 is zero. In a particularly preferred embodiment, m is zero and G is an alkene bond, as is depicted in Structure 21 :
  • alkene oligomer of structure 21 are generally depicted in the preferred trans configuration, although oligomers of cis or mixtures of trans and cis may also be used.
  • R groups may be added to alter the packing of the compositions on an electrode, the hydrophilicity or hydrophobicity of the oligomer, and the flexibility, i.e. the rotational, torsional or longitudinal flexibility of the oligomer.
  • n is as defined above.
  • R is hydrogen, although R may be also alkyl groups and polyethylene glycols or derivatives.
  • the conductive oligomer may be a mixture of different types of oligomers, for example of structures 13 and 20.
  • the monolayer comprises conductive oligomers, and the terminus of at least some of the conductive oligomers in the monolayer are electronically exposed.
  • electroconductive oligomers may or may not have terminal groups. Thus, in a preferred embodiment, there is no additional terminal group, and the conductive oligomer terminates with one of the groups depicted in the structures; for example, a B-D bond such as an acetylene bond.
  • a terminal group is added, sometimes depicted herein as "Q".
  • a terminal group may be used for several reasons; for example, to contribute to the electronic availability of the conductive oligomer for detection of ETMs, or to alter the surface of the SAM for other reasons, for example to prevent non-specific binding.
  • the target analyte is a nucleic acid
  • Preferred terminal groups include -NH 2 , -OH, -COOH, and alkyl groups such as -CH 3 , and (poly)alkyloxides such as (poly)ethylene glycol, with -OCH 2 CH 2 OH, -(OCH 2 CH 2 0) 2 H, -(OCH 2 CH 2 0) 3 H, and -(OCH 2 CH 2 0) 4 H being preferred.
  • the monolayer may comprise different conductive oligomer species, although preferably the different species are chosen such that a reasonably uniform SAM can be formed.
  • capture binding ligands such as nucleic acids are covalently attached to the electrode using conductive oligomers
  • preferred embodiments utilize conductive oligomers that terminate below the surface of the rest of the monolayer, i.e. below the insulator layer, if used, or below some fraction of the other conductive oligomers.
  • the use of different conductive oligomers may be done to facilitate monolayer formation, or to make monolayers with altered properties.
  • the monolayer may further comprise insulator moieties.
  • insulator herein is meant a substantially nonconducting oligomer, preferably linear.
  • substantially nonconducting herein is meant that the insulator will not transfer electrons at 100 Hz. The rate of electron transfer through the insulator is preferrably slower than the rate through the conductive oligomers described herein.
  • the insulators have a conductivity, S, of about 10 '7 ⁇ ⁇ 1 cm “1 or lower, with less than about 10 "8 ⁇ "1 cm “1 being preferred. See generally Gardner et al., supra.
  • insulators are alkyl or heteroalkyl oligomers or moieties with sigma bonds, although any particular insulator molecule may contain aromatic groups or one or more conjugated bonds.
  • heteroalkyl herein is meant an alkyl group that has at least one heteroatom, i.e. nitrogen, oxygen, sulfur, phosphorus, silicon or boron included in the chain.
  • the insulator may be quite similar to a conductive oligomer with the addition of one or more heteroatoms or bonds that serve to inhibit or slow, preferably substantially, electron transfer.
  • Suitable insulators include, but are not limited to, -(CH 2 ) n -, -(CRH) n -, and -
  • the insulators may be substituted with R groups as defined herein to alter the packing of the moieties or conductive oligomers on an electrode, the hydrophilicity or hydrophobicity of the insulator, and the flexibility, i.e. the rotational, torsional or longitudinal flexibility of the insulator.
  • R groups as defined herein to alter the packing of the moieties or conductive oligomers on an electrode, the hydrophilicity or hydrophobicity of the insulator, and the flexibility, i.e. the rotational, torsional or longitudinal flexibility of the insulator.
  • branched alkyl groups may be used.
  • the insulators may contain terminal groups, as outlined above, particularly to influence the surface of the monolayer. The length of the species making up the monolayer will vary as needed. As outlined above, it appears that binding of target analytes (for example, hybridization of nucleic acids) is more efficient at a distance from the surface.
  • the species to which capture binding ligands are attached may be basically the same length as the monolayer forming species or longer than them, resulting in the capture binding ligands being more accessible to the solvent for hybridization.
  • the conductive oligomers to which the capture binding ligands are attached may be shorter than the monolayer.
  • the actual combinations and ratios of the different species making up the monolayer can vary widely, and will depend on whether mechanism-1 or -2 is used.
  • three component systems are preferred for mechanism-2 systems, with the first species comprising a capture binding ligand containing species (termed a capture probe when the target analyte is a nucleic acid), attached to the electrode via either an insulator or a conductive oligomer.
  • the second species are the conductive oligomers, and the third species are insulators.
  • the first species can comprise from about 90% to about 1 %, with from about 20% to about 40% being preferred.
  • the target analytes are nucleic acids, from about 30% to about
  • the second species can comprise from about 1 % to about 90%, with from about 20% to about 90% being preferred, and from about 40% to about 60% being especially preferred.
  • the third species can comprise from about 1 % to about 90%, with from about 20% to about 40% being preferred, and from about 15% to about 30% being especially preferred.
  • Preferred ratios of first:second:third species are 2:2:1 for short targets, 1 :3:1 for longer targets, with total thiol concentration (when used to attach these species, as is more fully outlined below) in the 500 ⁇ M to 1 mM range, and 833 ⁇ M being preferred.
  • two component systems can be used.
  • the two components are the first and second species.
  • the first species can comprise from about 1% to about 90%, with from about 1% to about 40% being preferred, and from about 10% to about 40% being especially preferred.
  • the second species can comprise from about 1 % to about 90%, with from about 10% to about 60% being preferred, and from about 20% to about 40% being especially preferred.
  • the two components are the first and the third species.
  • the first species can comprise from about 1 % to about 90%, with from about 1 % to about 40% being preferred, and from about 10% to about 40% being especially preferred.
  • the second species can comprise from about 1 % to about 90%, with from about 10% to about 60% being preferred, and from about 20% to about 40% being especially preferred.
  • the covalent attachment of the conductive oligomers and insulators to the electrode may be accomplished in a variety of ways, depending on the electrode and the composition of the insulators and conductive oligomers used.
  • the attachment linkers (which can comprise insulators and conductive oligomers), which are used to covalently attach nucleic acid species (capture and detection probes) to the electrode, are attached in a similar manner. Thus, one end or terminus of the attachment linker is attached to the nucleoside or nucleic acid, and the other is attached to an electrode.
  • attachment linker may be desirable to have the attachment linker attached at a position other than a terminus, or even to have a branched attachment linker that is attached to an electrode at one terminus and to two or more nucleosides at other termini, although this is not preferred.
  • the attachment linker may be attached at two sites to the electrode, as is generally depicted in Structures 23-25.
  • some type of linker is used, as depicted below as "A" in Structure 22, where "X" is the conductive oligomer, "I” is an insulator and the hatched surface is the electrode:
  • A is a linker or atom.
  • A may be a sulfur moiety when a gold electrode is used.
  • A may be a silicon (silane) moiety attached to the oxygen of the oxide (see for example Chen et al., Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem. 78:195-201 (1977), both of which are expressly incorporated by reference).
  • A may be an amino moiety (preferably a primary amine; see for example Deinhammer et al., Langmuir 10:1306-1313 (1994)).
  • preferred A moieties include, but are not limited to, silane moieties, sulfur moieties (including alkyl sulfur moieties), and amino moieties.
  • epoxide type linkages with redox polymers such as are known in the art are not used.
  • the insulators and conductive oligomers may be attached to the electrode with more than one "A" moiety; the "A" moieties may be the same or different.
  • the electrode is a gold electrode
  • "A" is a sulfur atom or moiety
  • multiple sulfur atoms may be used to attach the conductive oligomer to the electrode, such as is generally depicted below in Structures 23, 24 and 25.
  • the A moiety is just a sulfur atom, but substituted sulfur moieties may also be used. Structure 23
  • a conductive oligomer terminating in a single carbon atom with three sulfur moities attached to the electrode may also comprise a "Q" terminal group.
  • the electrode is a gold electrode, and attachment is via a sulfur linkage as is well known in the art, i.e. the A moiety is a sulfur atom or moiety. Although the exact characteristics of the gold-sulfur attachment are not known, this linkage is considered covalent for the purposes of this invention.
  • a representative structure is depicted in Structure 26, using the Structure 15 conductive oligomer, although as for all the structures depicted herein, any of the conductive oligomers, or combinations of conductive oligomers, may be used. Similarly, any of the conductive oligomers or insulators may also comprise terminal groups as described herein.
  • Structure 26 depicts the "A" linker as comprising just a sulfur atom, although additional atoms may be present (i.e. linkers from the sulfur to the conductive oligomer or substitution groups).
  • Structure 26 shows the sulfur atom attached to the Y aromatic group, but as will be appreciated by those in the art, it may be attached to the B-D group (i.e. an acetylene) as well.
  • thiol linkages are preferred when either two sets of electrodes are used (i.e. the detection electrodes comprising the SAMs are not used at high electrophoretic voltages (i.e. greater than 800 or 900 mV), that can cause oxidation of the thiol linkage and thus loss of the SAM), or, if one set of electrodes is used, lower electrophoretic voltages are used as is generally described below.
  • the electrode is a carbon electrode, i.e. a glassy carbon electrode, and attachment is via a nitrogen of an amine group.
  • a representative structure is depicted in Structure 27. Again, additional atoms may be present, i.e. Z type linkers and/or terminal groups.
  • the oxygen atom is from the oxide of the metal oxide electrode.
  • the Si atom may also contain other atoms, i.e. be a silicon moiety containing substitution groups.
  • Other attachments for SAMs to other electrodes are known in the art; see for example Napier et al., Langmuir, 1997, for attachment to indium tin oxide electrodes, and also the chemisorption of phosphates to an indium tin oxide electrode (talk by H. Holden Thorpe, CHI conference, May 4-5, 1998).
  • the electrode comprises either capture probes to anchor the assay complexes to the electrode (used in either mechanism-1 or mechanism-2 systems), or detection probes (for mechanism-1 systems). Since the capture probes are not used for detection, the capture probes may be attached using attachment linkers, which may include either conductive oligomers or insulators, as described below. Detection probes used in mechanism-1 systems are attached using conductive oligomers.
  • the capture probe nucleic acid is covalently attached to the electrode, via an "attachment linker", that can be either a conductive oligomer (required for mechanism-1 systems) or an insulator.
  • attachment linker that can be either a conductive oligomer (required for mechanism-1 systems) or an insulator.
  • covalently attached herein is meant that two moieties are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.
  • nucleic acid or other binding ligand
  • the other end is attached to the electrode.
  • any of structures depicted herein may further comprise a nucleic acid effectively as a terminal group.
  • the present invention provides compositions comprising nucleic acids covalently attached to electrodes as is generally depicted below in Structure 29:
  • X is a conductive oligomer and I is an insulator as defined herein.
  • F 1 is a linkage that allows the covalent attachment of the electrode and the conductive oligomer or insulator, including bonds, atoms or linkers such as is described herein, for example as "A", defined below.
  • F 2 is a linkage that allows the covalent attachment of the conductive oligomer or insulator to the nucleic acid, and may be a bond, an atom or a linkage as is herein described.
  • F 2 may be part of the conductive oligomer, part of the insulator, part of the nucleic acid, or exogeneous to both, for example, as defined herein for "Z".
  • the SAMs of the invention can be made in a variety of ways, including deposition out of organic solutions and deposition out of aqueous solutions.
  • the methods outlined herein use a gold electrode as the example, although as will be appreciated by those in the art, other metals and methods may be used as well.
  • ITO indium-tin-oxide
  • a gold surface is first cleaned.
  • cleaning procedures may be employed, including, but not limited to, chemical cleaning or etchants (including Piranha solution (hydrogen peroxide/sulfuric acid) or aqua regia (hydrochloric acid/nitric acid), electrochemical methods, flame treatment, plasma treatment or combinations thereof.
  • the gold substrate is exposed to the SAM species.
  • the SAM species are phosphonate-containing species.
  • This can also be done in a variety of ways, including, but not limited to, solution deposition, gas phase deposition, microcontact printing, spray deposition, deposition using neat components, etc.
  • a preferred embodiment utilizes a deposition solution comprising a mixture of various SAM species in solution, generally thiol-containing species. Mixed monolayers that contain nucleic acids are usually prepared using a two step procedure.
  • the thiolated nucleic acid is deposited during the first deposition step (generally in the presence of at least one other monolayer-forming species) and the mixed monolayer formation is completed during the second step in which a second thiol solution minus nucleic acid is added.
  • the second step frequently involves mild heating to promote monolayer reorganization.
  • the deposition solution is an organic deposition solution.
  • a clean gold surface is placed into a clean vial.
  • a binding ligand deposition solution in organic solvent is prepared in which the total thiol concentration is between micromolar to saturation; preferred ranges include from about 1 ⁇ M to 10 mM, with from about 400 uM to about 1.0 mM being especially preferred.
  • the deposition solution contains thiol modified DNA (i.e. nucleic acid attached to an attachment linker) and thiol diluent molecules (either conductive oligomers or insulators, with the latter being preferred).
  • the ratio of nucleic acid to diluent is usually between 1000:1 to 1 :1000, with from about 10:1 to about 1 :10 being preferred and 1 :1 being especially preferred.
  • the preferred solvents are tetrahydrofuran (THF), acetonitrile, dimethylforamide
  • DMF dimethyl methacrylate
  • ethanol aqueous ethanol
  • nucleic acid deposition solution is added to the vial so as to completely cover the electrode surface.
  • the gold substrate is allowed to incubate at ambient temperature or slightly above ambient temperature for a period of time ranging from seconds to hours, with 5-30 minutes being
  • the deposition solution is removed and a solution of diluent molecule only (from about 1 ⁇ M to 10 mM, with from about 100 uM to about 1.0 mM being preferred) in organic solvent is added.
  • the gold substrate is allowed to incubate at room temperature or above room temperature for a period of time (seconds to days, with from about 10 minutes to about 24 hours being preferred).
  • the gold sample is removed from the solution, rinsed in clean solvent and used.
  • an aqueous deposition solution is used.
  • a clean gold surface is placed into a clean vial.
  • a nucleic acid deposition solution in water is prepared in which the total thiol concentration is between about 1 uM and 10 mM, with from about 1 ⁇ M to about 200 uM being preferred.
  • the aqueous solution frequently has salt present (up to saturation, with approximately 1 M being preferred), however pure water can be used.
  • the deposition solution contains thiol modified nucleic acid and often a thiol diluent molecule.
  • the ratio of nucleic acid to diluent is usually between 1000:1 to 1 :1000, with from about 10:1 to about 1 :10 being preferred and 1 :1 being especially preferred.
  • the nucleic acid deposition solution is added to the vial in such a volume so as to completely cover the electrode surface.
  • the gold substrate is allowed to incubate at ambient temperature or slightly above ambient temperature for 1-30 minutes with 5 minutes usually being sufficient. After the initial incubation, the deposition solution is removed and a solution of diluent molecule only (10 uM -1.0 mM) in either water or organic solvent is added.
  • the gold substrate is allowed to incubate at room temperature or above room temperature until a complete monolayer is formed (10 minutes-24 hours). The gold sample is removed from the solution, rinsed in clean solvent and used.
  • a circuit board is used as the substrate for the gold electrodes. Formation of the SAMs on the gold surface is generally done by first cleaning the boards, for example in a 10% sulfuric acid solution for 30 seconds, detergent solutions, aqua regia, plasma, etc., as outlined herein. Following the sulfuric acid treatment, the boards are washed, for example via immersion in two Milli-Q water baths for 1 minute each. The boards are then dried, for example under a stream of nitrogen. Spotting of the deposition solution onto the boards is done using any number of known spotting systems, generally by placing the boards on an X-Y table, preferably in a humidity chamber.
  • the size of the spotting drop will vary with the size of the electrodes on the boards and the equipment used for delivery of the solution; for example, for 250 ⁇ M size electrodes, a 30 nanoliter drop is used. The volume should be sufficient to cover the electrode surface completely.
  • the drop is incubated at room temperature for a period of time (sec to overnight, with 5 minutes preferred) and then the drop is removed by rinsing in a Milli-Q water bath.
  • the boards are then preferably treated with a second deposition solution, generally comprising insulator in organic solvent, preferably acetonitrile, by immersion in a 45°C bath. After 30 minutes, the boards are removed and immersed in an acetonitrile bath for 30 seconds followed by a milli-Q water bath for 30 seconds.
  • the boards are dried under a stream of nitrogen.
  • the electrode comprising the monolayer including conductive oligomers further comprises a nucleic acid capture probe.
  • the capture probe nucleic acid is covalently attached to the electrode. This attachment can be via a conductive oligomer or via an insulator.
  • capture probe or “anchor probe” herein is meant a component of an assay complex as defined herein that allows the attachment of a target sequence to the electrode, for the purposes of detection.
  • attachment of the target sequence to the capture probe may be direct (i.e. the target sequence hybridizes to the capture probe) or indirect (one or more capture extender probes are used).
  • the capture probes may have both nucleic and non-nucleic acid portions.
  • flexible linkers such as alkyl groups, including polyethylene glycol linkers, may be used to get the nucleic acid portion of the capture probe off the electrode surface. This may be particularly useful when the target sequences are large, for example when genomic DNA or rRNA is the target.
  • the capture probe nucleic acid is covalently attached to the electrode via a conductive oligomer.
  • the covalent attachment of the nucleic acid and the conductive oligomer may be accomplished in several ways.
  • the attachment is via attachment to the base of the nucleoside, via attachment to the backbone of the nucleic acid (either the ribose, the phosphate, or to an analogous group of a nucleic acid analog backbone), or via a transition metal ligand, as described below.
  • the techniques outlined below are generally described for naturally occuring nucleic acids, although as will be appreciated by those in the art, similar techniques may be used with nucleic acid analogs, and in some cases with other binding ligands.
  • the conductive oligomer is attached to the base of a nucleoside of the nucleic acid. This may be done in several ways, depending on the oligomer, as is described below. In one embodiment, the oligomer is attached to a terminal nucleoside, i.e. either the 3' or 5' nucleoside of the nucleic acid. Alternatively, the conductive oligomer is attached to an internal nucleoside.
  • the point of attachment to the base will vary with the base. Generally, attachment at any position is possible. In some embodiments, for example when the probe containing the ETMs may be used for hybridization (i.e. mechanism-1 systems) , it is preferred to attach at positions not involved in hydrogen bonding to the complementary base. Thus, for example, generally attachment is to the 5 or 6 position of pyrimidines such as uridine, cytosine and thymine. For purines such as adenine and guanine, the linkage is preferably via the 8 position. Attachment to non-standard bases is preferably done at the comparable positions.
  • the attachment is direct; that is, there are no intervening atoms between the conductive oligomer and the base.
  • conductive oligomers with terminal acetylene bonds are attached directly to the base.
  • Structure 30 is an example of this linkage, using a Structure 15 conductive oligomer and uridine as the base, although other bases and conductive oligomers can be used as will be appreciated by those in the art:
  • the pentose structures depicted herein may have hydrogen, hydroxy, phosphates or other groups such as amino groups attached.
  • the pentose and nucleoside structures depicted herein are depicted non-conventionally, as mirror images of the normal rendering.
  • the pentose and nucleoside structures may also contain additional groups, such as protecting groups, at any position, for example as needed during synthesis.
  • the base may contain additional modifications as needed, i.e. the carbonyl or amine groups may be altered or protected.
  • the attachment is any number of different Z linkers, including amide and amine linkages, as is generally depicted in Structure 31 using uridine as the base and a Structure 15 oligomer:
  • Z is a linker.
  • Z is a short linker of about 1 to about 10 atoms, with from 1 to 5 atoms being preferred, that may or may not contain alkene, alkynyl, amine, amide, azo, imine, etc., bonds.
  • Linkers are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • Preferred Z linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), with short alkyl groups, esters, amide, amine, epoxy groups and ethylene glycol and derivatives being preferred, with propyl, acetylene, and C 2 alkene being especially preferred.
  • Z may also be a sulfone group, forming sulfonamide linkages as discussed below.
  • the attachment of the nucleic acid and the conductive oligomer is done via attachment to the backbone of the nucleic acid. This may be done in a number of ways, including attachment to a ribose of the ribose-phosphate backbone, or to the phosphate of the backbone, or other groups of analogous backbones.
  • the site of attachment in this embodiment may be to a 3' or 5' terminal nucleotide, or to an internal nucleotide, as is more fully described below.
  • the conductive oligomer is attached to the ribose of the ribose-phosphate backbone. This may be done in several ways. As is known in the art, nucleosides that are modified at either the 2' or 3' position of the ribose with amino groups, sulfur groups, silicone groups, phosphorus groups, or oxo groups can be made (Imazawa et al., J. Org. Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem. 42(4):714 (1977); Verheyden et al., J. Orrg. Chem. 36(2):250 (1971 ); McGee et al., J. Org. Chem.
  • modified nucleosides are then used to add the conductive oligomers.
  • a preferred embodiment utilizes amino-modified nucleosides. These amino-modified riboses can then be used to form either amide or amine linkages to the conductive oligomers. In a preferred embodiment, the amino group is attached directly to the ribose, although as will be appreciated by those in the art, short linkers such as those described herein for "Z" may be present between the amino group and the ribose.
  • an amide linkage is used for attachment to the ribose.
  • m is zero and thus the conductive oligomer terminates in the amide bond.
  • the nitrogen of the amino group of the amino- modified ribose is the "D" atom of the conductive oligomer.
  • Structure 32 has the terminal bond fixed as an amide bond.
  • a heteroatom linkage is used, i.e. oxo, amine, sulfur, etc.
  • a preferred embodiment utilizes an amine linkage.
  • the nitrogen of the amino-modified ribose may be the "D" atom of the conductive oligomer when the Structure 3 conductive oligomer is used.
  • Structures 33 and 34 depict nucleosides with the Structures 13 and 21 conductive oligomers, respectively, using the nitrogen as the heteroatom, athough other heteroatoms can be used:
  • both m and t are not zero.
  • a preferred Z here is a methylene group, or other aliphatic alkyl linkers. One, two or three carbons in this position are particularly useful for synthetic reasons.
  • Suitable linkers include methylene and ethylene.
  • the conductive oligomer is covalently attached to the nucleic acid via the phosphate of the ribose-phosphate backbone (or analog) of a nucleic acid.
  • the attachment is direct, utilizes a linker or via an amide bond.
  • Structure 35 depicts a direct linkage
  • Structure 36 depicts linkage via an amide bond (both utilize the Structure 13 conductive oligomer, although Structure 20 conductive oligomers are also possible).
  • Structures 35 and 36 depict the conductive oligomer in the 3' position, although the 5' position is also possible.
  • both Structures 35 and 36 depict naturally occurring phosphodiester bonds, although as those in the art will appreciate, non-standard analogs of phosphodiester bonds may also be used.
  • Structure 36 depicts a preferred embodiment, wherein the terminal B-D bond is an amide bond, the terminal Y is not present, and Z is a linker, as defined herein.
  • the conductive oligomer is covalently attached to the nucleic acid via a transition metal ligand.
  • the conductive oligomer is covalently attached to a ligand which provides one or more of the coordination atoms for a transition metal.
  • the ligand to which the conductive oligomer is attached also has the nucleic acid attached, as is generally depicted below in Structure 37.
  • the conductive oligomer is attached to one ligand, and the nucleic acid is attached to another ligand, as is generally depicted below in Structure 38.
  • the conductive oligomer is covalently attached to the nucleic acid. Both of these structures depict Structure 13 conductive oligomers, although other oligomers may be utilized. Structures 37 and 38 depict two representative structures:
  • metal ions to connect the nucleic acids can serve as an internal control or calibration of the system, to evaluate the number of available nucleic acids on the surface.
  • metal ions are used to connect the nucleic acids to the conductive oligomers, it is generally desirable to have this metal ion complex have a different redox potential than that of the ETMs used in the rest of the system, as described below. This is generally true so as to be able to distinguish the presence of the capture probe from the presence of the target sequence. This may be useful for identification, calibration and/or quantification.
  • the amount of capture probe on an electrode may be compared to the amount of hybridized double stranded nucleic acid to quantify the amount of target sequence in a sample.
  • This is quite significant to serve as an internal control of the sensor or system. This allows a measurement either prior to the addition of target or after, on the same molecules that will be used for detection, rather than rely on a similar but different control system.
  • the actual molecules that will be used for the detection can be quantified prior to any experiment. This is a significant advantage over prior methods.
  • the capture probe nucleic acids are covalently attached to the electrode via an insulator.
  • insulators such as alkyl groups
  • the attachment of nucleic acids to insulators such as alkyl groups is well known, and can be done to the base or the backbone, including the ribose or phosphate for backbones containing these moieties, or to alternate backbones for nucleic acid analogs.
  • different capture probes, or one capture probes with a multiplicity of different capture extender probes can be used.
  • the present invention provides substrates comprising at least one detection electrode comprising monolayers and capture and/or detection probes, useful in target analyte detection systems.
  • the compositions of the invention are generally synthesized as outlined below, generally utilizing techniques well known in the art. As will be appreciated by those in the art, many of the techniques outlined below are directed to nucleic acids containing a ribose-phosphate backbone. However, as outlined above, many alternate nucleic acid analogs may be utilized, some of which may not contain either ribose or phosphate in the backbone. In these embodiments, for attachment at positions other than the base, attachment is done as will be appreciated by those in the art, depending on the backbone. Thus, for example, attachment can be made at the carbon atoms of the PNA backbone, as is described below, or at either terminus of the PNA.
  • compositions may be made in several ways.
  • a preferred method first synthesizes a conductive oligomer attached to a nucleoside, with addition of additional nucleosides to form the capture probe followed by attachment to the electrode.
  • the whole capture probe may be made and then the completed conductive oligomer added, followed by attachment to the electrode.
  • a monolayer of conductive oligomer (some of which have functional groups for attachment of capture probes) is attached to the electrode first, followed by attachment of the capture probe. The latter two methods may be preferred when conductive oligomers are used which are not stable in the solvents and under the conditions used in traditional nucleic acid synthesis.
  • compositions of the invention are made by first forming the conductive oligomer covalently attached to the nucleoside, followed by the addition of additional nucleosides to form a capture probe nucleic acid, with the last step comprising the addition of the conductive oligomer to the electrode.
  • the attachment of the conductive oligomer to the nucleoside may be done in several ways.
  • all or part of the conductive oligomer is synthesized first (generally with a functional group on the end for attachment to the electrode), which is then attached to the nucleoside. Additional nucleosides are then added as required, with the last step generally being attachment to the electrode.
  • oligomer units are added one at a time to the nucleoside, with addition of additional nucleosides and attachment to the electrode.
  • a number of representative syntheses are shown in the Figures of WO98/20162, expressly incorporated herein by reference.
  • the conductive oligomer is then attached to a nucleoside that may contain one (or more) of the oligomer units, attached as depicted herein.
  • attachment is to a ribose of the ribose-phosphate backbone.
  • attachment via amide and amine linkages are possible (see Figures 1 and 2 of WO98/20162).
  • a representative synthesis is shown in Figure 16 of WO98/20162.
  • attachment is via a phosphate of the ribose-phosphate backbone. Examples of two synthetic schemes are shown in Figure 4 and Figure 5 of WO98/20162. Although both Figures show attachment at the 3' position of the ribose, attachment can also be made via the 2' position.
  • Z is an ethylene linker, although other linkers may be used as well, as will be appreciated by those in the art.
  • attachment is via the base.
  • a general scheme is depicted in Figure 3 of WO98/20162, using uridine as the nucleoside and a phenylene-acetylene conductive oligomer.
  • amide linkages are also possible, using techniques well known in the art.
  • protecting groups may be added to the base prior to addition of the conductive oligomers, as is generally outlined in Figures 10 and 11 of WO98/20162.
  • the palladium cross-coupling reactions may be altered to prevent dimerization problems; i.e. two conductive oligomers dimerizing, rather than coupling to the base.
  • attachment to the base may be done by making the nucleoside with one unit of the oligomer, followed by the addition of others.
  • modified nucleosides Once the modified nucleosides are prepared, protected and activated, prior to attachment to the electrode, they may be incorporated into a growing oligonucleotide by standard synthetic techniques (Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK 1984; Eckstein) in several ways.
  • one or more modified nucleosides are converted to the triphosphate form and incorporated into a growing oligonucleotide chain by using standard molecular biology techniques such as with the use of the enzyme DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptase, and RNA polymerases.
  • DNA polymerase I DNA polymerase I
  • T4 DNA polymerase T4 DNA polymerase
  • T7 DNA polymerase T7 DNA polymerase
  • Taq DNA polymerase reverse transcriptase
  • RNA polymerases RNA polymerases.
  • terminal deoxynucleotidyltransferase may be used. (Ratliff, Terminal deoxynucleotidyltransferase. In The Enzymes, Vol 14A. P.D. Boyer ed. pp 105-118.
  • the present invention provides deoxyribonucleoside triphosphates comprising a covalently attached ETM.
  • Preferred embodiments utilize ETM attachment to the base or the backbone, such as the ribose (preferably in the 2' position), as is generally depicted below in Structures 40 and 41 :
  • nucleic acids comprising ETMs in situ.
  • a target sequence can hybridize to a capture probe (for example on the surface) in such a way that the terminus of the target sequence is exposed, i.e. unhybridized.
  • the addition of enzyme and triphosphate nucleotides labelled with ETMs allows the in situ creation of the label.
  • labeled nucleotides recognized by polymerases can allow simultaneous amplification and detection; that is, the target sequences are generated in situ.
  • the modified nucleoside is converted to the phosphoramidite or H- phosphonate form, which are then used in solid-phase or solution syntheses of oligonucleotides.
  • the modified nucleoside either for attachment at the ribose (i.e. amino- or thiol-modified nucleosides) or the base, is incorporated into the oligonucleotide at either an internal position or the 5' terminus. This is generally done in one of two ways.
  • the 5' position of the ribose is protected with 4',4-dimethoxytrityl (DMT) followed by reaction with either 2-cyanoethoxy-bis- diisopropylaminophosphine in the presence of diisopropylammonium tetrazolide, or by reaction with chlorodiisopropylamino 2'-cyanoethyoxyphosphine, to give the phosphoramidite as is known in the art; although other techniques may be used as will be appreciated by those in the art. See Gait, supra; Caruthers, Science 230:281 (1985), both of which are expressly incorporated herein by reference.
  • DMT 4',4-dimethoxytrityl
  • a preferred method utilizes the attachment of the modified nucleoside (or the nucleoside replacement) to controlled pore glass (CPG) or other oligomeric supports.
  • CPG controlled pore glass
  • the modified nucleoside is protected at the 5' end with DMT, and then reacted with succinic anhydride with activation.
  • the resulting succinyl compound is attached to CPG or other oligomeric supports as is known in the art.
  • Further phosphoramidite nucleosides are added, either modified or not, to the 5' end after deprotection.
  • the present invention provides conductive oligomers or insulators covalently attached to nucleosides attached to solid oligomeric supports such as CPG, and phosphoramidite derivatives of the nucleosides of the invention.
  • the invention further provides methods of making label probes with recruitment linkers comprising ETMs. These synthetic reactions will depend on the character of the recruitment linker and the method of attachment of the ETM, as will be appreciated by those in the art.
  • the label probes are generally made as outlined herein with the incorporation of ETMs at one or more positions.
  • a transition metal complex is used as the ETM, synthesis may occur in several ways.
  • the ligand(s) are added to a nucleoside, followed by the transition metal ion, and then the nucleoside with the transition metal complex attached is added to an oligonucleotide, i.e. by addition to the nucleic acid synthesizer.
  • the ligand(s) may be attached, followed by incorportation into a growing oligonucleotide chain, followed by the addition of the metal ion.
  • ETMs are attached to a ribose of the ribose-phosphate backbone. This is generally done as is outlined herein for conductive oligomers, as described herein, and in PCT publication WO 95/15971 , using amino-modified or oxo-modified nucleosides, at either the 2' or 3' position of the ribose.
  • the amino group may then be used either as a ligand, for example as a transition metal ligand for attachment of the metal ion, or as a chemically functional group that can be used for attachment of other ligands or organic ETMs, for example via amide linkages, as will be appreciated by those in the art.
  • the examples describe the synthesis of nucleosides with a variety of ETMs attached via the ribose.
  • ETMs are attached to a phosphate of the ribose-phosphate backbone.
  • this may be done using phosphodiester analogs such as phosphoramidite bonds, see generally PCT publication WO 95/15971 , or can be done in a similar manner to that depicted in Figures 4 and 5 of PCT US97/20014, where the conductive oligomer is replaced by a transition metal ligand or complex or an organic ETM, as well as is outlined in the Examples.
  • ETMs are attached to a base of the nucleoside. This may be done in a variety of ways. In one embodiment, amino groups of the base, either naturally occurring or added as is described herein (see the fiigures, for example), are used either as ligands for transition metal complexes or as a chemically functional group that can be used to add other ligands, for example via an amide linkage, or organic ETMs. This is done as will be appreciated by those in the art. Alternatively, nucleosides containing halogen atoms attached to the heterocyclic ring are commercially available.
  • Acetylene linked ligands may be added using the halogenated bases, as is generally known; see for example, Tzalis et al., Tetrahedron Lett. 36(34):6017-6020 (1995); Tzalis et al., Tetrahedron Lett. 36(2):3489-3490 (1995); and Tzalis et al., Chem. Communications (in press) 1996, all of which are hereby expressly incorporated by reference. See also the figures and the examples, which describes the synthesis of metallocenes (in this case, ferrocene) attached via acetylene linkages to the bases.
  • metallocenes in this case, ferrocene
  • the nucleosides are made with transition metal ligands, incorporated into a nucleic acid, and then the transition metal ion and any remaining necessary ligands are added as is known in the art. In an alternative embodiment, the transition metal ion and additional ligands are added prior to incorporation into the nucleic acid.
  • the attachment linker is attached to the electrode.
  • the method will vary depending on the type of electrode used.
  • the attachment linkers are generally made with a terminal "A" linker to facilitate attachment to the electrode.
  • a sulfur-gold attachment is considered a covalent attachment.
  • conductive oligomers, insulators, and attachment linkers are covalently attached via sulfur linkages to the electrode.
  • traditional protecting groups for use of attaching molecules to gold electrodes are generally not ideal for use in both synthesis of the compositions described herein and inclusion in oligonucleotide synthetic reactions.
  • the present invention provides novel methods for the attachment of conductive oligomers to gold electrodes, utilizing unusual protecting groups, including ethylpyridine, and trimethylsilylethyl as is depicted in the Figures.
  • unusual protecting groups including ethylpyridine, and trimethylsilylethyl as is depicted in the Figures.
  • traditional protecting groups such as acetyl groups and others may be used. See Greene et al., supra.
  • the subunit of the conductive oligomer which contains the sulfur atom for attachment to the electrode is protected with an ethyl-pyridine or trimethylsilylethyl group.
  • this is generally done by contacting the subunit containing the sulfur atom (preferably in the form of a sulfhydryl) with a vinyl pyridine group or vinyl trimethylsilylethyl group under conditions whereby an ethylpyridine group or trimethylsilylethyl group is added to the sulfur atom.
  • This subunit also generally contains a functional moiety for attachment of additional subunits, and thus additional subunits are attached to form the conductive oligomer.
  • the conductive oligomer is then attached to a nucleoside, and additional nucleosides attached.
  • the protecting group is then removed and the sulfur-gold covalent attachment is made.
  • all or part of the conductive oligomer is made, and then either a subunit containing a protected sulfur atom is added, or a sulfur atom is added and then protected.
  • the conductive oligomer is then attached to a nucleoside, and additional nucleosides attached.
  • the conductive oligomer attached to a nucleic acid is made, and then either a subunit containing a protected sulfur atom is added, or a sulfur atom is added and then protected.
  • the ethyl pyridine protecting group may be used as above, but removed after one or more steps and replaced with a standard protecting group like a disulfide.
  • the ethyl pyridine or trimethylsilylethyl group may serve as the protecting group for some of the synthetic reactions, and then removed and replaced with a traditional protecting group.
  • subunit of a conductive polymer herein is meant at least the moiety of the conductive oligomer to which the sulfur atom is attached, although additional atoms may be present, including either functional groups which allow the addition of additional components of the conductive oligomer, or additional components of the conductive oligomer.
  • a subunit comprises at least the first Y group.
  • a preferred method comprises 1 ) adding an ethyl pyridine or trimethylsilylethyl protecting group to a sulfur atom attached to a first subunit of a conductive oligomer, generally done by adding a vinyl pyridine or trimethylsilylethyl group to a sulfhydryl; 2) adding additional subunits to form the conductive oligomer; 3) adding at least a first nucleoside to the conductive oligomer; 4) adding additional nucleosides to the first nucleoside to form a nucleic acid; 5) attaching the conductive oligomer to the gold electrode. This may also be done in the absence of nucleosides, as is described in the Examples.
  • the above method may also be used to attach insulator molecules to a gold electrode.
  • a monolayer comprising conductive oligomers (and optionally insulators) is added to the electrode.
  • the chemistry of addition is similar to or the same as the addition of conductive oligomers to the electrode, i.e. using a sulfur atom for attachment to a gold electrode, etc.
  • compositions comprising monolayers in addition to the conductive oligomers covalently attached to nucleic acids may be made in at least one of five ways: (1 ) addition of the monolayer, followed by subsequent addition of the attachment linker-nucleic acid complex; (2) addition of theattachment linker-nucleic acid complex followed by addition of the monolayer; (3) simultaneous addition of the monolayer and attachment linker-nucleic acid complex; (4) formation of a monolayer (using any of 1 , 2 or 3) which includes attachment linkers which terminate in a functional moiety suitable for attachment of a completed nucleic acid; or (5) formation of a monolayer which includes attachment linkers which terminate in a functional moiety suitable for nucleic acid synthesis, i.e.
  • nucleic acid is synthesized on the surface of the monolayer as is known in the art.
  • suitable functional moieties include, but are not limited to, nucleosides, amino groups, carboxyl groups, protected sulfur moieties, or hydroxyl groups for phosphoramidite additions.
  • the examples describe the formation of a monolayer on a gold electrode using the preferred method (1 ).
  • the nucleic acid is a peptide nucleic acid or analog.
  • the invention provides peptide nucleic acids with at least one covalently attached ETM or attachment linker.
  • these moieties are covalently attached to an monomeric subunit of the PNA.
  • monomeric subunit of PNA herein is meant the -NH-CH 2 CH 2 -N(COCH 2 -Base)-CH 2 -CO- monomer, or derivatives (herein included within the definition of "nucleoside") of PNA.
  • the number of carbon atoms in the PNA backbone may be altered; see generally Nielsen et al., Chem. Soc. Rev.
  • the amide bond linking the base to the backbone may be altered; phosphoramide and sulfuramide bonds may be used.
  • the moieties are attached to an internal monomeric subunit.
  • internal herein is meant that the monomeric subunit is not either the N-terminal monomeric subunit or the C-terminal monomeric subunit.
  • the moieties can be attached either to a base or to the backbone of the monomeric subunit. Attachment to the base is done as outlined herein or known in the literature. In general, the moieties are added to a base which is then incorporated into a PNA as outlined herein.
  • the base may be either protected, as required for incorporation into the PNA synthetic reaction, or derivatized, to allow incorporation, either prior to the addition of the chemical substituent or afterwards. Protection and derivatization of the bases is shown in Figures 24-27 of WO98/20162. The bases can then be incorporated into monomeric subunits as shown in Figure 28 of WO98/20162.
  • Figure 29 depicts a representative synthesis of a PNA monomeric subunit with a ferrocene attached to a uracil base.
  • Figure 30 depicts the synthesis of a three unit conductive oligomer attached to a uracil base.
  • the moieties are covalently attached to the backbone of the PNA monomer.
  • the attachment is generally to one of the unsubstituted carbon atoms of the monomeric subunit, preferably the ⁇ -carbon of the backbone, although attachment at either of the carbon 1 or 2 positions, or the ⁇ -carbon of the amide bond linking the base to the backbone may be done.
  • other carbons or atoms may be substituted as well.
  • moieties are added at the ⁇ -carbon atoms, either to a terminal monomeric subunit or an internal one.
  • a modified monomeric subunit is synthesized with an ETM or an attachment linker, or a functional group for its attachment, and then the base is added and the modified monomer can be incorporated into a growing PNA chain.
  • Figure 31 of WO98/20162 depicts the synthesis of a conductive oligomer covalently attached to the backbone of a PNA monomeric subunit
  • Figure 32 of WO98/20162 depicts the synthesis of a ferrocene attached to the backbone of a monomeric subunit.
  • compositions of the invention may additionally contain one or more labels at any position.
  • label herein is meant an element (e.g. an isotope) or chemical compound that is attached to enable the detection of the compound.
  • Preferred labels are radioactive isotopic labels, and colored or fluorescent dyes.
  • the labels may be incorporated into the compound at any position.
  • the compositions of the invention may also contain other moieties such as cross-linking agents to facilitate cross-linking of the target-probe complex. See for example, Lukhtanov et al., Nucl. Acids. Res. 24(4):683 (1996) and Tabone et al., Biochem. 33:375 (1994), both of which are expressly incorporated by reference.
  • detection probes are covalently attached to the electrode, as above for capture probes.
  • the detection probes are either substantially complementary to a portion of the target sequence (direct detection), or to a portion of a label probe (sandwich assay), as is depicted in the Figures.
  • the first probe sequence may comprise a separation tag (e.g. biotin) or sequence (e.g.
  • compositions find use in a number of applications, as described herein.
  • compositions of the invention find use in hybridization assays.
  • electrodes can be made that have a single species of nucleic acid, i.e. a single nucleic acid sequence, or multiple nucleic acid species.
  • oligonucleotide arrays are well known in the art.
  • techniques are known for "addressing" locations within an electrode and for the surface modification of electrodes.
  • arrays of different nucleic acids are laid down on the electrode, each of which are covalently attached to the electrode via a conductive linker.
  • the number of different probe species of oligonucleotides may vary widely, from one to thousands, with from about 4 to about 100,000 being preferred, and from about 10 to about 10,000 being particularly preferred.
  • detection proceeds with electronic initiation. Without being limited by the mechanism or theory, detection is based on the transfer of electrons from the ETM to the electrode.
  • Detection of electron transfer i.e. the presence of the ETMs, is generally initiated electronically, with voltage being preferred.
  • a potential is applied to the assay complex. Precise control and variations in the applied potential can be via a potentiostat and either a three electrode system (one reference, one sample (or working) and one counter electrode) or a two electrode system (one sample and one counter electrode). This allows matching of applied potential to peak potential of the system which depends in part on the choice of ETMs and in part on the conductive oligomer used, the composition and integrity of the monolayer, and what type of reference electrode is used. As described herein, ferrocene is a preferred ETM.
  • a co-reductant or co-oxidant (collectively, co-redoxant) is used, as an additional electron source or sink.
  • co-reductant or co-oxidant (collectively, co-redoxant) is used, as an additional electron source or sink.
  • an input electron source in solution is used in the initiation of electron transfer, preferably when initiation and detection are being done using DC current or at AC frequencies where diffusion is not limiting.
  • preferred embodiments utilize monolayers that contain a minimum of "holes", such that short-circuiting of the system is avoided. This may be done in several general ways.
  • an input electron source is used that has a lower or similar redox potential than the ETM of the label probe. Thus, at voltages above the redox potential of the input electron source, both the ETM and the input electron source are oxidized and can thus donate electrons; the ETM donates an electron to the electrode and the input source donates to the ETM.
  • ferrocene as a ETM attached to the compositions of the invention as described in the examples, has a redox potential of roughly 200 mV in aqueous solution (which can change significantly depending on what the ferrocene is bound to, the manner of the linkage and the presence of any substitution groups).
  • Ferrocyanide an electron source, has a redox potential of roughly 200 mV as well (in aqueous solution). Accordingly, at or above voltages of roughly 200 mV, ferrocene is converted to ferricenium, which then transfers an electron to the electrode. Now the ferricyanide can be oxidized to transfer an electron to the ETM.
  • the electron source serves to amplify the signal generated in the system, as the electron source molecules rapidly and repeatedly donate electrons to the ETM attached to the nucleic acid.
  • the rate of electron donation or acceptance will be limited by the rate of diffusion of the co-reductant, the electron transfer between the co-reductant and the ETM, which in turn is affected by the concentration and size, etc.
  • input electron sources that have lower redox potentials than the ETM are used. At voltages less than the redox potential of the ETM, but higher than the redox potential of the electron source, the input source such as ferrocyanide is unable to be oxided and thus is unable to donate an electron to the ETM; i.e. no electron transfer occurs. Once ferrocene is oxidized, then there is a pathway for electron transfer.
  • an input electron source is used that has a higher redox potential than the ETM of the label probe.
  • luminol an electron source
  • the ferrocene is oxided, and transfers a single electron to the electrode via the conductive oligomer.
  • the ETM is unable to accept any electrons from the luminol electron source, since the voltages are less than the redox potential of the luminol.
  • the luminol then transfers an electron to the ETM, allowing rapid and repeated electron transfer.
  • the electron source serves to amplify the signal generated in the system, as the electron source molecules rapidly and repeatedly donate electrons to the ETM of the label probe.
  • Luminol has the added benefit of becoming a chemiluminiscent species upon oxidation (see Jirka et al., Analytica Chimica Acta 284:345 (1993)), thus allowing photo-detection of electron transfer from the ETM to the electrode.
  • luminol can only be oxidized by transferring an electron to the ETM on the label probe.
  • the ETM is not present, i.e.
  • the measure of luminol oxidation by photon emission is an indirect measurement of the ability of the ETM to donate electrons to the electrode.
  • photon detection is generally more sensitive than electronic detection, the sensitivity of the system may be increased. Initial results suggest that luminescence may depend on hydrogen peroxide concentration, pH, and luminol concentration, the latter of which appears to be non-linear.
  • Suitable electron source molecules are well known in the art, and include, but are not limited to, ferricyanide, and luminol.
  • output electron acceptors or sinks could be used, i.e. the above reactions could be run in reverse, with the ETM such as a metallocene receiving an electron from the electrode, converting it to the metallicenium, with the output electron acceptor then accepting the electron rapidly and repeatedly.
  • cobalticenium is the preferred ETM.
  • the presence of the ETMs at the surface of the monolayer can be detected in a variety of ways.
  • a variety of detection methods may be used, including, but not limited to, optical detection (as a result of spectral changes upon changes in redox states), which includes fluorescence, phosphorescence, luminiscence, chemiluminescence, electrochemiluminescence, and refractive index; and electronic detection, including, but not limited to, amperommetry, voltammetry, capacitance and impedence.
  • optical detection as a result of spectral changes upon changes in redox states
  • electronic detection including, but not limited to, amperommetry, voltammetry, capacitance and impedence.
  • These methods include time or frequency dependent methods based on AC or DC currents, pulsed methods, lock-in techniques, filtering (high pass, low pass, band pass), and time-resolved techniques including time-resolved fluoroscence.
  • the efficient transfer of electrons from the ETM to the electrode results in stereotyped changes in the redox state of the ETM.
  • ETMs including the complexes of ruthenium containing bipyridine, pyridine and imidazole rings
  • these changes in redox state are associated with changes in spectral properties.
  • Significant differences in absorbance are observed between reduced and oxidized states for these molecules. See for example Fabbrizzi et al., Chem. Soc. Rev. 1995 pp197-202). These differences can be monitored using a spectrophotometer or simple photomultiplier tube device.
  • possible electron donors and acceptors include all the derivatives listed above for photoactivation or initiation.
  • Preferred electron donors and acceptors have characteristically large spectral changes upon oxidation and reduction resulting in highly sensitive monitoring of electron transfer.
  • Such examples include Ru(NH 3 ) 4 py and Ru(bpy) 2 im as preferred examples. It should be understood that only the donor or acceptor that is being monitored by absorbance need have ideal spectral characteristics.
  • the electron transfer is detected fluorometrically.
  • Numerous transition metal complexes, including those of ruthenium, have distinct fluorescence properties. Therefore, the change in redox state of the electron donors and electron acceptors attached to the nucleic acid can be monitored very sensitively using fluorescence, for example with Ru(4,7-biphenyl 2 -phenanthroline) 3 2+
  • the production of this compound can be easily measured using standard fluorescence assay techniques. For example, laser induced fluorescence can be recorded in a standard single cell fluorimeter, a flow through "on-line” fluorimeter (such as those attached to a chromatography system) or a multi-sample "plate-reader” similar to those marketed for 96-well immuno assays.
  • fluorescence can be measured using fiber optic sensors with nucleic acid probes in solution or attached to the fiber optic. Fluorescence is monitored using a photomultiplier tube or other light detection instrument attached to the fiber optic. The advantage of this system is the extremely small volumes of sample that can be assayed.
  • scanning fluorescence detectors such as the Fluorlmager sold by Molecular Dynamics are ideally suited to monitoring the fluorescence of modified nucleic acid molecules arrayed on solid surfaces.
  • the advantage of this system is the large number of electron transfer probes that can be scanned at once using chips covered with thousands of distinct nucleic acid probes.
  • transition metal complexes display fluorescence with large Stokes shifts. Suitable examples include bis- and trisphenanthroline complexes and bis- and trisbipyridyl complexes of transition metals such as ruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84, p. 85-277, 1988). Preferred examples display efficient fluorescence (reasonably high quantum yields) as well as low reorganization energies.
  • electrochemiluminescence is used as the basis of the electron transfer detection.
  • ETMs such as Ru 2+ (bpy) 3
  • direct luminescence accompanies excited state decay. Changes in this property are associated with nucleic acid hybridization and can be monitored with a simple photomultiplier tube arrangement (see Blackburn, G. F. Clin. Chem. 37: 1534-1539
  • electronic detection is used, including amperommetry, voltammetry, capacitance, and impedence.
  • Suitable techniques include, but are not limited to, electrogravimetry; coulometry (including controlled potential coulometry and constant current coulometry); voltametry (cyclic voltametry, pulse voltametry (normal pulse voltametry, square wave voltametry, differential pulse voltametry, Osteryoung square wave voltametry, and coulostatic pulse techniques); stripping analysis (aniodic stripping analysis, cathiodic stripping analysis, square wave stripping voltammetry); conductance measurements (electrolytic conductance, direct analysis); time-dependent electrochemical analyses (chronoamperometry, chronopotentiometry, cyclic chronopotentiometry and amperometry, AC polography, chronogalvametry, and chronocoulometry); AC impedance measurement; capacitance measurement; AC voltametry; and photoelectrochemistry.
  • monitoring electron transfer is via amperometric detection.
  • This method of detection involves applying a potential (as compared to a separate reference electrode) between the nucleic acid-conjugated electrode and a reference (counter) electrode in the sample containing target genes of interest. Electron transfer of differing efficiencies is induced in samples in the presence or absence of target nucleic acid; that is, the presence or absence of the target nucleic acid, and thus the label probe, can result in different currents.
  • the device for measuring electron transfer amperometrically involves sensitive current detection and includes a means of controlling the voltage potential, usually a potentiostat. This voltage is optimized with reference to the potential of the electron donating complex on the label probe.
  • Possible electron donating complexes include those previously mentioned with complexes of iron, osmium, platinum, cobalt, rhenium and ruthenium being preferred and complexes of iron being most preferred.
  • potentiometric (or voltammetric) measurements involve non-faradaic (no net current flow) processes and are utilized traditionally in pH and other ion detectors. Similar sensors are used to monitor electron transfer between the ETM and the electrode.
  • other properties of insulators (such as resistance) and of conductors (such as conductivity, impedance and capicitance) could be used to monitor electron transfer between ETM and the electrode.
  • any system that generates a current (such as electron transfer) also generates a small magnetic field, which may be monitored in some embodiments.
  • one benefit of the fast rates of electron transfer observed in the compositions of the invention is that time resolution can greatly enhance the signal-to-noise results of monitors based on absorbance, fluorescence and electronic current.
  • the fast rates of electron transfer of the present invention result both in high signals and stereotyped delays between electron transfer initiation and completion. By amplifying signals of particular delays, such as through the use of pulsed initiation of electron transfer and "lock-in" amplifiers of detection, and Fourier transforms.
  • electron transfer is initiated using alternating current (AC) methods.
  • AC alternating current
  • ETMs bound to an electrode, generally respond similarly to an AC voltage across a circuit containing resistors and capacitors. Basically, any methods which enable the determination of the nature of these complexes, which act as a resistor and capacitor, can be used as the basis of detection.
  • traditional electrochemical theory such as exemplified in Laviron et al., J. Electroanal. Chem. 97:135 (1979) and Laviron et al., J. Electroanal. Chem.
  • Equation 1 Equation 1
  • Equation 1 n is the number of electrons oxidized or reduced per redox molecule, f is the applied frequency, F is Faraday's constant, N tota , is the total number of redox molecules, E 0 is the formal potential of the redox molecule, R is the gas constant, T is the temperature in degrees Kelvin, and E DC is the electrode potential.
  • the model fits the experimental data very well. In some cases the current is smaller than predicted, however this has been shown to be caused by ferrocene degradation which may be remedied in a number of ways.
  • the faradaic current can also be expressed as a function of time, as shown in Equation 2:
  • l F is the Faradaic current and q e is the elementary charge.
  • Equation 1 does not incorporate the effect of electron transfer rate nor of instrument factors. Electron transfer rate is important when the rate is close to or lower than the applied frequency. Thus, the true i AC should be a function of all three, as depicted in Equation 3.
  • Equation 3 i AC f(Nemst factors )f(k ET )f(instrument factors)
  • non-specifically bound label probes/ETMs show differences in impedance (i.e. higher impedances) than when the label probes containing the ETMs are specifically bound in the correct orientation.
  • the non-specifically bound material is washed away, resulting in an effective impedance of infinity.
  • frequency response when using AC initiation and detection methods, the frequency response of the system changes as a result of the presence of the ETM.
  • frequency response herein is meant a modification of signals as a result of electron transfer between the electrode and the ETM. This modification is different depending on signal frequency.
  • a frequency response includes AC currents at one or more frequencies, phase shifts, DC offset voltages, faradaic impedance, etc.
  • the first input electrical signal is then applied to the system, preferably via at least the sample electrode (containing the complexes of the invention) and the counter electrode, to initiate electron transfer between the electrode and the ETM.
  • the first input signal comprises at least an AC component.
  • the AC component may be of variable amplitude and frequency. Generally, for use in the present methods, the AC amplitude ranges from about 1 mV to about 1.1 V, with from about 10 mV to about 800 mV being preferred, and from about 10 mV to about 500 mV being especially preferred.
  • the AC frequency ranges from about 0.01 Hz to about 100 MHz, with from about 10 Hz to about 10 MHz being preferred, and from about 100 Hz to about 20 MHz being especially preferred.
  • the first input signal comprises a DC component and an AC component.
  • a DC offset voltage between the sample and counter electrodes is swept through the electrochemical potential of the ETM (for example, when ferrocene is used, the sweep is generally from 0 to 500 mV) (or alternatively, the working electrode is grounded and the reference electrode is swept from 0 to -500 mV).
  • the sweep is used to identify the DC voltage at which the maximum response of the system is seen. This is generally at or about the electrochemical potential of the ETM.
  • DC offset voltages of from about -1 V to about +1.1 V are preferred, with from about -500 mV to about +800 mV being especially preferred, and from about -300 mV to about 500 mV being particularly preferred. In a preferred embodiment, the DC offset voltage is not zero.
  • an AC signal component of variable amplitude and frequency is applied. If the ETM is present, and can respond to the AC perturbation, an AC current will be produced due to electron transfer between the electrode and the ETM.
  • a single input signal may be applied to differentiate between the presence and absence of the ETM (i.e. the presence of the target sequence) nucleic acid.
  • a plurality of input signals are applied. As outlined herein, this may take a variety of forms, including using multiple frequencies, multiple DC offset voltages, or multiple AC amplitudes, or combinations of any or all of these.
  • DC offset voltages are used, although as outlined above, DC voltage sweeps are preferred. This may be done at a single frequency, or at two or more frequencies .
  • the AC amplitude is varied. Without being bound by theory, it appears that increasing the amplitude increases the driving force. Thus, higher amplitudes, which result in higher overpotentials give faster rates of electron transfer. Thus, generally, the same system gives an improved response (i.e. higher output signals) at any single frequency through the use of higher overpotentials at that frequency. Thus, the amplitude may be increased at high frequencies to increase the rate of electron transfer through the system, resulting in greater sensitivity. In addition, this may be used, for example, to induce responses in slower systems such as those that do not possess optimal spacing configurations.
  • measurements of the system are taken at at least two separate amplitudes or overpotentials, with measurements at a plurality of amplitudes being preferred.
  • changes in response as a result of changes in amplitude may form the basis of identification, calibration and quantification of the system.
  • one or more AC frequencies can be used as well.
  • the AC frequency is varied.
  • different molecules respond in different ways.
  • increasing the frequency generally increases the output current.
  • higher frequencies result in a loss or decrease of output signal.
  • the frequency will be greater than the rate of electron transfer between the ETM and the electrode, and then the output signal will also drop.
  • detection utilizes a single measurement of output signal at a single frequency. That is, the frequency response of the system in the absence of target sequence, and thus the absence of label probe containing ETMs, can be previously determined to be very low at a particular high frequency. Using this information, any response at a particular frequency, will show the presence of the assay complex. That is, any response at a particular frequency is characteristic of the assay complex. Thus, it may only be necessary to use a single input high frequency, and any changes in frequency response is an indication that the ETM is present, and thus that the target sequence is present.
  • the use of AC techniques allows the significant reduction of background signals at any single frequency due to entities other than the ETMs, i.e.
  • one or more frequencies can be chosen that prevent a frequency response of one or more charge carriers in solution, whether or not a monolayer is present. This is particularly significant since many biological fluids such as blood contain significant amounts of redox active molecules which can interfere with amperometric detection methods.
  • measurements of the system are taken at at least two separate frequencies, with measurements at a plurality of frequencies being preferred.
  • a plurality of frequencies includes a scan. For example, measuring the output signal, e.g., the AC current, at a low input frequency such as 1 - 20 Hz, and comparing the response to the output signal at high frequency such as 10 - 100 kHz will show a frequency response difference between the presence and absence of the ETM.
  • the frequency response is determined at at least two, preferably at least about five, and more preferably at least about ten frequencies.
  • an output signal is received or detected.
  • the presence and magnitude of the output signal will depend on a number of factors, including the overpotential/amplitude of the input signal; the frequency of the input AC signal; the composition of the intervening medium; the DC offset; the environment of the system; the nature of the ETM; the solvent; and the type and concentration of salt.
  • the presence and magnitude of the output signal will depend in general on the presence or absence of the ETM, the placement and distance of the ETM from the surface of the monolayer and the character of the input signal. In some embodiments, it may be possible to distinguish between non-specific binding of label probes and the formation of target specific assay complexes containing label probes, on the basis of impedance.
  • the output signal comprises an AC current.
  • the magnitude of the output current will depend on a number of parameters. By varying these parameters, the system may be optimized in a number of ways.
  • AC currents generated in the present invention range from about 1 femptoamp to about 1 milliamp, with currents from about 50 femptoamps to about 100 microamps being preferred, and from about 1 picoamp to about 1 microamp being especially preferred.
  • the output signal is phase shifted in the AC component relative to the input signal.
  • the systems of the present invention may be sufficiently uniform to allow phase-shifting based detection. That is, the complex biomolecules of the invention through which electron transfer occurs react to the AC input in a homogeneous manner, similar to standard electronic components, such that a phase shift can be determined. This may serve as the basis of detection between the presence and absence of the ETM, and/or differences between the presence of target-specific assay complexes comprising label probes and non-specific binding of the label probes to the system components.
  • the output signal is characteristic of the presence of the ETM; that is, the output signal is characteristic of the presence of the target-specific assay complex comprising label probes and ETMs.
  • the basis of the detection is a difference in the faradaic impedance of the system as a result of the formation of the assay complex.
  • Faradaic impedance is the impedance of the system between the electrode and the ETM. Faradaic impedance is quite different from the bulk or dielectric impedance, which is the impedance of the bulk solution between the electrodes. Many factors may change the faradaic impedance which may not effect the bulk impedance, and vice versa.
  • the assay complexes comprising the nucleic acids in this system have a certain faradaic impedance, that will depend on the distance between the ETM and the electrode, their electronic properties, and the composition of the intervening medium, among other things.
  • the faradaic impedance between the ETM and the electrode is signficantly different depending on whether the label probes containing the ETMs are specifically or non-specifically bound to the electrode.
  • the present invention further provides apparatus for the detection of nucleic acids using
  • the apparatus includes a test chamber which has at least a first measuring or sample electrode, and a second measuring or counter electrode. Three electrode systems are also useful.
  • the first and second measuring electrodes are in contact with a test sample receiving region, such that in the presence of a liquid test sample, the two electrodes may be in electrical contact.
  • the first measuring electrode comprises a single stranded nucleic acid capture probe covalently attached via an attachment linker, and a monolayer comprising conductive oligomers, such as are described herein.
  • the apparatus further comprises an AC voltage source electrically connected to the test chamber; that is, to the measuring electrodes.
  • the AC voltage source is capable of delivering DC offset voltage as well.
  • the apparatus further comprises a processor capable of comparing the input signal and the output signal.
  • the processor is coupled to the electrodes and configured to receive an output signal, and thus detect the presence of the target nucleic acid.
  • compositions of the present invention may be used in a variety of research, clinical, quality control, or field testing settings.
  • the probes are used in genetic diagnosis.
  • probes can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art.
  • viral and bacterial detection is done using the complexes of the invention.
  • probes are designed to detect target sequences from a variety of bacteria and viruses.
  • current blood-screening techniques rely on the detection of anti- HIV antibodies.
  • the methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences. In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies.
  • viruses associated with leukemia, HTLV-I and HTLV-II may be detected in this way.
  • Bacterial infections such as tuberculosis, clymidia and other sexually transmitted diseases, may also be detected, for example using ribosomal RNA (rRNA) as the target sequences.
  • rRNA ribosomal RNA
  • the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples.
  • samples may be treated to lyse the bacteria to release its nucleic acid (particularly rRNA), and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E. coli, and Legionnaire's disease bacteria.
  • bioremediation strategies may be evaluated using the compositions of the invention.
  • the probes are used for forensic "DNA fingerprinting" to match crime-scene
  • the probes in an array are used for sequencing by hybridization.
  • the present invention provides for extremely specific and sensitive probes, which may, in some embodiments, detect target sequences without removal of unhybridized probe. This will be useful in the generation of automated gene probe assays.
  • compositions of the invention are useful to detect successful gene amplification in PCR, thus allowing successful PCR reactions to be an indication of the presence or absence of a target sequence.
  • PCR may be used in this manner in several ways.
  • the PCR reaction is done as is known in the art, and then added to a composition of the invention comprising the target nucleic acid with a ETM, covalently attached to an electrode via a conductive oligomer with subsequent detection of the target sequence.
  • PCR is done using nucleotides labelled with a ETM, either in the presence of, or with subsequent addition to, an electrode with a conductive oligomer and a target nucleic acid.
  • the nucleic acid attached to the electrode via a conductive polymer may be one PCR primer, with addition of a second primer labelled with an ETM. Elongation results in double stranded nucleic acid with a ETM and electrode covalently attached. In this way, the present invention is used for PCR detection of target sequences.
  • the arrays are used for mRNA detection.
  • a preferred embodiment utilizes either capture probes or capture extender probes that hybridize close to the 3' polyadenylation tail of the mRNAs. This allows the use of one species of target binding probe for detection, i.e. the probe contains a poly-T portion that will bind to the poly-A tail of the mRNA target. Generally, the probe will contain a second portion, preferably non-poly-T, that will bind to the detection probe (or other probe). This allows one target-binding probe to be made, and thus decreases the amount of different probe synthesis that is done.
  • the use of restriction enzymes and ligation methods allows the creation of "universal" arrays.
  • monolayers comprising capture probes that comprise restriction endonuclease ends as is generally depicted in Figure 7.
  • an array comprising any number of restriction endonuclease sites is made.
  • Treating a target sample with one or more of these restriction endonucleases allows the targets to bind to the array. This can be done without knowing the sequence of the target.
  • the target sequences can be ligated, as desired, using standard methods such as ligases, and the target sequence detected, using either standard labels or the methods of the invention.
  • the present invention provides methods which can result in sensitive detection of nucleic acids.
  • less than about 10 X 10 s molecules are detected, with less than about 10 X 10 5 being preferred, less than 10 X 10 4 being particularly preferred, less than about 10 X 10 3 being especially preferred, and less than about 10 X 10 2 being most preferred.
  • this assumes a 1 :1 correlation between target sequences and reporter molecules; if more than one reporter molecule (i.e. electron transfer moeity) is used for each target sequence, the sensitivity will go up.
  • kits useful in the practice of the invention.
  • the kits include the compositions including the primers and enzymes, along with any number of reagents or buffers, including additional enzymes and primers, dNTPs and/or NTPs (including substituted nucleotides), buffers, salts, inhibitors, etc.
  • the kits can optionally include instructions for the use of the kits.
  • the column was eluted with 20%ethyl acetate/dichloromethane, 50 % ethyl acetate/dichloromethane, 70 % ethyl acetate/dichloromethane, ethyl acetate, 1 % methanol/ethyl acetate, 3 % methanol/ethyl acetate, and
  • N17 The synthesis of N17 is described, as depicted in Figure 11 A.
  • the aqueous layer was further extracted by ethyl acetate ( 3 x 200 mL). The combined organic layer was dried over Na 2 S0 4 and concentrated.
  • the crude product was further purified on a flash column of 300 g of silica gel packed with 1% TEA in dichloromethane. The eluting solvents were dichloromethane (500 mL), 3% MeOH in dichloromethane (500 mL), 5% MeOH in dichloromethane (500 mL), and 8% MeOH in dichloromethane (2000 mL). The desired fractions were collected and concentrated to afford 11.70 g of the title product (19%).
  • N16 N15 (11.50 gm, 27.17 mmol) in 300 mL dry pyridine cooled at 0°C, was added trimethylsily chloride (13.71 mL, 0.11 mol, 4.0). The mixture was stirred at 0 °C for 40 min. Phenoxyacetyl chloride (9.38 mL, 67.93 mmol) was added. The reaction was stirred at 0 °C for
  • the reaction was stirred at room temperature overnight.
  • the solution was transferred to a mixture of 700 mL of dichloromethane and 500 mL of 5% NaHC0 3 . After shaking well, the organic layer was separated, further washed with 5% NaHC0 3 (2 x 200 mL), and then concentrated.
  • the crude product was purified on a flash column of 270 gm of silica gel packed with 1% TEA/30% CH 2 CI 2 /Hexane.
  • the eluting solvents were 1 % TEA/ 50% CH 2 CI 2 /Hexane (1000 mL), and 1% TEA /CH 2 CI 2 (2000 mL).
  • the fractions containing the desired product were collected and concentrated to afford 10.0 g of the title product (43%).
  • the crude product was purified on a flash column of 120 gm of silica gel packed with 1 % TEA and 10% dichloromethane in hexane.
  • the eluting solvents were 1 % TEA and 10% dichloromethane in hexane (500 mL), 1 % TEA and 20% dichloromethane in hexane (500 mL), and 1 % TEA and 40% dichloromethane in hexane (1500 mL).
  • the right fractions were collected and concentrated to afford the final product (7.37gm, 60%).
  • N3 (1.00 g,1.97 mmol) was dissolved in 15 mL of triethyl phosphate, followed by adding diisopropylethylamine (0.69 mL, 3.9 mmol). While the mixture was kept at 0 °C, and phospherous oxychloride (0.45g, 2.93 mmol) was added. The reaction mixture was stirred at 0 °C for 4 hours, then at 4 °C overnight. Bis(tributyl)ammonium phosphate (3.24 g, 5.91 mmol.) was added, and the reaction mixture was stirred at 0 °C for six hours, and at 4 °C overnight. The white precipitate produced in the reaction was removed by filtration. The filtrate was treated with water (20 mL), and yellow precipitate was formed. The precipitate was filtrated and was dried under high vacuum to afford 0.63 g of the title product as yellow solid.
  • Synthesis of Y63 A reaction mixture of 1.0 gm (1.45 mmol) of N-PAC deoxy-adenosine, 1.77g of the crude Y62, and 125mg of N, N-diisopropylammonium tetrazolide, and 100 ml of dichloromethane. The reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted by adding 100ml of CH 2 CI 2 and 100 mL of 5% NaHC0 3 solution. The organic phase was separated and dried over sodium sulfate. The solution was then concentrated for column chromatography. Silica gel (35 gm) was packed with 1 % TEA /Hexane. The crude material was eluted with 1 % TEA /10-40% CH 2 CI 2 / Hexane. The fractions containing product were pooled and concentrated to afford 0.25 gm of the title product.
  • ETM attachments as depicted in Figure 1 were compared. As shown in Table 1 , a detection probe was attached to the electrode surface (the sequence containing the wire in the table). Positive (i.e. probes complementary to the detection probe) and negative (i.e. probes not complementary to the detection probe) control label probes were added.
  • Electrodes containing the different compositions of the invention were made and used in AC detection methods. The experiments were run as follows. A DC offset voltage between the working (sample) electrode and the reference electrode was swept through the electrochemical potential of the ferrocene, typically from 0 to 500 mV. On top of the DC offset, an AC signal of variable amplitude and frequency was applied. The AC current at the excitation frequency was plotted versus the DC offset.
  • a capture probe D112 comprising a 25 base sequence, was mixed with the Y5 conductive oligomer and the
  • a capture extender probe D179 comprising a 24 base sequence perfectly complementary to the D112 capture probe, and a 24 base sequence perfectly complementary to the 2tar target, separated by a single base, was added, with the 2tar target.
  • the D179 molecule carries a ferrocene (using a C15 linkage to the base) at the end that is closest to the electrode.
  • the attachment linkers are conductive oligomers, the use of an ETM at or near this position allows verification that the D179 molecule is present.
  • a ferrocene at this position has a different redox potential than the ETMs used for detection.
  • a label probe D309 (dendrimer) was added, comprising a 18 base sequence perfectly complementary to a portion of the target sequence, a 13 base sequence linker and four ferrocenes attached using a branching configuration.
  • a representative scan is shown in Figure 20A.
  • a representative scan is shown in Figure 20B.
  • Example A A capture probe D94 was added with the Y5 and M44 conductive oligomer at a 2:2:1 ratio with the total thiol concentration being 833 ⁇ M on the electrode surface, as outlined above.
  • a target sequence (D336) comprising a 15 base sequence perfectly complementary to the D94 capture probe, a 14 base linker sequence, and 6 ferrocenes linked via the N6 compound was used.
  • a representative scan is shown in Figure
  • Example B A capture probe D94 was added with the Y5 and M44 conductive oligomer at a 2:2:1 ratio with the total thiol concentration being 833 ⁇ M on the electrode surface, as outlined above.
  • a target sequence (D429) comprising a 15 base sequence perfectly complementary to the D94 capture probe, a C131 ethylene glycol linker hooked to 6 ferrocenes linked via the N6 compound was used.
  • a representative scan is shown in Figure 20E.
  • the capture probe D112, Y5 conductive oligomer, the M44 insulator, and capture extender probe D179 were as outlined above.
  • Two label probes were added: D295 comprising an 18 base sequence perfectly complementary to a portion of the target sequence, a 15 base sequence linker and six ferrocenes attached using the N6 linkage depicted in Figure 23.
  • D297 is the same, except that it's 18 base sequence hybridizes to a different portion of the target sequence.
  • a representative scan is shown in Figure 20G. When the 2tar target was not added, a representative scan is shown in Figure 20H.
  • the capture probe D112, Y5 conductive oligomer, the M44 insulator, and capture extender probe D179 were as outlined above.
  • Two label probes were added: D296 comprising an 18 base sequence perfectly complementary to a portion of the target sequence, a 5 base sequence linker and six ferrocenes attached using the N6 linkage depicted in Figure 23.
  • D298 is the same, except that it's 18 base sequence hybridizes to a different portion of the target sequence.
  • a representative scan is shown in Figure 20I. When the 2tar target was not added, a representative scan is shown in Figure 20J.
  • the Y5 conductive oligomer, the M44 insulator, and one surface probe D350 that was complementary to 2 capture sequences D417 and EU1 were used as outlined herein.
  • the D350, Y5 and M44 was added at a 0.5:4.5:1 ratio.
  • Two capture extender probes were used; D417 that has 16 bases complementary to the D350 capture probe and 21 bases complementary to the target sequence, and EU1 that has 16 bases complementary to the D350 capture probe and 23 bases complementary to a different portion of the target sequence.
  • D468 comprising a 30 base sequence perfectly complementary to a portion of the target sequence
  • a linker comprising three glen linkers as shown in Figure 19 (comprising polyethylene glycol) and six ferrocenes attached using N6.
  • D449 is the same, except that it's 28 base sequence hybridizes to a different portion of the target sequence, and the polyethylene glycol linker used (C131) is shorter.
  • a representative scan is shown in Figure 20K.
  • Example A A capture probe D112, Y5 conductive oligomer and the M44 insulator were put on the electrode at
  • a target sequence MT1 was added, that comprises a sequence complementary to D112 and a 20 base sequence complementary to the label probe D358 were combined; in this case, the label probe D358 was added to the target sequence prior to the introduction to the electrode.
  • the label probe contains six ferrocenes attached using the N6 linkages depicted in Figure 23.
  • a representative scan is shown in Figure 20L. The replacment of MT1 with NC112 which is not complementary to the capture probe resulted in no signal; similarly, the removal of MT1 resulted in no signal.
  • Example B A capture probe D334, Y5 conductive oligomer and the M44 insulator were put on the electrode at 2:2:1 ratio with the total thiol concentration being 833 ⁇ M.
  • a target sequence LP280 was added, that comprises a sequence complementary to the capture probe and a 20 base sequence complementary to the label probe D335 were combined; in this case, the label probe D335 was added to the target prior to introduction to the electrode.
  • the label probe contains six ferrocenes attached using the N6 linkages depicted in Figure 23. A representative scan is shown in Figure 20M. Replacing LP280 with the LN280 probe (which is complementary to the label probe but not the capture probe) resulted in no signal.
  • the sense primer contained the ETMs (using the N6 linkage described herein), although as will be appreciated by those in the art, triphosphate nucleotides containing ETMs could be used to label non-primer sequences.
  • the surface probe was designed to hybridize to 16 nucleotides of non-primer sequences, immediately adjacent to the primer sequence; that is, the labeled primer sequence will not bind to the surface probe. Thus, only if amplification has occured, such that the amplified sequence will bind to the surface probe, will the detection of the adjacent ETMs proceed.
  • the target sequence in this case was the plasmid pBKBHIOS (NIH AIDS Research and Reference Reagent program - McKesson Bioservices, Rockville MD) which contains an 8.9 kb Sstl fragment of pBH10-R3 dervied from the HXB2 clone which contains the entire HIV-1 genome and has the Genbank accession code K03455 or M38432) inserted into the Sstl site on pBluescript ll-KS(+). The insert is oriented such that transcription from the T7 promoter produces sense RNA.
  • pBKBHIOS NIH AIDS Research and Reference Reagent program - McKesson Bioservices, Rockville MD
  • the “sense” primer, D353 was as follows: 5'-(N6)A(N6)AGGGCTGTTGGAAATGTGG-3'.
  • the "antisense” primer, D351 was as follows: 5'-TGTTGGCTCTGGTCTGCTCTGA-3'.
  • the following is the expected PCR product of the reaction, comprising 140 bp:
  • the surface capture probe (without any overlap to the sense primer) D459 was as follows: 5'- TTGGTGTCCTTCCTTU-4 unit wire(C11 )-3'.
  • PCR reaction conditions were standard: TAQ polymerase at TAQ 10X buffer. 1 ⁇ M of the primers was added to either 6 X 10 3 , 6X 10 ⁇ or 6 X 10 7 molecules of template. The reaction conditions were 90 ° C for 30 sec, 57 ° C for 30 sec, and 70°C for 1 minute.
  • the electrodes were prepared by melting 0.127 mm diamter pure gold wire on one end to form a ball. The electrodes were dipped in aqua regia for 20 seconds and tehn rinse with water. The SAM was deposited by dipping the electrode into a deposition solution of 1.3:4.0:7 D459:H6:M44 in 37:39:24 THF:ACN:water at 1 mM total thiol which was heated at 50 ° C for five minutes prior to the introduction of the electrodes. The electrodes were added and then removed immediately to room temperature to sit for 15 minutes. Electrodes were then transferred to M44 (in 37:39:24 THF:ACN:water at 400 ⁇ M total thiol concentration).
  • the electrodes sat in M44 at room tern for 5 minutes, then the following heat cycling was applied: 70 ° C for 1 minute, followed by 55°C for 30 sec, repeating this cycle 2 more times followed by a 0.3 ° C ramp down to RT with soaking at RT for 10 minutes.
  • the electrodes were taken out of M44 solution, rinsed in 2XSSC, and hybridized as follows.
  • the PCR products were adjusted to 6XSSC (no FCS).
  • the control was also adjusted to 6XSSC Hybridization was carried out at RT after rinsing twice in 6XSSC for at least 1.5 hours.
  • ACV conditions were as follows: Ag/AgCI reference electrode and Pt auxiliary electrodes were used, and NaCI0 4 was used as the electrolyte solution.
  • a ligation probe HIVLIG (5'-CCACCAGATCTTCCCTAA AAAATTAGCCTGTCTCTCAGTACAATCTTTCATTTGGTGT-3') and the target sequence HIVCOMP (5'- ATGTGGAAAGAAAGGACACCAATTGAAAGATTGTACTGAGAGACAGGCTAATTTTTTAGGGAAGATCTG G-3') was added, with ligase and the reaction allowed to proceed.
  • the reaction conditions were as follows: 10 ⁇ M of HIVLIG annealed to HIVCOMP were hybridized to the electrode surface (in 6XSSC) for 80 min. The surface was rinsed in ligase buffer. The ligase (T4) and buffer were added and incubated for 2 hours at RT.
  • Triton X at 10 "4 M was added at 70 ° C to allow the denaturation of the newly formed hybridization complex, resulting in the newly formed long surface probe (comprising D368 ligated to the HIVLIG probe).
  • the addition of the D456 signalling probe (5'-(N6)G(N6)CT(N60C(N60G(N6)C(N6)TTCTGCACCGTAAGCCA TCAAAGATTGTACTGAG-3') allowed detection (results not shown).
  • the D456 probe was designed such that it hybridizes to the HIVLIG probe; that is, a surface probe that was not ligated would not allow detection.
  • the capture probe for a rRNA assay containing 0, 4 and 8 ethylene glycol units was tested on four separate electrode surfaces.
  • Surface 1 contained 2:1 ratio of H6:M44, with a total thiol concentration of 500 ⁇ M.
  • Surface 2 contained a 2:2:1 ratio of D568/H6/M44 with a total thiol concentration of 833 ⁇ M.
  • Surface 3 contained a 2:2:1 ratio of D570/H6/M44 with a total thiol concentration of 833 ⁇ M.
  • D568 was a capture probe comprising 5'-GTC AAT GAG CAA AGG TAT TAA (P282)-3 ⁇ P282 was a thiol.
  • D569 was a capture probe comprising 4 ethylene glycol units: 5'-GTC AAT GAG CAA AGG TAT TAA (C131 ) (P282)-3 ⁇
  • D570 was a capture probe comprising 8 ethylene glycol units: 5'-GTC AAT GAG CAA AGG TAT TAA (C131) (C131) (P282)-3 ⁇
  • the H6 (in the protected form) was as follows: (CH 3 ) 3 Si-(CH 2 ) 2 -S-(C 6 H 5 )-C5C-(C 6 H 5 )-C :H.
  • M44 is the same as M43 and was as follows: HS-(CH 2 ) ⁇ r (0CH 2 CH 3 ) 3 -0H.
  • the D483 label probe hybridizes to a second portion of the rRNA target, and was as follows: 5'-(N6)C(N6) G(N6C (N6)GG CCT (N6)C(N6) G(N6)C (N6)(C131)(C131) (C131 )(C131)T TAA TAC CTT TGC TC-3'.
  • the D495 is a negative control and was as follows: 5'-GAC CAG CTA GGG ATC GTC GCC TAG GTGAG(C131 ) (C131 )(C131 )(C131 ) (N6)G(N6) CT(N6) C(N6)G (N6)C(N6)-3 ⁇ The results were as follows:
  • D350/H6/M44 surfaces mixed in a ration of 1 :3.5:1.5 deposited at a 833 ⁇ M total thiol concentration.
  • D350 is a 4 unit wire with a 15mer DNA
  • H6 is a 2 unit wire
  • M44 is an ethylene glycol terminated alkane chain.
  • a D417 tether sequence (42mer) and a EU1 capture sequence (62mer) bound the 16S rRNA to the D350 on the surface.
  • a series of 9 label probes (D449, D469, D489, D490, D491, D476, D475 and D477) pre- annealed to the rRNA gave the electrochemical signal.
  • These label probes (signalling molecules) have 6 or 8 N6 or Y63 type ferrocenes.
  • the label probes that flank the tack-down regions were replaced (one end at a time) with label probes containing either 20 or 40 ferrocenes.
  • a label probe that binds to a region in the middle of the tack-down regions was replaced with label probes containing either 20 or 40 ferrocenes.
  • Hybridization solutions were annealed by heating at 70°C for 30 seconds and then cooling to 22 ° C over ⁇ 38 seconds.
  • the molecules were all in 4X SSC at twice the targeted concentrations, with the rRNA at 35 U.S.C ⁇ ⁇ M, the capture sequence at 1.0 ⁇ M, and the label probes at 3 ⁇ M.
  • the solution was diluted 1 :1 with fetal calf serum, halving the concentrations and changing the solvent to 2X SSC with 50% FCS.
  • Hybridization was done as follows: After the 2X SSC dip described above, the derivatized electrode was placed into an eppendorf tube with 20 ⁇ L hybridization solution. It was allowed to hybridize at room temperature for 10 minutes.
  • D94 was as follows: 5'- ACC ATG CAC ACA GA(C11 )-3'.
  • D109 was as follows: 5'-CTG CGG TTA TTA AC(C11 )-3'.
  • the "+" surface was a 2:2:1 ratio of D94:H6:M44, with a total thiol concentration of 833 ⁇ M.
  • the "-" surface was a 2:2:1 ratio of D109:H6:M44, with a total thiol concentration of 833 ⁇ M.
  • the D548 structure was as follows: 5'- (N38)(N38)(N38) (N38)(N38)(N38)(N38)(N38)(N38) ATC TGT GTC CAT GGT-3'. On each N38 was a 5'- (H2)(C23)-3 ⁇
  • the D549 structure was as follows: 5'-(N38)(N38)(N38) (N38)(N38)(N38)(N38)(N38)(N38) ATC
  • the D550 structure was as follows: 5'-(N38)(N38)(N38) (N38) AT CTG TGT CCA TGG T-3'. On each N38 was a 5'-(H2)(C23)(C23)-3'.
  • the D551 structure was as follows: 5'-(n38)(N38)(N38) (N38)ATCTG TGT CAA TGG T-3'. On each N38 was a 5'-(H2)(C23)(C23)(C23)(C23)-3'.
  • a 5' N38 has two sites for secondary modification.
  • a representative schematic is shown in Figure 23E.
  • D405 has the structure: 5'-(C23)(C23)(C23) (C23)(C23)(C23)(C23)(C23)
  • the objective was to develop methods for the detection of rRNA from a bacterial pathogen. Initially, purified RNA was used to optimize the strategy. Subsequently, crude lysates were analyzed. E. coli served as a non- infectious model bacterium target in this study.
  • rRNA is characterized by highly defined secondary and tertiary structure that makes capturing a specific sequence within the structure difficult.
  • the 16S rRNA transcript of E. Coli is 1542 bases in length, and is present at 2X10 4 copies/cell.
  • Helper sequences have been characterized that act to specifically unfold portions of the rRNA and facilitate hybridization of probes to adjacent regions.
  • 50nM and 10nM rRNA solutions were heated to 70°C in the presence of helper sequences A and B (2.5uM), and signaling probes B and C (125nM), in 1 M NaCI0 4 and lysed blood for 3 minutes. After cooling, the solution was added to small cartridge chips and hybridized for 4 hours. The chips were deposited with capture probe/H6/M44 (2:2:1) in 0.9%TEA/6XSSC in the following pattern.
  • Capture probe A acts as an additional tack down point only, there is no signaling molecule directly adjacent to its position as there is associated with capture probes B and C.
  • the following graph displays the peak heights of signals generated from the 50nM and 10nM rRNA solutions after 4 hours with both signaling B and C added at 125nM. Purified rRNA was clearly detected at both 50nM and 10nM, while control surfaces, containing a randomer capture probe (R), yielded no detectable signals. Electrodes bearing triple capture probes (A, B, and C) gave rise to the largest signals, and on average, pads bearing double capture probes gave larger signals than the pads with only a single capture probe. Although there is no signaling molecule adjacent to capture probe A, the extra tack-down point in close proximity to signaling moieties B and C allows, in some cases, increased signaling (compare C alone to AC).
  • the cell lysate was made according to Qiagen RNeasy protocol. A 50mL overnight culture. was centrifuged and the pellet was resuspended in 500uL of 1 mg/ml lysozyme in a Tris-EDTA buffer and incubated at room temperature for 5 minutes. 1.9mL of the guanadium isothiocynate based lysis buffer, buffer RLT, with 0.145M Beta mercaptoethanol (B-Me, to inactivate RNase ) was added.
  • arrays were hybridized in the presence of bacterial lysate such that the lysate comprised from 6% down to 0.1875% of the final solution.
  • a 50mL overnight E. coli culture, lysed in buffer AL without the addition of B-Me (guanidinium hydrochloride) was prepared in the manner described above.
  • Another set of arrays were hybridized in a NaCI04/lysed blood solution containing purified RNA in amounts that correspond to that found in the range of lysates tested, 6% to 0.1875%. Peaks were detectable on arrays with 6% crude lysate and with 6% purified RNA after 2 hours hybridization without a significant size difference between crude and purified. Peaks were also detectable on 0.75% and 0.375% of a 50mL culture after overnight hybridization, and barely detectable with 0.1875% of a 50mL culture.
  • the E. coli cell concentration in the culture used to prepare purified RNA and crude lysates was determined to be 4.7x10 8 /mL by a plating dilution series. Therefore 0.1875% of a 50mL culture corresponds to 4x107 E. Coli cells. Assuming 1X10 4 16S rRNA molecules per cell, we detected 4.4x10 11 16S rRNA molecules. That corresponds to a 200pM concentration in our large cartridges. Thus, our detection limit for 16SrRNA from pure RNA or crude lysates is close to our current detection limit for well-studied oligos and isolated RNA (e.g. HIV 840).
  • the pads baring the triple capture probe, A, B, and C were the only pads to give measurable signals, while double and single capture probes on the surface gave signals equivalent to the negative pads (too small to measure).
  • the incorporation of multiple capture and signaling sequences on a long target, such as rRNA increases signal size and lowers the detection limit.
  • Beta mercaptoethanol was added to both lysis buffers to inactivate RNase and thereby protect RNA from degradation after cell lysis.
  • concentration recommended by this protocol, 34mM resulted in complete inhibition of electrochemical signaling.
  • Beta mercaptoethanol added at concentrations as low as 8.5mM yielded signal size degradation over time.
  • lysis buffer alone provides adequate protection from RNase in cell lysate to allow detection of RNA. The tolerable amount of B-Me will be established and used when assaying crude lysates.
  • Guanadinium isothiocynate in buffer RLT also causes inhibition of electrochemical signaling on the CMS sensor.
  • Buffer RLT is recommended by Qiagen for use in the RNeasy Kit because guanadinium isothiocynate is a harsh denaturant and effectively inactivates RNase.
  • This buffer is incompatible with our current system as it causes pad or signal disintegration.
  • Whole blood lysis buffer AL currently used as the hybridization buffer standard, uses guanadinium hydrochloride to lyse cells. It is not as stringent as guanadinium isothiocynate, but it is still effective enough in inactivating RNase as to allow detection of 93.75 uL of a 50 mL overnight culture of E. coli. It is possible that detection limits could be increased by more effectively inactivating RNase present in cell lysate.

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Abstract

L'invention concerne des compositions et des procédés utiles dans la détection d'acides nucléiques au moyen de diverses techniques d'amplification, y compris l'amplification de signal et l'amplification de cible. La détection est mise en oeuvre à l'aide d'une fraction de transfert d'électrons (ETM) qui est associée à l'acide nucléique, directement ou indirectement, pour permettre une détection électronique de ladite ETM par une électrode.
EP00950511A 1999-07-20 2000-07-20 Amplification d'acides nucleiques avec detection electronique Withdrawn EP1194593A2 (fr)

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CA2379693A1 (fr) 2001-01-25
WO2001006016A9 (fr) 2002-07-11
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WO2001006016A3 (fr) 2001-08-23
JP2003530822A (ja) 2003-10-21
WO2001006016A2 (fr) 2001-01-25

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