JP2002533694A - Tissue collection device with biosensor - Google Patents

Abstract

  (57) [Summary] The present invention provides a tissue collection device having electrodes, particularly a blood collection device. The electrode may further comprise a self-assembled monolayer and a capture binding ligand, especially a nucleic acid capture probe. The monolayer may have insulators and / or electron transport path forming species (EFS). These devices may also have at least one reagent, including an anticoagulant, a probe nucleic acid and a lysis reagent. In yet another aspect, the invention provides a method for detecting a target analyte in a sample, comprising sending an initiation signal to a tissue collection device having an electrode. The electrode may have a self-assembled monolayer and an assay complex that includes a capture binding ligand, a target analyte and an electron transport. These methods further include detecting electron transfer between the electrode and the electron transfer unit. These methods may further include methods of collecting the specimen, for example, using a blood collection device.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a tissue collection device having a biosensor, such as a blood collection device, for detecting a target analyte such as a nucleic acid and a protein including an antibody and an enzyme.

BACKGROUND OF THE INVENTION The fields of molecular pathology, molecular oncology and molecular diagnostics are developing rapidly with the development and validation of novel molecular technologies of paramount importance in clinical settings. . The speed of discovery of new etiological genes is rapidly accelerating, and the opportunity to become aware of the role of genetic components in many disease states is increasing, and the demand for fast, inexpensive, and accurate test methods is increasing exponentially.

[0003] In general, these assays fall into several categories, including nucleic acid assays (for pathogens, oncogenes, genetic disorders, etc.) and immunoassays (for various antigens and For antibodies). In addition, the need to test large numbers of people to diagnose and confirm the risk for these genetic diseases by accumulating knowledge about pathogenic mutations and single nucleotide polymorphisms (SNPs) in human genes And a request has been invited.

[0004] Clinically, these tests are specimens from patients, often blood, but are described in US Patents 4,991,104; 3,974,930; 4,301,936; 4,187,952; 4,186,840;
65,200; 4,111,326; 6,001,087; 6,001,429; 5,975,343 and 5,968,620. The sample is then generally processed through various steps to perform the assay.

At the same time, the area of biosensors is rapidly expanding. In many of these, the mechanism of detection is based on a specific ligand / antiligand reaction. That is, certain combinations of substances (ie, binding pairs or ligands / antiligands) are known to bind to each other, but bind little or no other substances. This has been the subject of numerous studies that utilize these binding pairs to detect complexes. These generally allow one component of the complex to be detected in any way that makes the entire complex detectable, such as a radioisotope,
This is done by labeling using fluorescent or other optically active molecules, enzymes, and the like.

[0006] Other assays use electronic signals for detection. Numerous systems have been reported, including: U.S. Patent Nos.5,591,578; 5,824,473; 5,770,369; 5,705,348;
5,780,234 and 5,952,172; WO98 / 20162; WO99 / 37819; PCT / US98 / 12430; PCT /
US99 / 01703; PCT / US98 / 12082; PCT / US99 / 14191; PCT / US99 / 21683; PCT / US99 / 254
64 and PCT / US99 / 10104 and U.S. Patent Application 09 / 135,183; 09 / 295,691 and
See 09 / 306,653.

[0007] In general, it would be desirable to provide the ability to test tissue samples with a minimum number of operational steps. One object of the present invention is to provide a tissue collection device incorporating a biosensor, particularly an electrode, for use in diagnosis and screening.

SUMMARY OF THE INVENTION In accordance with the foregoing objects, the present invention provides a tissue collection device, particularly a blood collection device, having electrodes. The electrode may further comprise a self-assembled monolayer and a capture binding ligand, especially a nucleic acid capture probe. This single layer contains an insulator
And / or may include electroconduit-forming species (EFS). The device may further include at least one reagent, including an anticoagulant, a probe nucleic acid and a lysing agent.

In another aspect, the invention provides a method for detecting a target analyte present in a sample, comprising sending an initiation signal to a tissue collection device having an electrode. The electrode may include a self-assembled monolayer and an assay complex that includes a capture binding ligand, a target analyte and an electron transport. The method further comprises an electrode and an electron transfer unit (ETM).
Detecting the transfer of electrons between them. The method may further include collecting the specimen using, for example, a blood collection device or the like.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A, 1B, 1C and 1D depict a tissue collection device with a container disposed.
FIG. 1A illustrates a tube 10 having a cap 20. The cap 20 has a container 30 containing a reagent 40. FIG. 1B is similar except that it has two containers 30 and 31 containing two reagents 40 and 41. FIG. 1C
A similar configuration is depicted, except that the container 30 is not the cap 20 but a part of the tube 10. FIG. 1D depicts an arrangement utilizing a membrane 50 at the end of the tube. It should be noted that the biosensor is not shown in any of the systems depicted in FIG.

FIGS. 2A and 2B depict a tissue collection device having a container for testing a portion of a specimen. FIG. 2A depicts a device utilizing the cap 20, but as those skilled in the art will recognize, devices having inlet ports, membranes, and other configurations may be used as well. The container 30 (which may contain the reagent 40) is separated from the sample portion 70 by a valve or seal 60. FIG. 2B shows a detachable connector 6.
5, a similar system is depicted, except that the container 30 can be separated from the sample chamber 70.
It should be noted that the biosensor is not shown in any of the systems depicted in FIG.

FIGS. 3A, 3B and 3C depict some possible configurations of biosensors and external leads for the composition of the present invention. FIG. 3A depicts the electrodes of the biosensor 80 in the cap 20 with the interconnect device 90. 3B and 3
C depicts the electrodes of biosensor 80 as a component of tube 10.
FIG. 3B depicts a single biosensor; FIG. 3C illustrates multiple biosensors 8.
Draw 0. Although FIG. 3C depicts a set of external leads for all electrodes, multiple leads may be used as well, as will be appreciated by those skilled in the art. In addition, a physical septum may be provided between each section of the tube, with each section having one biosensor. FIG. 3D depicts the biosensor 80 in the container 30 of the device 10.

FIGS. 4A, 4B and 4C show a preferred embodiment 3 for attaching a target sequence to an electrode.
Draw the seed. Although generally depicted as nucleic acids, embodiments other than nucleic acids are also useful. FIG. 4A depicts the target sequence 120 hybridized to the capture probe 100 linked via the attachment linker 106. Attachment linker 106 may be either a conductive oligomer or an insulator, as outlined herein.
For all of the drawings and configurations outlined herein, the capture probe may be the same, or the capture probe at any particular pad may be different and include sequences that hybridize to different parts of the target. It may be. Electrode 1
05 includes a single layer of protective film agent 107, which may include a conductive oligomer (herein described as 108) and / or an insulator (herein described as 109). For all of the embodiments depicted in the figures, n represents an integer of at least 1, but as one skilled in the art will recognize, this system may not utilize any capture probes (n is zero), Generally, this aspect is not preferred. The upper limit for n depends on the length of the target sequence and the sensitivity required. FIG. 4B
Depicts the use of a single capture extension probe 110. This probe has target sequence 1
20 has a first portion 111 that will hybridize to the first portion and a second portion that will hybridize to the capture probe 100. FIG. 4C depicts the use of two capture extension probes 110 and 130. One capture extension 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 the first portion 102 of the capture probe 100. Have. The second capture extension probe 130 will hybridize to a second portion of the target sequence 120 first portion 132
And a second portion 131 that will hybridize to the second portion 101 of the capture probe 100.

FIGS. 5A, 5B, 5C, 5D and 5E depict the binding of an ETM (Electron Transfer Section) with different possible configurations of labeled probes. These figures depict a second binding ligand that is a hybridizable nucleic acid, as will be appreciated by those of skill in the art.
The second binding ligand can be non-nucleic acid as well. In FIGS. 5A-C, the recruitment linker is a nucleic acid, but not in FIGS. 5D and 5E. A =
B = linkage to base; C = linkage to ribose; D = linkage to phosphate; E = metallocene polymer linked to base, ribose or phosphate (or other backbone analog). This can be another ETM polymer, as described herein); F = attached via a base, ribose or phosphate (or other backbone analog) G = attachment of a “branched” structure via a base, ribose or phosphate ester (or other backbone analog); H = attachment of a metallocene (or other ETM) polymer; I = dendritic structure J = bond using a standard linker.

FIGS. 6A, 6B, 6C, 6D, 6E, 6F and 6G depict some aspects of the present invention. The monolayers depicted in these figures all show the presence of both conductive oligomer 108 and insulator 107 in an approximately 1: 1 ratio. However, various ratios may be used as described herein, or both may be completely absent,
Further, another single layer forming material may be used. In addition, as one of ordinary skill in the art would recognize, any one of these structures may be repeated for a particular target sequence. That is, multiple assay complexes may be formed for a long target sequence. In addition, any of the electrode-coupling aspects of FIG. 4 may be used in any of these systems. FIGS. 6A, 6B and 6D have a target sequence 120 containing each ETM135; these may be added enzymatically as described herein. For example, during a PCR reaction using nucleotides modified with ETMs, an essentially random insertion into the entire target sequence occurs or is added to the end of the target sequence. FIG.
D depicts a method using two different capture probes 100 and 100 'that hybridize to different portions of the target sequence 120. As will be appreciated by those skilled in the art, in this embodiment the orientation of the 5'-3 'to which the two capture probes point is different. FIG. 6C shows a labeled probe 145 that hybridizes directly to the labeled sequence 120.
Depict the use of. FIG. 6C shows a labeled probe 14 including a first portion 141 that hybridizes to a portion of the target sequence 120, a second portion 142 including an ETM 135.
5 is shown. Although not depicted, a particularly preferred embodiment for use in a tissue collection device involves direct capture of a target sequence to a capture probe using at least one labeled probe that hybridizes to a different portion of the target. Use. 6E, 6F and 6G do not hybridize directly to the target, but rather to an amplification probe that has hybridized directly (FIG. 6E) or indirectly (FIGS. 6F and 6G) to the target sequence. 7 depicts a system utilizing a marker probe 145. FIG. 6E shows the first portion 1 hybridizing to the target sequence 120.
Amplification probe 150 having at least one second portion 152, that is, an amplification sequence, that hybridizes to the first portion 141 of the labeled probe.
Use 6F is similar, except that a first labeled extension probe 160 having a first portion 161 that hybridizes to the target sequence 120 and a second portion 162 that hybridizes to the first portion 151 of the amplification probe 150 is shown in FIG. It differs in using it. The second portion 152 of the amplification probe 150 hybridizes to the first portion 141 of the labeled probe 140. The label probe 140 also includes a recruitment linker 142 having an ETM 135. FIG. 6G adds a second labeled extension probe 170 having 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 amplification probe. FIG. 6H depicts a system utilizing a multi-labeled probe. Labeled probe 14
The first portion 141 of the zero can hybridize to all or a portion of the recruitment linker 142. Figures 7A, 7B, 7C, 7D, 7E and 7F show some "Mechanism-1".
Draw the detection system. FIG. 7A shows a portion of the target sequence 120 that has hybridized to the detection probe 300 attached to the electrode 105 via the conductive oligomer 108. The hybridization complex comprises an ETM 135 that can be covalently or non-covalently bound to either the target sequence or the detection probe 300 (ie, a hybridization indicator). This monolayer is indicated by insulator 107, but any type of non-reactive component (passivation agent) may be used, as described herein. FIG.
B is a capture probe 100 connected to an electrode 105 via a binding linker 106.
, Which will serve as one type of capture probe, as the labeled probe 145 hybridizes to the detection probe 300, as will be appreciated. Labeled probe 145 has a first portion 141 that hybridizes to a portion of target sequence 120 and a second portion 142 that hybridizes to detection probe 300. FIG. 7C shows a branched amplification probe 150 having a first portion 151 that hybridizes to the target sequence 120 (although labeled extension probes can be used as well) and an amplification sequence 152 that hybridizes directly to the detection sequence 300. Depict the use of. This target sequence may be further attached to an electrode using a capture probe as outlined in FIG. FIG. 7D shows a linear amplification probe 1
Draw the same using 50. FIG. 7E depicts a similar system, which includes a first portion 141 that hybridizes to the amplification sequence 152, and a detection probe 300.
A labeled probe 145 that includes a second portion 142 that hybridizes to
FIG. 7F depicts the same utilizing a linear amplification probe 150.

DETAILED DESCRIPTION OF THE INVENTION The present invention seeks to use a biosensor in a tissue collection device. In general, the ability to collect a tissue sample, eg, a blood sample, into a container containing a biosensor (and optionally assay reagents) allows for a one-step molecular diagnostic assay. It can be used in various ways. For example, in some embodiments, by placing blood in a container with a biosensor, with minimal blood handling, for example, with respect to contaminants including pathogens such as viruses (herpes strains, HIV, etc.) or bacteria, fast and inexpensive. Blood testing becomes possible. Alternatively, tissues such as blood or vaginal samples collected for testing, particularly for diseases transmitted by sexual activity, for example, can also be rapidly tested with minimal blood handling.

Thus, the present invention provides a tissue collection device. Where "organization"
Or grammatical equivalents herein mean any tissue capable of performing a bioassay. This includes, but is not limited to, blood, lymph, saliva, vaginal and anal excrement, urine, stool, sweat, tears, and other bodily fluids. As used herein, "tissue collection device" generally refers to a container that is sealable and can be used to collect, contain, and store tissue, including body fluids. Tissue collection devices are generally sterilized. Particularly described herein are blood collection devices, such as blood collection tubes and bags, as is well known in the art,
This includes the VACTAINER ™ vacuum tube. These may be configured in various structures. For example, US Patents 4,991,104; 3,974,930; 4,301,936; 4,
187,952; 4,186,840; 4,136,794; 4,465,200; 4,111,326; 6,001,087; 6,001,42
9; 5,975,343; and 5,966,620. These are all incorporated herein by reference in their entirety.

As will be appreciated by those skilled in the art and as outlined herein,
The tissue collection device of the present invention can be of various configurations. In one aspect, the device has a single compartment for containing a specimen. Here, reagents may be added if necessary before collection. Alternatively, the tissue collection device is configured to have more than one compartment. This is done in various ways depending on the intended use of the device. For example, if the entire sample is used in the assay, each compartment may be used to store assay reagents. Alternatively, if only a portion of the collected analyte is to be assayed, each compartment may be used to partition portions or both for subsequent assays.

In one preferred embodiment, all of the analyte is used in the assay. In this aspect, it may be desirable to have one or more storage compartments for reagents. For example, US Pat. No. 6,001,087 discloses a collection assembly in which the cap of the device has a container containing a reagent as defined below. Insertion of a needle, such as occurs during blood collection, breaks the container and causes the addition of reagent to the specimen. This is illustrated generally in FIGS. 1A and 1B, where one and multiple compartments breaking in this manner are shown. For example, centrifugation at a first speed ruptures the first membrane to perform the first step of the assay; faster second centrifugation ruptures the second membrane to perform the second step of the assay; Various other membranes can be used in a similar manner. In addition, liquid "compartments" or "phases" can be created and used to separate the different components of the assay by using substances of different specific gravity. Similarly, the container may be part of a tube instead of a cap as depicted in FIG. 1C. Alternatively, the container may be at the end of the tube and utilize a membrane that can be broken by centrifugation as shown in FIG. 1D. Alternatively, a gel septum as used in the VACUTAINER ™ PPT ™ system may be used.

In one preferred embodiment, only a portion of the collected analyte is used in the assay. In this aspect, the device can have a compartment that allows testing of only a portion of the specimen. For example, as shown in FIG. 2A, a separate container is placed, containing reagents if necessary, and a stopper or valve, especially a one-way valve such as, for example, a check valve, is attached to allow a portion of the sample to be removed. It can also be possible to transfer to a container.
The separation container may be removable from the specimen compartment to allow for a single collection from the patient, as illustrated in FIG. 2B, followed by separation of the portion to allow for separate testing.

In one preferred embodiment, the tissue collection tube contains a reagent. This reagent includes: for example, an anticoagulant for blood, and to facilitate optimal binding and detection of the target analyte to the sensor composition and / or to reduce non-specific or background interactions Also included are reagents used in tissue handling such as salts that may be used, buffers, neutral proteins such as albumin, detergents, and various other alternative reagents. Further, depending on the preparation method of the sample and the purity of the target, for example, a protease inhibitor, a nuclease inhibitor, an antimicrobial agent,
Reagents that improve the efficiency of the assay in other ways, such as for example, may be used.
For example, it may contain reagents or other components to promote the attachment of the target analyte to the biosensor as generally described in PCT / US99 / 14191. This patent document is incorporated herein by reference in its entirety.

In addition, prior to, after, or during the introduction of the sample tissue, US Pat.
, 578; 5,824,473; 5,770,369; 5,705,348; 5,780,234 and 5,952,172; WO98 /
20162; WO99 / 37819; PCT / US98 / 12430; PCT / US99 / 01703; PCT / US98 / 12082; PCT /
US99 / 14191; PCT / US99 / 21683; PCT / US99 / 25464 and PCT / US99 / 10104 and
Other reagents including components of bioassays, including, for example, amplification probes, labeled probes, capture extension probes, and the like, as generally outlined in U.S. Patent Applications 09 / 135,183; 09 / 295,691 and 09 / 306,653. Can be placed in a tube.

In one preferred embodiment, the reagent is a cell (including patient cells, bacteria, etc.)
Or a cell lysing agent for lysing a pathogen such as a virus. Suitable lysis buffers include, but are not limited to, guanidium chloride, chaotropic salts, enzymes such as lysozyme, and the like. In certain embodiments, such as for blood cells, low pressure lysis can be effected simply by dilution with water or buffer. The lysing agent may be in the form of a solution or a solid that is incorporated into the solution upon introduction of the analyte.

In one preferred embodiment, one or more containers of the tissue collection device contain the reagent.

The tissue collection device has a biosensor as outlined further above. As outlined in more detail below, this biosensor uses an array forma
During t), a single substrate may be included with a single electrode, multiple substrates for each of one or more electrodes, or a single substrate with multiple electrodes.

As those skilled in the art will appreciate, placement of the biosensor in the device can be accomplished in a variety of ways, as generally depicted in FIG. In one preferred embodiment,
The biosensor is in the cap or stopper of the device as shown in FIG. 3A. In this embodiment, there are leads or interconnects outside the cap, with the biosensor contacting the analyte inside the device. In all of the various configurations,
What is important is that some contact is made with some or all of the analyte to be assayed.

In a preferred embodiment, the location of the biosensor (s) is generally as shown in FIGS. 3B and 3C.
On the internal surface of the specimen compartment, as depicted in FIG. In this embodiment, the external leads may be on the specimen compartment or on the cap. As the skilled artisan will appreciate, if the biosensor is in a cap, the device will need to be inverted when performing the assay. In this aspect, different biosensors may be located on different parts of the device. For example, the detection of the genomic sequence may be performed at one location in the tube, for example, near the end where heavier blood cells are present after centrifugation, and the detection of pathogenic sequences (especially viruses) may be performed, for example, by centrifugation. It may be performed in another part, such as where plasma is present after separation.

Alternatively, different biosensors may be located in different physical compartments of the device; for example, if a gel septum or other membrane is used to separate certain cell types at the specimen surface, It will be possible to reduce the complexity of analytes in a compartment.

The present invention provides a tissue collecting device including a biosensor. As used herein, "biosensor" refers to a sensor that can detect the presence of a target analyte in an analyte. Here, biosensors include electrochemical sensors, optical fiber sensors, wave-dissipating sensors, surface plasmon resonance sensors (surface pla
smon resonance sensor) and sound wave sensors.

Thus, the method of the present invention is directed to the detection of a target analyte. As used herein, a "target analyte" or "analyte" or grammatical equivalents thereof is any molecule or compound to be detected that is capable of binding to a binding species as defined below. Means Suitable subjects include, but are not limited to, small or contaminants or biomolecules such as, for example, environmental or clinical chemicals, but are not limited to pest control. Includes agents, pesticides, toxins, therapeutic and abused drugs, hormones, antibiotics, antibodies, organic materials, and the like. Suitable biomolecules include, but are not limited to, proteins (including enzymes, immunoglobulins and glycoproteins), nucleic acids, lipids, lectins, carbohydrates, hormones, whole cells (including prokaryotic (eg, pathogenic bacterial And eukaryotic cells (including mammalian tumor cells), viruses, spores, and the like. Particularly preferred analytes are proteins, including enzymes; drugs; cells;
Antibody; antigen; cell membrane antigens and receptors (neural, hormonal, trophic, and cell surface receptors) or their ligands.

As used herein, “protein” or grammatical equivalents thereof refers to proteins, oligopeptides and peptides, and analogs thereof, as well as protein and peptide analog structures, including non-naturally occurring amino acids and amino acid analogs. Include.

As will be apparent to those skilled in the art, the majority of analytes will be detected using the present method; that is, essentially any target analyte capable of constructing the binding ligands described below. Could be detected using the method of the present invention.

[0033] In a preferred embodiment, the target analyte is a nucleic acid. As used herein, “nucleic acid” or “
“Oligonucleotide” or grammatical equivalents thereof means at least two nucleotides covalently linked together. In general, the nucleic acids of the invention contain phosphodiester linkages, but may optionally include other backbone structures, such as phosphoramides (Beaucage et al., Tetrahedron, 49 (1), as outlined below.
0): 1925 (1993) and references therein; Letsinger, J. Org. Chem., 35: 3800.
(1970); Sprinzl et al., Eur. J. Biochem., 81: 579 (1977); Letsinger et a.
l., Nucl. Acids Res., 14: 3487 (1986); Sawai et al., Chem. Lett., 805 (1
984); Letsinger et al., J. Am. Chem. Soc., 110: 4470 (1988); and Pauwe.
ls et al., Chemica Scripta, 26: 141 91986); phosphorothioates (Mag et a
l., Nucleic Acids Res., 19: 1437 (1991); and U.S. Patent No. 5,644,048)
Phosphorodithioate (Briu et al., J. Am. Chem. Soc., 111: 2321 (1989))
); O-methyl phosphoramidite linkage (Eckstein, "Oligonucleotides and An
alogues: A Practical Approach, ", Oxford University Press); and peptide nucleic acid backbones and linkages (Egholm, J. Am. Chem. Soc., 114:
1895 (1992); Meier et al., Chem. Int.Ed.Engl., 31: 1008 (1992); Nielse
n, Naure, 365: 566 (1993); Carlsson et al., Nature, 380; 207 (1996); all of which are incorporated by reference). . Other analog nucleic acids have a positively charged backbone (De
Natl. Acad. Sci. USA, 92: 6097 (1995)); with a nonionic backbone (US Patent Nos. 5,386,023; 5,637,684; 5,602,240; 5,21).
6,141 and 4,469,863; Kiedrowshi et al., Angew. Chem., Intl. Ed. En
glish, 30: 423 (1991); Letsinger et al., J. Am. Chem. Soc., 110; 4470 (1
988); Letsinger et al., Nucleoside & Nucleotide, 13; 1597 (1994); Chapte
rs 2 and 3, ASC Symposium Series 580 "Carbohydrate Modifications in Anti
sense Research ", Ed. YS Sanghui and P. Dan Cook; Mesmaeker et al., Bi
oorganic & Medicinal Chem. Lett., 4: 395 (1994); Jeffs et al., J. Biomol.
ecular NMR, 34; 17 (1994); Tetrahedron Lett., 37; 743 (1996)); and those with non-ribose backbones (US Pat. Nos. 5,235,033 and 5,034,506).
Issue; and Chapters 6 and 7, ASC Symposium Series 580 "Carbohydrate Mod
ifications in Antisense Research ", Ed. YS Sanghul and P. Dan Cook); nucleic acids containing one or more carbocyclic saccharides are also included in the definition of nucleic acids (Jenkins et al., (See Chem. Soc. Rev. (1995) pp. 169-176.) Several nucleic acid analogs are described in Rawls, C & E News, June 2, 1997, page 35. All of these references are referenced. Modifications in these ribose-phosphate backbones enhance ETM binding, or enhance stability, and extend the half-life of these molecules in a physiological environment. Can be done for purpose.

As will be apparent to those skilled in the art, all of these nucleic acid analogs can be used in the present invention.
In addition, mixtures of naturally occurring nucleic acids and analogs can be made; analog structures, eg, at surface species or ETM binding sites, may be used. Alternatively, mixtures of heterologous nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs are possible.

Particularly preferred is peptide nucleic acid (PNA), including analogs of peptide nucleic acid. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This offers two advantages. First, the PNA backbone shows an improved hybridization mechanism. PNA shows a large change in melting point (Tm) between mismatched and perfectly matched base pairs. DNA and RN
A typically shows a 2-4 ° C drop in Tm for internal mismatches. For a non-ionic PNA backbone, this drop is close to 7-9 ° C. Similarly, due to their non-ionic nature, hybridization of bases attached to these backbones is relatively insensitive to salt concentration. This is because the low salt hybridization solution has a lower Faraday current than the saline solution (in the range of 150 mM),
It is particularly advantageous in the system according to the invention.

The nucleic acid may be single-stranded or double-stranded, or may be a sequence containing both double-stranded and single-stranded portions. This nucleic acid is DNA, including genomic DNA and cDNA.
, RNA or hybrid. Here, the nucleic acid can be any combination of deoxyribo- and ribo-nucleotides and any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xantan, hypoxanine, isocytosine, isoguanine, and the like. As described generally in U.S. Patent No. 5,681,702, the preferred embodiment is for nucleic acids designed to be complementary to other probes rather than the target sequence, since they reduce non-specific hybridization. Utilizes isocytosine and isoguanine. As used herein, the term "nucleoside" includes nucleotides and nucleosides and nucleotide analogs, and modified nucleosides, such as, for example, amino-modified nucleosides. In addition, "nucleoside" also includes non-naturally occurring analog structures. Thus, for example, individual units of a peptide nucleic acid, each containing a base, are described herein as nucleosides.

Thus, in one preferred embodiment, the target analyte is a target sequence. As used herein, the term "target sequence" or "target nucleic acid" or grammatical equivalents thereof means a nucleic acid sequence that is on a single strand of nucleic acid. The target sequence is a gene, a regulatory sequence, a genomic DN.
A, RNA including cDNA, mRNA, rRNA, and other portions may be used. As outlined herein, the target sequence may be a target sequence obtained from an analyte, or may be a secondary target such as, for example, the product of an amplification reaction, or the like. It can be of any length, but it is understood that longer sequences are more specific. As the skilled artisan will appreciate, the complementary target sequence can be in a variety of forms. For example, it may be contained within a large nucleic acid sequence, such as all or part of a gene or mRNA, restriction fragment of a plasmid or genomic DNA, among others. As outlined in detail below, probes are made to hybridize to a target sequence to determine the presence or absence of the target sequence in a sample. Generally speaking, this term will be understood by those skilled in the art. The target sequence may also include different target domains. For example, the first target domain of the analyte target sequence will hybridize to a portion of a capture probe or capture extension probe. Also,
The second target domain will hybridize to an amplification probe, a labeled probe, or various capture or capture extension probes, and other portions. The target domains can be contiguous or remote. Unless otherwise indicated, the terms "first" and "second" are not meant to indicate the orientation of the sequence with respect to the 5'-3 'direction of the target sequence. For example, assuming the 5'-3 'direction of the complementary target sequence, the first domain may be located either 5' or 3 'of the second domain.

In one preferred embodiment, the method of the invention is used to detect a pathogen, for example a bacterium. In this embodiment, suitable target sequences include rRNA, which is generally described in U.S. Pat. No. 4,851,3, which is hereby fully incorporated by reference.
30; 5,288,611; 5,723,597; 6,641,632; 5,738,987; 5,830,654; 5,763,163; 5,
738,989; 5,738,988; 5,723,597; 5,714,324; 5,582,975; 5,747,252; 5,567,58
7; 5,558,990; 5,622,827; 5,514,551; 5,501,951; 5,656,427; 5,352,579; 5,6
83,870; 5,374,718; 5,292,874; 5,780,219; 5,030,557; and 5,541,308.

In addition, in one preferred embodiment, the composition of the present invention comprises a biosensor having an assay complex in a tissue collection device and is suitable for a variety of standard diagnostic tests. Can be used. For example, R hereby incorporated by reference in its entirety.
eference Guide for Diagnostic Molecular Pathology / Flow Cytometry: Fasc
icle VII, 1996 College of American Pathologists, ISBN 0-930304-63-2. These assays include, but are not limited to: the adenosine deaminase gene; adenovirus; adenoleukodystrophy gene; adult renal cysts; Aeromonas bacteria; Plesiomonas bacteria; α1-antitrypsin deficiency; α-Mediterranean anemia; Alzheimer's disease; Amoeba; Androgen receptor gene; Determination of antimicrobial resistance; Arylsulfatase A gene; Aspartoacylase gene; β-thalassemia; Babesia microti; Bartonella; Basal cell carcinoma; bcl-2 gene translocation; Bordetella; Borrelia burgdorferi; B / T
C-myc; c-src proto-oncogene; Campylobacter; Alcobacter; Helicobacter; Candida albicans; carbonic anhydrase II gene; fetal carcinoma antigen; Charcot-Marie-Tooth neuropathy; Chlamydia trachomatis Clostridium difficile; colorectal cancer gene profile; copper transport P-type ATPase gene; cystic fibrosis gene; cytomegalovirus; DNA repair disease; DNA storage bank; Down syndrome gene; dystrophin gene; Enterotoxic Escherichia coli; enterovirus;
Epstein-Barr virus; Factor V (leiden); Factor VIII c gene;
Factor IX gene; familial breast cancer; familial hypertrophic cardiomyopathy gene; familial filamentous fungus; ferrochelatase gene; fibrillin gene; forensic test; fragile chromosome X syndrome; protein G gene; Gardnerella vaginalis; Giardia; Glucokinase gene; Glucose transporter gene; Haemophilus;
Hepatitis B virus; Hepatitis B virus; Hepatitis C virus; Hepatitis delta virus; Hepatitis E virus; HER-2 / neu gene amplification; Hereditary amyloidosis; Herpes simplex virus; Hexose amidase-α-sub HIV / I; HLA test; human herpesvirus A and B-6; human papillomavirus; Huntington's disease gene; Hurler-Shayer syndrome gene; insulin gene; insulin receptor gene; laminin laminin receptor Leberneria pneumophila; Leishmania bacteria; leukemia / lymphoma molecular prognostic marker; low-density lipoprotein receptor and ligand, apolipoprotein B and apolipoprotein E; luteinizing hormone gene; medium-chain acyl-coenzyme A-dehydrogenase gene; minimal residual disease detection; mitochondrial ATPase 6 gene; mitochondrial DNA; mitochondrial DNA deletion; mitochondrial DNA loss; mitochondrial t-RNA gene; Molecular epidemiology of infection; multidrug resistance gene; multiple endocrine tumors of type 1; multiple endocrine tumors of type 2A and 2B; leprosy; mycobacterium; mycoplasma; myotonic dystrophy gene; gonorrhea; N-myc Gene amplification; ornithine transcarbamylase gene; p53 tumor suppressor gene; parvovirus B19; paternity test; plasmodium; porphobilinogen deaminase gene; porphobilinogen synthase gene; Host disease; protein C gene; pyruvate dehydrogenase gene; ravies virus; ras family oncogene; respiratory rash virus; retinal germ cell tumor gene; retinoic acid receptor gene; RhD genotyping; rotavirus; Schistosoma; Sickle cell disease; Thyroid stimulating hormone receptor gene; Toxoplasma gondii; Treponema pallidum; Trichomonas; Trypanosoma; Tumor translocation suppressor gene-NM23; Uroporphyrinogen III synthase gene; Herpes virus; von Willebrand disease and Wilms tumor;

As will be apparent to those skilled in the art, the method can be used to detect many types of analytes;
Basically, any target analyte that can produce a binding ligand as described below can be detected using the method of the present invention. Many of the techniques described below describe nucleic acids as target analytes, but it will be apparent to those skilled in the art that other target analytes can be detected using the same system.

The present invention is a method for detecting a target analyte in a tissue collection device, preferably using electrodes. Generally, there are two basic detection mechanisms. In one preferred embodiment, ETM detection is based on the transfer of electrons through overlapping π orbitals of double stranded nucleic acid. This underlying mechanism is described in U.S. Patent Nos. 5,591,578; 5,770,369 and 5,705,
348 and PCT / US97 / 20014.
". Briefly, previous studies have demonstrated that electron transfer can proceed rapidly through overlapping π orbitals of double-stranded nucleic acid, and clearly more slowly through single-stranded nucleic acid. Thus, it serves as the basis for the assay. Therefore, ETM is applied to the nucleic acid attached to the detection electrode via a conductive oligomer.
(Covalently attached to one strand using a hybridization indicator described below, or non-covalently attached to the hybridization complex) to allow the nucleic acid and the ETM to pass through the conductive oligomer and the electrode. Electron transfer between them can be detected.

This can be done if the analyte is a nucleic acid; or, if a non-nucleic acid target analyte is used, optionally for capture binding ligand (to attach the target analyte to the detection electrode) and for detection This is accomplished with a soluble binding ligand having a nucleic acid "tail" that can bind directly or indirectly to the surface detection probe.

Alternatively, ETMs can be detected without necessarily passing through electron transport through the nucleic acid. Rather, it can be detected directly on the surface of a monolayer. That is, in order to generate a signal, electrons from the ETM do not need to move via overlapping π orbitals. As mentioned above, in this embodiment, the detection electrode preferably comprises a self-assembled monolayer (SAM) that serves to protect the electrode from redox species in the analyte. In this embodiment, the ETM on a SAM surface shaped to have few "defects" (sometimes referred to herein as "microchannels", "nanochannels" or "electron channels")
Can be detected directly. This basic philosophy is referred to herein as “mechanism-2”.
". Essentially, this electron conduit is a specific ET to the surface.
M can be reached. While not being bound by theory, it should be noted that the configuration of this electron transport path depends in part on the ETM chosen. For example, the use of relatively hydrophobic ETMs allows the use of hydrophobic electron transport path forming species. This effectively eliminates hydrophilic or charged ETMs. Similarly, the use of more hydrophilic or more charged species in the SAM helps to eliminate hydrophobic ETMs.

It should be noted that this defect should be distinguished from a “hole” where the analyte component is in direct contact with the detection electrode. As detailed below, the electron transfer path can be formed in several general ways. Without limitation, these include, for example, rough electrode surfaces such as gold electrodes molded on PC circuit boards; or the incorporation of at least two different molecular species within a single layer, ie, "mixed" The use of "monolayers", at least one of which is an electron transport path forming species (EFS). Thus, when the target analyte binds, the soluble binding ligand containing the ETM is carried to the surface, and presumably the detection of the ETM can proceed via an "electron transfer path" to the electrode. The role of this defective SAM is essentially to bring the ETM into contact with the electrical surface of the electrode, while still protecting the electrode from solution components, and
It is to maintain the advantage of reducing the amount of non-specific binding to the electrode. From another perspective, the role of the binding ligand is to provide specificity to recruit the ETM to a surface where the ETM can be directly detected.

In general, the SAMs of the present invention can be formed in a number of ways and contain a wide variety of components depending on the electrode surface and system used. For the "Mechanism-1" embodiment, the preferred embodiment utilizes two monolayer-forming species: a monolayer-forming species (insulator or conductive oligomer) and a conductive oligomer species containing a capture binding ligand. However, as those skilled in the art will recognize, additional monolayer-forming species can be included as well. For the “mechanism-2” system, the composition of the SAM depends on the detection electrode surface. In general, two basic "mechanism-2" systems have been announced: sensing electrodes consisting of "smooth" surfaces, such as gold ball electrodes, and commercial electrodes, e.g., on PC circuit boards. It consists of a "rough" surface, such as that produced using a process. Generally, without being bound by theory, a monolayer formed on an imperfect surface, i.e., having a "rough" surface, will automatically have sufficient electron transport paths, even in the absence of exogenous EFS. It appears to form a monolayer containing This is probably due to the difficulty of forming a homogeneous monolayer on the "rough" surface. However, a homogenous surface allows for the formation of a more homogenous monolayer, so a "smooth" surface has a sufficient number of E's to create electron transport paths.
It would be necessary to include FS. Again, without being bound by theory, the inclusion of molecular species that hinders the homogeneity of the monolayer, such as the inclusion of a more flexible but rigid molecule in the background, creates an electron transport path. Thus, a "smooth" surface includes a monolayer that includes three components: an insulator species, EFS, and a species that includes a capture ligand. However, in some cases, such as when the capture ligand species is present at a high density, the capture ligand species may also serve as EFS. "Smoothness" is not physically measured in this context, but rather EF
It is measured as a function of the signal increase measured when S is included. That is, a comparison between the signal from the detection electrode coated with the monolayer-forming molecular species and the signal from the detection electrode coated with the monolayer-forming molecular species containing EFS.
An increase indicates that the surface is relatively smooth. The reason is that the addition of EFS promotes the ETM to reach the electrode. Also noteworthy is that
Although the discussion herein is primarily directed to gold electrodes and thiol-containing monolayer-forming species, other types of electrodes and monolayer-forming species can be used.

The “electron channel” of the mechanism-2 system does not cause direct contact between the analyte component and the electrode surface, ie, the electron channel allows physical access to the electrode. It should be noted that it is not a stoma or hole sized to
Rather, without being bound by theory, the electron transport path is a type of ETM
It appears that the ETMs, especially the hydrophobic ETMs, are well penetrated into the monolayer to allow detection. However, other types of redox-active species, including certain hydrophilic species, do not penetrate to the monolayer, despite the presence of electron transport pathways. Thus, redox-active species that may be present in the analyte generally do not show a substantial signal as a result of the electron transport pathway. The exact system will generally vary depending on the composition of the SAM and the choice of ETM, but on the other hand, for an appropriate SAM to reduce non-specific binding, which also has sufficient electron transfer paths for ETM detection. Is to add ferrocene or ferrocyanide to the SAM. The former must emit a signal, but the latter must not.

Thus, in the mechanism-1 system, the monolayer, as described in more detail below,
A first molecular species comprising a conductive oligomer comprising a capture binding ligand; and a second molecular species comprising a monolayer-forming species comprising one or both of an insulator or a conductive oligomer. The monolayer in the mechanism-2 system comprises a first species comprising an attachment linker containing a capture binding ligand, a second species comprising an electron transport path forming species (EFS), and optionally an insulator. Including the third molecular species. In addition, for a "coarse" mechanism-2 system, the following two-component system is desirable: a first species that includes an attached linker that includes a capture binding ligand, and a second species that includes an insulator.

[0048] The biosensor of the present invention preferably includes an electrode. As used herein, "electrode" means a component that can sense a current or voltage when coupled to an electrical device and convert this into a signal. Alternatively, an electrode can be defined as a component that can be energized in solution and / or can transfer electrons to or from molecular species in solution. Thus, the electrodes are ETMs as described below. Suitable electrodes are known to those of skill in the art and include, but are not limited to, certain metals and their oxides, including metals including gold, platinum, palladium, silicon, aluminum; and oxides. Platinum, titanium oxide, tin oxide, mixed oxide of indium and tin,
Palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo2O6),
Metal oxides including tungsten oxide (WO3) and ruthenium oxide; and carbon (
Glassy carbon electrodes, including graphite and carbon paste). Preferred electrodes are electrodes such as gold, platinum, carbon and metal oxides, with gold being particularly preferred.

Although the electrodes described herein are shown as flat surfaces, this is one of the possible shapes of the electrodes and is for illustration only. The shape of the electrode varies depending on the detection method used. For example, planar electrodes are suitable for photodetection methods, or where a series of nucleic acids need to be prepared and require a position that can be processed for synthesis and detection. Alternatively, for single probe analysis, the electrode with the SAM attached to the inner surface can be in the form of a tube. Electrode coils or meshes are similarly preferred in some embodiments. This allows to maximize the surface area containing the target analyte to be contacted with a small volume of analyte.

In a preferred embodiment, the detection electrode is formed on a substrate. In addition, while the discussion herein is generally directed to the formation of gold electrodes, other electrodes may be used as well, as one skilled in the art will recognize. Substrates include a variety of materials, as will be appreciated by those skilled in the art, but printed circuit board (PCB) materials are particularly preferred. Thus, in general, suitable substrates include, but are not limited to, fiberglass, Teflon, ceramic, glass, silicon, mica, plastic (acrylic, polystyrene and copolymers of polystyrene and other materials, polypropylene, Polyethylene, polybutylene, polycarbonate, polyurethane, Teflon (trademark) and derivatives thereof, GETEK (a mixture of propylene oxide and fiberglass) and the like.

In general, suitable substrate materials include printed circuit board materials. Circuit board material is a material that includes an insulating substrate covered with a conductive layer and is processed using lithographic techniques, particularly exposure lithography, to form electrodes and interconnect molds (interactive in the art). Connection or lead wire). The insulating substrate is generally, but not exclusively, a polymer. Using one or more layers as is known in the art, a "two-dimensional" substrate (e.g., all electrodes and interconnects are coplanar) or a "three-dimensional" substrate (where the electrodes are On one surface, the interconnect may penetrate the substrate and exit to the other side). In three-dimensional systems, drilling or etching is often used, followed by electroplating with a metal, such as copper, to provide a "through-substrate" interconnect. Circuit board materials often have foils, such as copper foil, already adhered to the board, and add additional copper as needed (eg, for interconnects), for example, by electroplating. The copper surface may then need to be roughened, for example by etching, to allow the adhesion layer to adhere.

Thus, in a preferred aspect, the present invention provides a biosensor or biochip (referred to herein as a “chip”) comprising a substrate having a plurality of electrodes, preferably gold electrodes. The number of electrodes is as outlined below for the array. Each electrode preferably has a self-assembled monolayer as outlined herein. In a preferred embodiment, one of the monolayer-forming species includes a capture ligand as outlined herein. In addition, each electrode is interconnected and attached to the electrode at one end, and ultimately to a device that can control the electrode. That is, each electrode is independently addressable.

The substrate may generally be part of a tissue collection device, and the device may be configured as a part of a cap or tube, or as a part of a container for contacting a fixed volume of sample with a detection electrode. . Generally, the detection chamber is about 1 nL
To 1 mL. As will be apparent to those skilled in the art, smaller or larger volumes may be used depending on the experimental conditions and assays.

In a preferred embodiment, all or part of the tissue collection device is designed as a “cartridge”. It is located in a device with electronic components (AC / DC voltage source, ammeter, processor, readout display, temperature controller, light source, etc.). In this aspect, the interconnections from each electrode are arranged such that a cartridge is inserted into the device to establish a connection between the electrode and the electronic device.

The detection electrodes on the circuit board material (or other substrate) are generally prepared in a variety of ways. Generally, high purity gold may be used to coat the surface by vacuum evaporation (sputtering and evaporation) or solution deposition (electroplating or electroless). When performing electroplating, the substrate must first contain a conductive material; fiberglass circuit boards often include copper foil. In many cases, an adhesive layer is used between the substrate and the gold to ensure good mechanical stability on some substrates. Thus, the preferred embodiment utilizes an adherent layer of an adhesive metal, such as chromium, titanium, titanium / tungsten, tantalum, nickel or palladium, which metal is used for gold as described above. Can be attached. When using electroplated metals (either sticky or electrode metals), granular refining additives, often referred to in the industry as brighteners, can optionally be added to alter the nature of surface deposition. Suitable brighteners are mixtures of organic and inorganic substances, with cobalt and nickel being preferred.

Generally, the adhesive layer is from about 100 ° to about 25 microns (1000 microinches)
Thickness. If the adhesive metal is electrochemically active, the electrode metal must be coated to a thickness that prevents "spills"; if the adhesive metal is not electrochemically active,
The electrode metal may be thin. Generally, the electrode metal (preferably gold) is deposited at a thickness in the range of about 500 ° to about 5 microns (200 microinches), with a thickness of about 30 to about 50 microinches being preferred. Generally, gold is deposited to electrodes having a size of about 5 microns to about 5 mm in diameter, preferably about 100 to 250 microns. The detection electrode thus formed is
Then, preferably, it is washed and SAM is added as described below.

As described above, the present invention provides a method for manufacturing a substrate including a plurality of gold electrodes. The method includes first coating the substrate with an adhesive metal, such as nickel or palladium (optionally with a brightener). Electroplating is preferred. The electrode metal, preferably gold, is then coated on the adhesive metal (again, preferably by electroplating). The device type, including the electrodes and their associated interconnects, is then made using lithographic techniques, particularly exposure lithographic techniques known in the art, and wet chemical etching. A non-conductive, chemically resistive insulating material, such as a solder mask or plastic, is laid using exposure lithography techniques, leaving a connection point to the electrodes and leads; generally, the leads are themselves coated.

In the method, the SAM is added subsequently. In a preferred embodiment, the required chemicals, i.e., monolayer-forming species, are added using a drop deposition technique, one of which is preferably a species that includes a capture ligand. Drip deposition techniques are well known for producing "spot" arrays. This is done to add a different composition to each electrode, ie, to create an array containing various capture ligands.
Alternatively, the SAM species may be the same for each electrode, in which case it may be implemented by drip deposition techniques or by dipping the entire substrate or substrate surface in a solution.

The utility of this system is evident in the specific case of an array format; that is, a matrix of addressable sensing electrodes (herein generally “pads”, “addresses” or “addresses”). "Micro area"). That is, each biosensor can be manufactured by an array method. As used herein, "array" means that the microregions are in an array format; the size of the array depends on the configuration and end use of the array. Arrays containing from about two different capture ligands to thousands of ligands can be made. Generally, the array will consist of from 2 to 100,000 or more, depending on the size of the electrodes and the end use of the array. A preferred range is from about 2 to about 10,000, preferably from about 5 to about 1000, and particularly preferred is from about 10 to about 100. In some embodiments, the constructs of the invention may not be in an array format; that is, in some embodiments, an arrangement comprising a single capture ligand may be made as well. In addition, certain arrays may use multiple substrates of the same or different configurations. Thus, for example, a large array may be composed of multiple small substrates,
Alternatively, different substrates may be placed at various locations within the tissue collection device.

Thus, in a preferred embodiment, the tissue collection device of the present invention includes an electrode.

In a preferred embodiment, the biosensor electrode comprises a self-assembled monolayer (SAM). As used herein, "monolayer" or "self-assembled monolayer" or "SAM" refers to a relatively ordered assemblage of molecules that automatically chemisorb onto a surface, where the molecules are mutually favored. It has an orientation (eg, substantially parallel to each other) and also has a preferred orientation with respect to the surface (eg, substantially perpendicular to the surface). Each molecule has a functional group attached to the surface and has a portion that interacts with adjacent molecules in a monolayer to form a relatively ordered arrangement. A "mixed" monolayer consists of a heterogeneous monolayer; that is, at least two different molecules make up the monolayer. The SAMs may include a variety of EFSs including, but not limited to, EFSs such as, for example, conductive oligomers, insulators, attached linkers, and the like (collectively, “surface species” or “monolayer-forming species”). It may contain a molecular species. In general, the attachment chemistries outlined herein can be used for any surface species, even if specifically depicted for conductive oligomers or other species. As outlined herein, the efficiency of target analyte binding (eg, oligonucleotide hybridization) may be improved when the analyte is at a certain distance from the electrode. Similarly, non-specific binding of biomolecules, including target analytes, to electrodes is generally reduced in the presence of a monolayer. Thus, the monolayer facilitates maintaining the isolation of the analyte from the electrode surface. In addition, the monolayer helps to isolate extraneous electroactive species from the electrode surface. Thus, this layer helps to prevent electrical contact between each electrode and each ETM or between the electrodes and exogenous electroactive species in the solvent. Such contact can cause a direct "short circuit" or an indirect short circuit through charged species that may be present in the sample. Thus, in one embodiment, the monolayer is preferably tightly packed into a uniform layer on the electrode surface, minimizing the presence of "holes". In this embodiment, the monolayer acts as a physical barrier that blocks solvent access to the electrodes.

In a preferred embodiment, the monolayer comprises an electron transport path forming species. As used herein, "electron transport path forming species" or "EFS" allows for ETM detection at the surface, generally consisting of an insulator such as, for example, an alkyl group and having sufficient electron transport path in a monolayer. Means a molecule that can be produced. Generally, EFS has one or more of the following properties: it is a relatively rigid molecule (eg, as compared to an alkyl chain); it has a different conformation than other monolayer-forming molecules. An alkyl chain attached to the electrode surface (eg, an alkyl chain attached to the gold surface via a thiol group would attach at an angle of about 45 ° and a phenylacetylene chain attached to the gold surface at about 90 °)
Structures that sterically hinder or block the formation of a densely packed monolayer (eg, a branching group such as an alkyl group; or a structure such as a polyethylene glycol unit). Or it is activated to form an electron transport pathway (eg, a photoactivatable molecule, hereby incorporated by reference in its entirety). Which can be selectively removed from the surface by optical activation, leaving an electron transfer path, as generally described in US patent application Ser. No. 60 / 145,912.

Suitable EFSs include conductive oligomers and phenyl-acetylene-polyethylene glycol molecular species as described below. However, in some embodiments, E
FS is not a conductive polymer.

[0064] In a preferred embodiment, the EFS comprises a conductive oligomer. As used herein, a "conductive oligomer" is a substantially conductive oligomer, preferably a linear one, and some embodiments of which are described in the literature as "electric wires". Have been. As used herein, "substantially conductive" means that the oligomer is at 100 Hz
Means that electrons can be transmitted. Generally, conductive oligomers have substantially overlapping π-orbitals, ie, conjugated π-orbitals, such as between the monomer units of the conductive oligomer, whereas conductive oligomers have one or more sigma (σ). Also includes binding. In addition, the conductive oligomer may be capable of injecting electrons into the associated ETM or ET.
It can also be defined functionally by the ability to accept from M. In addition, conductive oligomers are more conductive than insulators as defined herein. Further, the conductive oligomers of the present invention are to be distinguished from electroactive polymers, which are capable of donating or accepting electrons by themselves.

In a preferred embodiment, the conductive oligomer has a conductivity S of about 10 ⁻⁶ to 10 ⁴ Ω ⁻¹ cm ⁻¹ , preferably about 10 ⁻⁵ to 10 ³ Ω ⁻¹ cm ^−1. Thus, these S values are calculated from molecules in the range of about 20 ° to about 200 °. As described below, the insulator has a conductivity S of about 10 ⁻⁷ Ω ⁻¹ cm ⁻¹ or less, preferably less than 10 ⁻⁸ Ω ⁻¹ cm ⁻¹ . In general, Gardner et al., Sensors and Actuat
ors A51 (1995) 57-66 (incorporated herein by reference).

The desired properties of the conductive oligomer include high conductivity, sufficient solubility in organic solvents and / or water for the synthesis and use of the compositions of the present invention, and favorable chemical resistance to the reaction. However, the reaction takes place in 1) during the synthesis of the bound ligand (
That is, it is a nucleic acid synthesis, and a nucleoside containing a conductive oligomer can be added to a nucleic acid synthesizer in the synthesis of the composition of the present invention), 2) when the conductive oligomer is attached to an electrode, or 3) when a binding assay is performed. It is. Further, conductive oligomers that promote the formation of a self-assembled monolayer are preferred.

The oligomer of the invention comprises at least two monomer subunits, as described herein. As described in detail below, oligomers include homo- and hetero-oligomers, and also include polymers.

In a preferred embodiment, the conductive oligomer has the structure shown in Structure 1: Structure 1

Embedded image As will be appreciated by those skilled in the art, any of the structures shown herein may have additional atoms or structures. That is, the conductive oligomer of structure 1 can be used for electrodes, transition metal complexes, organic ETMs, ETMs such as metallocenes, and binding ligands such as nucleic acids,
Alternatively, it can be bound to several of these. Unless otherwise specified, the conductive oligomers shown here are attached to the electrode on the left side. That is, in the case of the structure 13,
The "Y" on the left is connected to the electrodes described herein. When attaching a conductive oligomer to a binding ligand, the "Y" on the right, if present, is attached to the binding ligand, such as a nucleic acid, either directly or using a linker according to the invention.

In this embodiment, Y is an aromatic group, n is an integer from 1 to 50, and g is 1
Or e, e is an integer from 0 to 10, and m is 0 or 1. When g is 1, BD is a bond that can be shared with an adjacent bond (hereinafter referred to as A-conjugated bond ＠), and is preferably acetylene (BD is preferably selected from acetylene. Alkene, substituted alkene, amide, azo, -C = N- (including -N = C-, -CR = N- and -N = CR-), -Si =
Si- and -Si = C- (including -C = Si-, -Si = CR- and -CR = Si-). When g is 0, e is preferably 1, and D is preferably a carbonyl or heteroatom moiety, wherein the heteroatom is selected from oxygen, sulfur, nitrogen, silicon or phosphorus. Thus, suitable heteroatoms include -NH and -NR, where R is as defined herein; substituted sulfur; sulfonyl
(-SO ₂ -) sulfoxide (-SO-); phosphine oxide (-PO- and -RPO
-); And thiophosphines (-PS- and -RPS-), but are not limited thereto. However, in the case where a conductive oligomer is bound to a gold electrode, a sulfur derivative is not preferred, as outlined below.

An “aromatic group” or equivalent thereof, as used herein, refers to an aromatic monocyclic or polycyclic hydrocarbon moiety generally containing from 5 to 14 carbon atoms (making larger polycyclic ring structures And carbocyclic ketone or thioketone derivatives thereof, wherein the carbon atom with its free valency is a member of an aromatic ring. The aromatic ring includes an arylene group and an aromatic group excluding two or more atoms. For the purposes of this application, aromatics include heterocycles. “Heterocycle” or “heteroaryl” refers to the replacement of one to five specified carbon atoms with a heteroatom selected from nitrogen, oxygen, sulfur, phosphorus, boron and silicon, and estimating the free valency thereof. Aromatic groups whose atoms are members of an aromatic ring, and their heterocyclic ketone and thioketone derivatives are meant. Thus, heterocycles include thienyl, furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl, isoquinolyl, thiazolyl,
Imidosyl and the like.

What is important is that the Y-aromatic groups of the conductive oligomer can be different, that is, the conductive oligomer can be a hetero-oligomer. That is, the conductive oligomer may include a single type of Y group or a plurality of types of Y group oligomers.

An aromatic group can be substituted with a substituent generally represented by R herein. The R group can optionally be added to affect the packing of the conductive oligomer, ie,
The R group can be used to alter the association of oligomers in a monolayer. R groups can also be added to 1) alter the solubility of the oligomer or composition containing the oligomer; 2) alter the conjugation or electrochemical potential of the system; and 3) alter the charge or properties of the monolayer surface.

In a preferred embodiment, when the conductive oligomer is larger than three subunits, the R groups are preferred to increase solubility when performing solution synthesis. However, the R groups and their locations are chosen to minimize the effect on the packing of the conductive oligomer on the surface, especially within a monolayer, as described below. Generally, only small R groups are used in a monolayer, and large R groups are generally on the surface of the monolayer. for that reason,
For example, it is preferable to add a methyl group to the conductive oligomer portion in the monolayer to increase the solubility. For example, it is preferable to attach a longer alkoxy group from C3 to C10 on the monolayer surface. In general, in the systems described herein, this generally means that the sterically important R groups depend on the average length of the molecules forming the monolayer, but not on the first two or three oligomer subunits. It means not binding to any.

Suitable R groups include hydrogen, alkyl, alcohol, aromatic, amino, amide, nitro, ether, ester, aldehyde, sulfonyl, silicon, halogen, sulfur containing, phosphorus containing, and ethylene glycol. But not limited to them. In the structures shown herein, R is hydrogen if that position is not substituted. At certain positions, two substituents, R and R ', are possible, in which case the R and R' groups may either be the same or different.

“Alkyl group” or its equivalent means a straight-chain or branched alkyl group, with a straight-chain alkyl group being preferred. If it is branched, it is branched at one or more places, unless otherwise specified. The alkyl group has from about 1 to about 30 carbon atoms
(C1-C30), and in a preferred embodiment utilizes from about 1 to about 20 carbon atoms (C1-C20), with about C1 to about C12 to C15 being preferred;
Although 1 to C5 are particularly preferred, in some embodiments, the alkyl group can be larger. Also included within the definition of alkyl group are cycloalkyl groups, such as C5 and C6 rings, and heterocyclic rings having nitrogen, oxygen, sulfur, or phosphorus. Alkyl also includes heteroalkyl, preferably having sulfur, oxygen, nitrogen and silicon heteroatoms. Alkyl also includes substituted alkyl groups. “Substituted alkyl group” refers to an alkyl group that also contains one or more substituent “R” as defined above.

“Amino group” or its equivalent means a —NH ₂ , —NHR and —NR ₂ group, wherein R is as defined herein.

“Nitro group” refers to the group —NO ₂ .

“Sulfur-containing moiety” means a compound containing a sulfur atom, such as thia-, thio- and sulfo-compounds, thiols (—SH and —SR), sulfides (—RSR—)
, Sulfoxide (—R—SO—R—), sulfone (—R—SO ₂ —R—), disulfide (—R—S—S—R—) and sulfonyl ester (—R—SO ₂ —O—R
-), But are not limited to these. “Phosphorus-containing moiety” means a compound that contains phosphorus and includes, but is not limited to, phosphines and phosphates. “Silicon-containing part” means a silicon-containing compound.

“Ether” refers to the group —O—R—. Preferred ethers are alkoxy _{groups, -O- (CH 2) 2 CH} 3 and -O- (CH _{2) 4} CH ₃ being preferred.

“Ester” refers to the group —COOR.

“Halogen” refers to bromine, iodine, chlorine or fluorine. Preferred substituted alkyls are partially or fully halogenated alkyls, such as CF ₃
And so on.

“Aldehyde” refers to a —RCOH group.

“Alcohol” means an —OH group and an alkyl alcohol —ROH.

“Amido” refers to an —RCONH— or RCONR— group.

“Ethylene glycol” or “(poly) ethylene glycol” refers to — (O-
CH_Two-CH_Two)_nGroup, but each carbon atom of an ethylene group may be single or double
May be heavily substituted, ie,-(O-CR_Two-CR_Two)_n-, Where R is as defined above
It may be justice. Ethylene with another heteroatom at the oxygen position
Glycol derivatives (i.e.,-(N-CH_Two-CH_Two)_n-Or-(S-CH_Two-CH_Two) _n -Or having a substituent).

Preferred substituents include methyl, ethyl, propyl, alkoxy groups such as —O— (CH ₂ ) ₂ CH ₃ and —O— (CH ₂ ) ₄ CH ₃ and ethylene glycol and derivatives thereof. However, the present invention is not limited to these.

Preferred aromatic groups include, but are not limited to, phenyl, naphthyl, naphthalene, anthracene, phenanthroline, pyrrole, pyridine, thiophene, porphyrin and their respective derivatives, including fused ring derivatives.

In the conductive oligomers shown herein, when g is 1, BD is a bond connecting two atoms or chemical moieties. In a preferred embodiment, BD is a conjugated bond containing overlapping or conjugated orbitals.

Preferred BD bonds are acetylene (—C≡C—, also called alkyne or ethyne), alkene (—CH ＝ CH—, also called ethylene), substituted alkene (
-CR = CR-, -CH = CR- and -CR = CH-), amide (-NH-CO
-And -NR-CO- or -CO-NH- and -CO-NR-), azo (-
N = N-), esters and thioesters (-CO-O-, -O-CO-, CS-
O- and -O-CS-) and other conjugated bonds (-CH = N-, -CR = N-,-
N = CH- and -N = CR-), (-SiH = SiH-, -SiR = SiH-, -S
iH = SiR- and SiR = SiR-), (-SiH = CH-, -SiR = CH-,
-SiH = CR-, -SiR = CR-, -CH = SiH-, -CR = SiH-, -C
H = SiR- and -CR = SiR-). Particularly preferred BD
The linkages are acetylene, alkenes, amides and these three substituted derivatives and azo. Particularly preferred BD bonds are acetylene, alkenes and amides. The oligomer component attached to the double bond may be in the trans or cis configuration, or in a mixed form. As such, either B or D can include carbon, nitrogen or silicon. The substituents are as defined above for R.

When g = 0 in the Structure 1 conductive oligomer, e is preferably 1, and the D moiety can be a carbonyl or heteroatom moiety as defined above.

As in the Y ring above, within any single conductive oligomer, a BD bond (or g =
D) when 0) may be all the same or at least one may be different. For example, when m is 0, the terminal BD bond can be an amide bond and the remaining BD bonds can be acetylene bonds. Generally, when an amide bond is present, it is preferred that the amide bond be as small as possible, but in some embodiments, all BD bonds are amide bonds. Thus, as outlined above at the Y ring, one type of BD bond can be present in the conductive oligomer in the monolayer as described below, and another type of B-D bond on the monolayer level. -D bonds, for example when nucleic acids are linked via conductive oligomers, can be present to give greater flexibility to nucleic acid hybridization.

In the structures shown herein, n is an integer from 1 to 50, but longer oligomers can also be used (eg, Schumm et al., Angew. Chem. Int. Ed. Engl. 19
94 33 (13): 1360). Without being bound by theory, for efficient nucleic acid hybridization on a surface, the hybridization should occur at a distance from the surface, and the rate of hybridization may vary, especially for long oligonucleotides of 200 to 300 base pairs. , It seems to rise as a function of distance from the surface. Thus, when a nucleic acid is bound via a conductive oligomer, as described in more detail below, the length of the conductive oligomer is such that the nearest nucleotide of the nucleic acid is from about 6 ° to about 100 ° from the electrode surface (provided that (A distance of up to 500 ° can be used), preferably about 15 ° to about 60 °, and about 2 °.
5 ° to about 60 ° is also preferred. Thus, n varies according to the size of the aromatic group,
Generally, from about 1 to about 20, with about 2 to about 15 being preferred, and about 3 to about 10
Is particularly preferred.

In the structures shown herein, m is either 0 or 1. That is, m is 0
At the end of the conductive oligomer, there is a BD bond or a D moiety at the end,
The D atom is attached to the nucleic acid, either directly or via a linker.
In some embodiments, when the conductive oligomer is attached to the phosphate of the ribose-phosphate backbone of the nucleic acid, there may be another atom such as a linker attached between the conductive oligomer and the nucleic acid. Further, as outlined below,
The D atom can be the nitrogen atom of an amino-modified ribose. Alternatively, when m is 1, the terminal of the conductive oligomer has Y and an aromatic group, that is, the aromatic group is bonded to a nucleic acid or a linker.

As will be apparent to those skilled in the art, a number of conductive oligomers are available. These include, for example, fused aromatic rings or-(CF ₂ ) _n -,-(CHF) _n- and-(CFR
) _N - as with ^Teflon other known in the art such as compounds comprising like oligomeric conductive oligomer such as, including conductive oligomers falling within the scope of Structure 1 or Structure 8 formulas. For example, Schumm et al., Angew. Chem. Int. Ed. Engl. 33:
1361 (1994); Grosshenny et al., Platinum Metals Rev. 40 (1): 26-35 (199
6); Tour, Chem. Rev. 96: 537-553 (1996); Hsung et al., Organometallics 1
4: 4808-4815 (1995); and references cited therein, all of which are expressly incorporated herein by reference.

Particularly preferred conductive oligomers of this embodiment are: Structure 2

Embedded image Structure 2 is structure 1 when g is 1. Preferred embodiments of structure 2 include e
Is 0 and Y is pyrrole or substituted pyrrole; e is 0 and Y is thiophene or substituted thiophene; e is 0 and Y is furan or substituted furan; e is 0 E is 0, Y is pyridine or substituted pyridine; e is 1, BD is acetylene, Y is phenyl or substituted phenyl some stuff(
For example, see Structure 4 below). A preferred embodiment of Structure 2 is also for Structure 3 below, where e is 1.

Structure 3

Embedded image A preferred embodiment of structure 3 is where Y is phenyl or substituted phenyl and B-
D is azo; Y is phenyl or substituted phenyl; BD is acetylene; Y is phenyl or substituted phenyl and BD is alkene; Y is pyridine or substituted pyridine Y is thiophene or substituted thiophene and BD is acetylene; Y is furan or substituted furan, and BD is acetylene; Y is thiophene or substituted thiophene;
Is thiophene or furan (or substituted thiophene or furan), and BD
Is an alternate bond of alkene and acetylene.

Most of the structures shown herein utilize a conductive oligomer of Structure 3. However, any Structure 3 oligomer can be substituted with other structures herein, ie, Structure 1 or 8 oligomers, or other conductive oligomers, and the use of such Structure 3 has the meaning to limit the scope of the invention. do not have.

Particularly preferred embodiments of Structure 3 include Structures 4, 5, 6 and 7 below: Structure 4

Embedded image Particularly preferred embodiments of Structure 4 include those wherein n is 2, m is 1 and R is hydrogen; n is 3 and m is 0 and R is hydrogen; There is the use of R groups to enhance

Structure 5

Embedded image When the BD bond is an amide bond, as in Structure 5, the conductive oligomer is a pseudo-peptide oligomer. The amide bond in Structure 5 is shown with the carbonyl on the left, ie, CONH-, but vice versa, ie, -NHCO- can also be used. Structure 17
Particularly preferred embodiments of are those wherein n is 2, m is 1 and R is hydrogen; n is 3, m is 0 and R is hydrogen (in this embodiment , The terminal nitrogen (the D atom) can be the nitrogen of an amino-modified ribose), and the use of R groups to increase solubility.

Structure 6

Embedded image In a preferred embodiment of Structure 6, the first n is 2 and the second n is 1.
There are those where m is 0 and all R groups are hydrogen, or the use of R groups to increase solubility.

Structure 7

Embedded image Preferred embodiments of Structure 7 include those wherein the first n is 3, the second n is 1-3, m is either 0 or 1, and the R group for increasing solubility. There is use.

In a preferred embodiment, the conductive oligomer has the structure shown in Structure 8: Structure 8

Embedded image In this embodiment, C is a carbon atom, n is an integer from 1 to 50, m is 0 or 1, and J is selected from the group consisting of oxygen, nitrogen, silicon, phosphorus, sulfur, carbonyl, or sulfoxide. G is a bond selected from an alkane, alkene or acetylene, and together with two carbon atoms, a C—G—C group is an alkene (—CH ＝ CH—), a substituted alkene (-CR = CR-) or a mixture thereof (-CH = CR or -CR = CH-), acetylene (-C≡
C-) or alkane (-CR ₂ -CR ₂ -, R is as hydrogen or is any substituent described herein). The G bond of each subunit may be the same or different from the G bond of the other subunit, that is, an oligomer having alternating alkene and acetylene bonds can be used. However, when G is an alkane bond, the number of alkane bonds in the oligomer should be kept to a minimum, with about 6 or less sigma bonds per conductive oligomer being preferred. Alkene bonds are preferred,
Although shown generally herein, alkane and acetylene linkages can be substituted for any of the structures or embodiments described herein, as will be apparent to those skilled in the art.

In certain embodiments, for example, in the absence of ETM, if m = 0, at least one G bond is an alkane bond.

In a preferred embodiment, m in Structure 8 is 0. In a particularly preferred embodiment,
As shown in Structure 9, m is 0 and G is an alkene bond: Structure 9

Embedded image The alkene oligomers of structure 9 and others shown herein are generally shown in the preferred trans configuration, but cis or mixed trans and cis oligomers can also be used. As described above, R groups can be added to alter the packing of the composition on the electrode, the hydrophilic or hydrophobic nature of the oligomer, and the flexibility of the oligomer, ie, rotation, twist, or longitudinal flexibility. . n is as defined above.

In a preferred embodiment, R is hydrogen, but R may be an alkyl group and polyethylene glycol or a derivative.

In another embodiment, the conductive oligomer may be a different type of oligomer, for example, a mixture of structures 1 and 8.

The conductive oligomer may or may not have a terminal group. Thus, in a preferred embodiment, there are no extra end groups, and the conductive oligomer exhibits structures 1 to 9 whose ends end in one of the groups, such as a BD bond, such as an acetylene bond. Alternatively, in a preferred embodiment, a terminal group, often referred to herein as "Q", is added. The end groups may be used for several reasons, for example to improve the electronic availability of the conductive oligomer for the detection of ETMs, or to alter the surface of the SAM for other reasons, such as preventing non-specific binding. Can be used. For example, if the target analyte is a nucleic acid, a negatively charged surface is formed as a negatively charged group at the terminus to promote hybridization, and if the nucleic acid is DNA or RNA, the nucleic acid lands on the surface May be repelled or impeded. Preferred passivating agents terminal groups, -NH _2, -OH, -COOH, alkyl group of -CH _3, etc., and includes a (poly) alkyl oxides such as (poly) ethylene glycol, -OCH ₂ CH _{2 OH, - (OCH 2 CH 2} O) 2 H, - (OCH 2 C
H ₂ O) ₃ H, and _{- (OCH 2 CH 2 O)} 4 H being preferred.

In one embodiment, it is possible to use a mixture of conductive oligomers having different types of end groups. Thus, for example, some end groups may facilitate detection and some may prevent non-specific binding.

[0109] In a preferred embodiment, the monolayer may further include an insulating portion. As used herein, "insulator" means a substantially non-conductive oligomer, preferably linear. As used herein, "substantially non-conductive" means that the insulator does not transmit electrons at 100 Hz. The rate of electron transfer through the insulator is preferably slower than the rate through the conductive oligomers described herein.

In a preferred embodiment, the insulator has a conductivity S of less than or equal to about 10 ^-7 Ω ^-1 cm ^-1 and is preferably less than about 10 ^-8 Ω ^-1 cm ^-1 . See generally Gardner et al. Supra.

Typically, the insulator is an alkyl or heteroalkyl oligomer or moiety having a sigma bond, but any particular insulator molecule may include an aromatic group or one or more conjugated bonds. As used herein, “heteroalkyl” refers to at least one
An alkyl group having two heteroatoms, ie, nitrogen, oxygen, sulfur, phosphorus, silicon or boron contained in the chain. Alternatively, the insulator can be very similar to a conductive oligomer, preferably with the addition of one or more heteroatoms or bonds that substantially inhibit or retard electron transfer.

Suitable insulators are known to those skilled in the art and include-(CH ₂ ) n-,-(CRH) n- and-(CR2) n-, ethylene glycol or other heteroatoms instead of oxygen, ie Including but not limited to derivatives using nitrogen or sulfur (sulfur derivatives are not preferred when the electrode is gold).

For conductive oligomers, the insulator may be substituted with an R group as defined herein, the packing of the moiety or conductive oligomer at the electrode, the hydrophilic or hydrophobic nature of the insulator, and the flexibility of the insulator. Gender, ie, rotation, torsional or longitudinal flexibility. For example, a branched alkyl group may be used. Similarly, as outlined above, the insulator may include end groups that act, inter alia, on the surface of the monolayer.

The length of the species from which the monolayer is made will vary as needed. As outlined above, binding of the target analyte (eg, nucleic acid hybridization) appears to be more effective away from the surface. The species to which the nucleic acid binds (which can be either an insulator or a conductive oligomer, as outlined below) is essentially the same length or longer than the species forming the monolayer, and the capture binding ligand Are more accessible to the solvent for hybridization. In some embodiments, the conductive oligomer to which the capture binding ligand binds is shorter than a monolayer.

Although the monolayer can include different surface species, in various configurations, a reasonably identical SAM
It will be appreciated that it is preferable to select different species so that can be formed. Thus, for example, when a capture binding ligand, such as a nucleic acid, is covalently attached to the electrode using a conductive oligomer, it is not possible to use one type of conductive oligomer to bind the nucleic acid and use another type in the SAM. It is possible. Similarly, it is desirable to have a mixture of conductive oligomers of different lengths in a monolayer to facilitate the reduction of non-specific signals. Thus, for example, a preferred embodiment is an EFS comprising conductive oligomers that terminate below the surface of the remainder of the monolayer, ie, if used, under an insulating layer or under a specific fraction of other conductive oligomers. Use surface species such as Similarly, different species, such as conductive oligomers, can be used to facilitate the formation of a monolayer or to create a monolayer with different properties.

As will be apparent to those skilled in the art, the actual combinations and ratios of the different species that make up the monolayer can vary widely, and it depends on whether mechanism-1 or mechanism-2 is used. It may vary depending on whether a rough or smooth surface is used. In general, a three-component system is preferred for the mechanism-2 system, wherein the first species comprises a capture binding ligand containing the species (called a capture probe if the target analyte is a nucleic acid) and a binding linker (e.g., an insulator Or any of the conductive oligomers). The second species is E
FS, and a third species is an insulator. In this embodiment, the first species is about 9
It can include 0% to about 1%, with about 20% to about 40% being preferred. When the target analyte is a nucleic acid, about 30% to about 40% are particularly preferred for short oligonucleotide targets, and about 10% to about 20% are preferred for long targets. The second species is from about 1% to about 90
%, With about 20% to about 90% being preferred, with about 40% to about 60% being particularly preferred. The third species can include from about 1% to about 90%, with about 20% to about 40% being preferred, and about 15% to about 30% being particularly preferred. Preferred ratios of the first: second: third species in the SAM constituent solvent to achieve these approximate ratios are 2: 2: 1 for short targets and 1: 3: 1 for long targets. Yes, the total thiol concentration (when used for binding to these species, detailed below) ranges from 500 μM to 1 mM and preferably 833 μM.

Alternatively, a two-component system comprising a first and a second type can be used. In some embodiments, the two components used in either mechanism-1 or mechanism-2 systems are first and second species. In this embodiment, the first species may comprise from about 1% to about 90%, preferably from about 10% to about 40%, with about 1%
0% to about 40% is particularly preferred. The second species may comprise from about 1% to about 90%, with about 10% to about 60% being preferred, and about 20% to about 40% being particularly preferred. Alternatively, in a mechanism-1 system or a "coarse" mechanism-2 system, the two components are the first and third species. In this embodiment, the first species may comprise from about 1% to about 90%, with about 10% to about 40% being preferred, and about 10% to about 40% being particularly preferred. The second species may comprise from about 1% to about 90%, with about 10% to about 60% being preferred, and about 20% to about 40% being particularly preferred.

In a preferred embodiment, the SAM is precipitated in an aqueous solution. Steel et al.
, Anal. Chem. 70: 4670 (1998), Herne et al., J. Am. Chem. Soc. 119: 8916.
(1997) and AJ Bard, Electroanalytical Chemistry: A Series of Advance
s, Vol. 20. Dekker NY 1996- Finklea, Electrochemistry of Organized mo
As outlined in the nolayers of Thiols and Related molecules on Electrodes, precipitation of SAM-forming species can be performed from aqueous solutions (often including salts), all of which are incorporated herein by reference.

The covalent attachment of the monolayer-forming species and the capture binding ligand species to the electrode can be achieved in various ways and depends on the composition of the electrode and each part. In a preferred embodiment, a binding linker covalently linked to a nucleoside or nucleic acid described herein is covalently linked to the electrode. Thus, one end or end of the binding linker is bound to a nucleoside or nucleic acid and the other is bound to an electrode. In certain embodiments, it is desirable for the linking linker to be attached at a position other than the terminus, or further for a branched linker to be attached to the electrode at one end and to two or more nucleosides at the other end. This is undesirable. Similarly, generally the structure 11
As described in -13, a binding linker can bind to an electrode at two sites. Generally, certain types of linkers are used, as shown below in Structure 10 as A. In structure 10, "X" is EFS (eg, a conductive oligomer), "I" is an insulator, and the hatched surface is an electrode:

[0120]

Embedded image

In this embodiment, A is a linker or an atom. The choice of "A" depends in part on the characteristics of the electrode. Therefore, for example, A is a sulfur part when a gold electrode is used. Alternatively, when using a metal oxide electrode, A is a silicon (silane) moiety bonded to the oxygen of the oxide (eg, Chen et al., Langmuir 10: 3332-3337).
(1994); Lenhard et al., J. Electroanal.Chem. 78: 195-201 (1977);
Both are hereby incorporated by reference). When using a carbon-based electrode, A is an amino moiety (preferably a primary amine; for example, Deinhammer et al.
l., Langmuir 10: 1306-1313 (1994)). Thus, preferred A parts include, but are not limited to, a silane moiety, a sulfur moiety (including an alkyl sulfur moiety), and an amino moiety. In a preferred embodiment, no epoxide-type linkage with a redox polymer as known in the art is used.

Although depicted herein as only one portion, these portions may be associated with the electrode at one or more “A” portions; the “A” portions may be the same or different. Therefore,
For example, when the electrode is a gold electrode and "A" is a sulfur atom or moiety, a plurality of sulfur atoms may be added to the electrode as depicted generally for the conductive oligomers of Structures 11, 12 and 13 below. Can be used to combine species of As will be appreciated by those skilled in the art, such other structures can be manufactured. Structures 11, 12 and 1
In 3, the A portion is only a sulfur atom, but a substituted sulfur portion may be used.

[0123]

Embedded image

[0124]

Embedded image

[0125]

Embedded image

It should also be noted that, like structure 13, it is possible to have a species that has three sulfur moieties attached to the electrode and terminates at one carbon atom. Further, although not necessarily described herein, these species may also include "Q" end groups.

In a preferred embodiment, the electrode is a gold electrode and the bond is through a sulfur bond as is known in the art, ie, part A is a sulfur atom or moiety. Although the exact nature of the gold-sulfur bond is not known, this bond is considered a covalent bond for the purposes of the present invention. A representative structure is depicted in Structure 14 using the conductive oligomer of Structure 3, but for all structures described herein, any surface species may be used.
Similarly, any surface species may include the terminal groups described herein. The structure 14 is composed of “A
"Although linkers are described as containing only sulfur atoms, other atoms may be present.
(Ie, a linker from sulfur to a conductive oligomer or to a substituent). Further, while structure 14 represents a sulfur atom attached to the Y aromatic group, it will be apparent to those of skill in the art that the structure 14 can be similarly attached to the BD group (i.e., acetylene). Good.

[0128]

Embedded image Generally, a thiol bond is preferred.

In a preferred embodiment, the electrode is a carbon electrode, ie a vitreous carbon electrode, and the bond is through the nitrogen atom of the amine group. A representative structure is shown in Structure 15. Yet another atom may be present, ie, a Z-type linker and an X or end group.

[0130]

Embedded image

[0131]

Embedded image

In structure 16, the oxygen atoms are from the oxide of the metal oxide electrode. S
The i-atom may include other atoms, ie, may be a substituent-containing silicon moiety. Other attachments of SAM to other electrodes are known in the art; see, for example, Napier et al., Langmuir, 1997 for attachment to indium tin oxide electrodes and chemisorption of phosphate esters on indium tin oxide electrodes. )
(Holden Thorpe at the CHI Conference (May 4-5, 1998)
H. Holden Thorpe).

The SAMs of the present invention can be made by various methods, for example, precipitation from organic solutions and precipitation from aqueous solutions. The methods outlined herein use gold electrodes as an example, but other metals and methods may be used as well as those skilled in the art will appreciate. In a preferred embodiment, indium-tin-oxide (ITO) is used as the electrode.

In a preferred embodiment, the gold surface is first cleaned. Various cleaning techniques can be employed, including, but not limited to, chemical cleaning or etching reagents (Piranha solution (hydrogen peroxide / sulfuric acid) or aqua regia (hydrochloric acid / nitric acid)) ), An electrochemical method, a flame treatment, a plasma treatment or a combination thereof.

Following the cleaning, the gold substrate is exposed to SAM species. SA when electrode is ITO
Species M is a phosphate ester containing species. This treatment can also be performed in various ways, for example, solution deposition, vapor deposition, microcontact printing, spray deposition,
Examples include, but are not limited to, deposition using a neat component. A preferred embodiment utilizes a precipitation solution comprising a mixture of various SAM species, generally thiol-containing species, in the solution. A target analyte, particularly a mixed monolayer containing DNA, is usually prepared using a two-step procedure. Thiolated DNA precipitates during the first precipitation step (generally in the presence of at least one other monolayer-forming species) and mixed monolayer formation is completed during the second step of adding a second thiol solution without DNA. I do. The second step often involves mild heating to promote monolayer reconstruction.

In a preferred embodiment, the deposition solution is an organic deposition solution. In this embodiment, the clean gold surface is placed in a clean vial. The bound ligand deposition solution in an organic solvent is prepared such that the total thiol concentration is between micromolar and saturation; a preferred range is from about 1 μM to 10 mM, particularly preferred from about 400 μM to 1.0 mM. In a preferred embodiment, the precipitation solution contains thiol-modified DNA (ie, nucleic acids attached to an attachment linker) and thiol-diluted molecules (which are conductive oligomers or insulators, the latter being preferred). The ratio of DNA to diluent (if any) is usually between 1000: 1 and 1: 1000, preferably about 10:
It is from 1 to about 1:10, particularly preferred is 1: 1. Suitable solvents are tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF)
, Ethanol, or mixtures thereof; generally, any solvent of sufficient polarity to dissolve the capture ligand can be used, as long as the solvent does not have a functional group that will react with the surface. Sufficient DNA precipitation solution is added to the vial to completely cover the surface of the electrode. The gold substrate is incubated at ambient temperature or slightly above ambient temperature for a few seconds to a few hours, preferably 5 to 30 minutes. After the first incubation, the precipitation solution is removed, and an organic solvent solution containing only diluted molecules (
About 1 μM to 10 mM, preferably about 100 μM to about 1.0 mM). The gold substrate is incubated at room temperature or a temperature higher than room temperature for a predetermined time (several seconds to several days, preferably about 10 minutes to about 24 hours). A gold sample is removed from the solution and rinsed in a clean solvent for use.

In a preferred embodiment, an aqueous precipitation solution is used. Place the clean gold surface in a clean vial as described above. The DNA precipitation aqueous solution is prepared such that the total thiol concentration is between about 1 μM and 10 mM, preferably between about 1 μM and 200 μM. Aqueous solutions are often present with salts (until saturation, preferably about 1M), but purified water can be used. The precipitation solution contains thiol-modified DNA and often thiol-diluted molecules. The ratio of DNA to diluent is usually 1000: 1 and 1:10
00, preferably from about 10: 1 to about 1:10, particularly preferably 1: 1. The DNA deposition solution is added to the vial in a volume that completely covers the surface of the electrode. This gold substrate is heated at an ambient temperature or slightly higher
Incubate for 30 minutes, usually 5 minutes is sufficient. After the first incubation, the precipitation solution is removed and a dilute molecule only (10 μM to 1.0 mM) aqueous or organic solvent solution is added. The gold substrate is incubated at or above room temperature until a complete monolayer is formed (10 minutes to 24 hours). A gold sample is removed from the solution and rinsed in a clean solvent for use.

In a preferred embodiment, a circuit board is used as a substrate for gold electrodes, as described herein. The formation of a SAM on a gold surface is generally performed by first cleaning the substrate as described herein, for example, in a 10% sulfuric acid solution for 30 seconds with a detergent solution, aqua regia, plasma, or the like. Following the sulfuric acid treatment, the substrate is
For example, washing is performed by immersing each in two milli-Q water baths for one minute. The substrate is then dried, for example, under a stream of nitrogen. To spot the deposition solution on the substrate, a number of known spot systems are used, typically placing the substrate on an XY table and performed in a humidity chamber. The size of the spot drop depends on the size of the electrode on the substrate and the instrument used for solution delivery; for example, for a 250 μM size electrode, a 30 nanoliter drop is used. This capacitance must be sufficient to completely cover the electrode surface. The droplets are incubated at room temperature for a period of time (seconds to overnight, preferably 5 minutes), and then the droplets are removed by rinsing in a Milli-Q water bath. The substrate is then preferably treated with a second deposition solution, generally a solution comprising the insulator in an organic solvent, preferably acetonitrile, by immersion in a 45 ° C. bath. After 30 minutes, the substrate is removed and immersed in an acetonitrile bath for 30 seconds and then in a Milli-Q water bath for 30 seconds. The substrate is dried under a stream of nitrogen.

[0139] In a preferred embodiment, the detection electrode further comprises a capture binding ligand, preferably a covalently attached ligand. As used herein, "binding ligand" or "binding species" is a compound used to probe for the presence of a target analyte, and refers to a compound that binds to the target analyte. In general, for most of the embodiments described herein, there will be at least two binding ligands per molecule of the target analyte; ie, "capture" or "anchor" binding to the detection electrodes described herein. Ligand (also referred to herein as a "capture probe", particularly with respect to nucleic acid binding ligands), and soluble binding ligand (if a nucleic acid is used, also referred to herein as "labeled probe" or "signaling probe"). Wherein the ligand independently binds to the target analyte and comprises, directly or indirectly, at least one ETM. However, as described herein, in some cases, the target analyte may include an ETM, thus using only one capture binding ligand.

In general, capture binding ligands allow a target analyte to attach to a biosensor electrode for detection. As described in more detail below, the target analyte attaches to the capture binding ligand directly (ie, the target analyte attaches to the capture binding ligand) or indirectly (using one or more capture extender ligands). May be possible).

In a preferred embodiment, the binding is specific and the binding ligand is part of a binding pair. As used herein, "specifically binds" means that the ligand binds to the analyte in the test sample with sufficient specificity to distinguish the analyte from other components or contaminants. However, it will be apparent to one skilled in the art that analytes can be detected using less specific binding; for example, the system uses an array of different binding ligands, eg, different ligands. Yes, detection of any particular analyte is via the "sign" of binding of the bound ligand to the panel, similar to the manner in which the "electronic nose" works. This binding must be sufficient for the analyte to remain bound under the conditions of the assay, including a washing step to remove non-specific binding. In some embodiments, for example, in the detection of certain biomolecules, the binding constants of the analyte to the binding ligand is at least about 10 ⁴
〜１０10 ⁶ M ⁻¹ , preferably about 10 ⁵ to 10 ⁹ M ⁻¹ , and particularly preferred is about 10 ⁷ to 10 ⁹ M ⁻¹ .

As will be apparent to one of skill in the art, the composition of the binding ligand will depend on the composition of the target analyte. Binding ligands for a wide range of analytes are known or can be readily found using known techniques. For example, if the analyte is a single-stranded nucleic acid, the binding ligand will generally be a substantially complementary nucleic acid. Alternatively, U.S. Pat.
3, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and the nucleic acid "aptomer" as described in any of the related patents incorporated herein by reference. It can be generated to substantially combine. Similarly, the analyte may be a nucleic acid binding protein and the capture binding ligand is a single-stranded or double-stranded nucleic acid; alternatively, the binding ligand may be where the analyte is a single-stranded or double-stranded nucleic acid. , A nucleic acid binding protein. When the analyte is a protein, the binding ligand comprises a protein (including, inter alia, antibodies or fragments thereof (such as FAbs)), a small molecule, or an aptamer as described above. Suitable binding ligand proteins include peptides. For example, where the analyte is an enzyme, suitable binding ligands include substrates, inhibitors, and other proteins that bind the enzyme (ie, components of a multiple enzyme (or protein) complex). As will be apparent to those skilled in the art, any two molecules that associate may be used as analytes or as binding ligands, preferably specifically. Suitable analyte / binding ligand pairs include antibodies / antigens, receptors / ligands, proteins / nucleic acids; nucleic acids / nucleic acids, enzymes / substrates and / or inhibitors, carbohydrates (including glycoproteins and glycolipids) / lectins, carbohydrates And other binding partners, proteins / proteins; and proteins / small molecules, and the like. These may be wild type or derived sequences. In a preferred embodiment, the binding ligand is a cell surface receptor known to multimerize, for example,
Growth hormone receptor, glucose transporter (especially GLUT4 receptor), transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, high density lipoprotein receptor, leptin receptor, interleukin receptor (IL- 1, IL
-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-11, IL-12, IL-13, IL-15, and IL-17 receptors), VEGF receptor, PDGF receptor, EPO receptor, TPO receptor,
It is part of the ciliary neurotrophic factor receptor, prolactin receptor, and T cell receptor. Similarly, there is a great deal of literature relating to the development of binding partners based on binding chemistry methods.

In this embodiment, where the binding ligand is a nucleic acid, preferred compositions and techniques are described in US Pat. Nos. 5,591,578; 5,824,473; 5,770,369; 5,705,348; 12430; PCT / US99 / 017
03; PCT / US98 / 12082; PCT / US99 / 14191; PCT / US99 / 21683; PCT / US99 / 25464; PCT /
And USSNs 09 / 135,183; 09 / 295,691 and 09 / 306,653, all of which are hereby incorporated by reference.

The method of attaching the capture binding ligand to the attachment linker (either insulator or conductive oligomer) is generally performed as is known in the art, but the composition of the attachment linker and the capture binding ligand Depends on both. Generally, the capture binding ligand is attached to the attachment linker via the use of a functional group, but each functional group is then available for attachment. Suitable functional groups for attachment are amino, carboxy, oxo and thiol groups. These functional groups may then be attached, directly or indirectly, through the use of a linker designated herein as "Z". Linkers are well known in the art; for example, homo- or hetero-bifunctional linkers are well-known (see: Pierce Chemical Company, 1994, Catalog of Crosslinking Techniques, 155-155). 200 pages; incorporated herein by reference.) Suitable Z linkers include alkyl groups (substituted alkyl groups and alkyl groups containing heteroatom moieties), esters with short chain alkyl groups, amides, amines,
Epoxy groups and ethylene glycol, and preferred derivatives and the like, propyl, acetylene, and C ₂ alkenes are particularly preferred, but not limited thereto. Z may be a sulfone group forming a sulfonamide.

In this manner, capture binding ligands can be added, including proteins, lectins, nucleic acids, small organic molecules, carbohydrates, and the like.

A preferred embodiment utilizes a proteinaceous capture binding ligand. As is known in the art, a number of techniques can be used to attach a proteinaceous capture binding ligand to an attachment linker. A wide variety of techniques are known for adding a moiety to a protein.

[0147] A preferred embodiment utilizes nucleic acids as capture binding ligands. As will be apparent to those skilled in the art, some of the methods outlined below can be similarly applied to non-nucleic acid systems in a similar manner.

The nucleic acid capture binding ligand is covalently attached to the electrode via an “attachment linker”, which may be a conductive oligomer (required in a mechanism-1 system) or any surface species including insulators are doing. As used herein, "covalently attached" means that the two moieties are attached by at least one bond, which includes sigma, pi, and coordination bonds.

Thus, one end of the attachment linker is attached to the nucleic acid (or other binding ligand) and the other end (as will be appreciated by those skilled in the art, but need not be the exact end in any case). Is attached to the electrode. Thus, any of the structures depicted herein may further include a nucleic acid that is effective as a terminal group. Thus, the present invention
Provided is a composition comprising a nucleic acid covalently attached to an electrode, as generally depicted in Structure 17 below.

[0150]

Embedded image

In the structure 17, the hatched symbols on the left represent electrodes. X is a conductive oligomer and I is an insulator, as defined herein, but still other surface species may be used. F ₁ is a bond that results in a covalent bond between the electrode and the conductive oligomer or insulator, and includes a bond, atom, or linker as described herein, for example, as defined below as “A”. F ₂ is a bond covalently linking the surface species to a nucleic acid, binding as described herein, may be atoms or a bond. F ₂
Can be part of a conductive oligomer, part of an insulator, part of a nucleic acid, or can be exogenous to both, for example as defined herein for “Z”.

In a preferred embodiment, for example, in a “mechanism-1 system”, the capture probe nucleic acid is covalently attached to the electrode via a conductive oligomer. Covalent attachment of the nucleic acid to the conductive oligomer can be achieved in several ways. In preferred embodiments, the attachment is by attachment to a nucleoside base, as described below, by attachment to a nucleic acid backbone (a ribose, phosphate ester, or analogous group of a nucleic acid analog backbone), or by a transition metal. Due to attachment to ligand. Although the techniques outlined below generally describe naturally occurring nucleic acids, similar techniques can be used with nucleic acid analogs, and as will be appreciated by those skilled in the art, and in some cases use other binding ligands. Can be used.

In a preferred embodiment, the attachment linker is attached to a nucleoside base of the nucleic acid.
This can be accomplished in several ways depending on the type of attachment linker, as described below. In one embodiment, the attachment linker is a terminal nucleoside, ie, 3
Attached to the 'or 5' nucleoside. Alternatively, an attachment linker is attached to the internal nucleoside.

The point of attachment to a base differs depending on the base. Generally, attachment at any location is possible. In some embodiments, for example, when a probe comprising an ETM is used for hybridization (ie, the mechanism-1 system), it is preferably attached to a position that does not participate in hydrogen bonding of the complementary base. Thus, for example, the attachment is generally 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 suitably made at the corresponding locations.

In one embodiment, the attachment is direct; that is, there are no intervening atoms between the attachment linker and the base. In this embodiment, for example, a conductive oligomer attachment linker having a terminal acetylene bond is attached directly to the base. Structure 18 is an example of this linkage, where uridine is used as the conductive oligomer of Structure 3 and a base, but as will be appreciated by those skilled in the art, the use of a conductive oligomer or an attached linker with other bases. Can also.

[0156]

Embedded image

It should be noted that other groups such as hydrogen, hydroxy, phosphate or amino groups are attached to the pentose structures shown herein. Further, the pentose and nucleoside structures depicted herein are non-conventionally shown as mirror images of the usual expression. In addition, the pentose and nucleoside structures can also contain new groups at any position, for example, as needed during synthesis, such as protecting groups.

In addition, the base may optionally contain further modifications, ie alter or protect the carbonyl or amine group.

In another embodiment, the linkage is generally through a number of different Z-linkers, including amide and amine linkages, as shown in Structure 19 using uridine and the oligomer of Structure 3 as bases:

[0160]

Embedded image

[0161] In this embodiment, Z is a linker. Preferably, Z is a short linker of about 1 to about 10 atoms, preferably 1 to 5 atoms, which may or may not contain a bond such as an alkene, alkynyl, amine, amide, azo, imine and the like. Good. Linkers are known in the art, e.g., as is well known, are homo- or heterobifunctional linkers (see 1994 Pierce Chemical Company catalog, technical section on cross-linker, pages 155-200; citation source). As part of the present specification). Preferred Z linkers include, but are not limited to, alkyl groups (including substituted alkyl groups and alkyl groups containing a heteroatom moiety), and are preferably short alkyl groups, esters, amides, amines, epoxy groups. And ethylene glycol and derivatives, particularly preferred are propyl,
Acetylene and C2 alkenes. Z may also be a sulfone group,
A sulfonamide bond is formed as described below.

In a preferred embodiment, attachment of the nucleic acid to the attachment linker is through attachment to the nucleic acid backbone. This can be accomplished in a number of ways, including binding the ribose-phosphate backbone to ribose or the backbone to phosphate, or to other groups of the similar backbone.

As a preliminary matter, it should be understood that the binding site in this embodiment is the 3 ′ or 5 ′ terminal nucleotide, or an internal nucleotide, as described more fully below.

In a preferred embodiment, an attachment linker is attached to the ribose of the ribose-phosphate backbone. This can be implemented in several ways. As is known in the art, an amino group, a sulfur group, a silicon group, at either the 2 'or 3' position of ribose,
Nucleosides modified with phosphorus 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. Org.Chem. 36 (2): 250 (1971); McGee et al.,
J. Org. Chem. 61: 781-785 (199); Mikhailopulo et al., Liebigs. Ann. Che.
m. 513-519 (1993); McGee et al., Nucleosides & Nucleotides 14 (6): 1329
(1995), all of which are incorporated herein by reference). These modified nucleosides are then used to add an attachment linker.

A preferred embodiment utilizes amino-modified nucleosides. At this time, these amino-modified ribose can be used to form an amide or amine bond to the attached linker. In a preferred embodiment, the amino group is attached directly to the ribose, but as will be apparent to those skilled in the art, a short linker as described for "Z" can be provided between the amino group and the ribose.

[0166] In a preferred embodiment, an amide bond is used to attach to ribose.
Preferably, if a conductive oligomer having a structure 1 to 3 is used, since m is 0, the terminal of the conductive oligomer is an amide bond. In this embodiment, the nitrogen of the amino group of the amino-modified ribose is the "D" atom of the conductive oligomer. Therefore,
A preferred linkage of this embodiment is shown in Structure 20 (using a conductive oligomer of Structure 3).

[0167]

Embedded image

As will be apparent to those skilled in the art, Structure 20 has terminal bonds fixed as amide bonds.

In a preferred embodiment, a heteroatom bond is used, ie, oxo, amine, sulfur, and the like. A preferred embodiment utilizes an amine linkage. Also, as outlined above for the amide bond, the amine of amino-modified ribose may be the "D" atom of the conductive oligomer when a conductive oligomer of structure 3 is used for the amine bond as well. Thus, for example, structures 21 and 22 show nucleosides with conductive oligomers of structures 3 and 9, respectively, using nitrogen as a heteroatom, but other heteroatoms can be used:

[0170]

Embedded image

In structure 21, preferably neither m nor t is 0. Preferred Z here is a methylene group or other aliphatic alkyl linker. One, two or three carbons in this position are particularly useful during synthesis.

[0172]

Embedded image

In Structure 22, Z is as defined above. Suitable linkers include methylene and ethylene.

In another embodiment, the attachment linker is covalently attached to the nucleic acid via the phosphate of the ribose-phosphate backbone (or analog) of the nucleic acid. In this embodiment, the linkage is direct or utilizes a linker or amide linkage. Structure 23 is
A direct bond is shown and structure 24 shows a bond via an amide bond (both utilizing conductive oligomers of structure 3, but also conductive oligomers of structure 8 and other attached linkers). Is possible). Structures 23 and 24 show the conductive oligomer at the 3 'position, but the 5' position is also possible. Further, structures 23 and 24
Both show a natural phosphodiester linkage, but as will be apparent to those skilled in the art, non-standard analogs of the phosphodiester linkage can also be used.

[0175]

Embedded image

In structure 23, when Y exists at the terminal (ie, m = 1), Z does not exist (ie,
t = 0) is preferable. If Y is not present at the end, Z is preferably present.

Structure 24 shows a preferred embodiment where the terminal BD bond is an amide bond, there is no terminal Y, and Z is a linker as defined above.

[0178]

Embedded image

[0179] In a preferred embodiment, the attachment linker is covalently attached to the nucleic acid via a transition metal ligand. In this embodiment, the attachment linker is covalently attached to a ligand that provides one or more coordination atoms to the transition metal. In one embodiment, the nucleic acid is also attached to the ligand to which the attachment linker is attached, as shown generally in Structure 25 below. Alternatively, the attachment linker is attached to one ligand and the nucleic acid is attached to another ligand, as shown generally in Structure 26 below using a conductive oligomer as the attachment linker. Thus, in the presence of the transition metal, the attachment linker is covalently linked to the nucleic acid. Each of these structures shows the conductive oligomer of Structure 3, but other oligomers can be used. Structures 25 and 26 show two representative structures for nucleic acids:

[0180]

Embedded image

[0181]

Embedded image

[0182] In the structures described herein, M is a metal atom, with transition metals being preferred. Transition metals suitable for use in the present invention are cadmium (Cd), copper (Cu), cobalt (C
o), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru),
Rhodium (Rh), Osmium (Os), Rhenium (Re), Platinum (Pt), Scandium (Sc), Titanium (Ti), Vanadium (V), Chromium (Cr)
, Manganese (Mn), nickel (Ni), molybdenum (Mo), technetium (
Tc), tungsten (W), and iridium (Ir), but are not limited thereto. That is, the first series of transition metals, the platinum group (Ru
, Rh, Pd, Os, Ir and Pt) and Fe, Re, W, Mo and Tc
Is preferred. Particularly preferred are ruthenium, rhenium, osmium, platinum, cobalt and iron.

L is an auxiliary ligand, which provides a coordination atom for metal ion binding. As those skilled in the art will recognize, the number and nature of the auxiliary ligands will depend on the coordination number of the metal ion. The monodentate, bidentate or multidentate auxiliary ligand may be used at any position. Thus, for example, if the metal has a coordination number of 6, L from the end of the conductive oligomer, L and r provided from the nucleic acid are added up to 6. Therefore, if the metal has six coordination numbers,
r may range from 0 (when all coordinating atoms are provided by the other two ligands) to 4 (when all auxiliary ligands are monodentate). Thus, in general, r will be between 0 and 8, depending on the coordination number of the metal ion and the choice of other ligands.

In certain embodiments, the metal ion has a coordination number of 6, and the ligand attached to the conductive oligomer and the ligand attached to the nucleic acid are both at least dimers; ie, r is Preferably, it is 0, 1 (ie the remaining auxiliary ligand is a dimer) or 2 (two monodentate auxiliary ligands are used).

As will be recognized in the art, the auxiliary ligands can be the same or different.
Suitable ligands fall into two categories: coordinating atoms (generally, Sigma (σ
) As a donor) (depending on the metal ion), ligands using nitrogen, oxygen, sulfur, carbon or phosphorus atoms, and organometallic ligands such as metallocene ligands (generally, pi (π) donors in the literature) It called the body, which is herein depicted with 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; phenanthroline, especially 1,10
Phenanthroline (abbreviated as phen) and substituted derivatives of phenanthroline,
For example, 4,7-dimethylphenanthroline and dipyrido [3,2-a: 2 ′
, 3'-c] phenazine (abbreviated as dppz); dipyridophenazine; 1,4,5
1,8,9,12-hexaazatriphenylene (abbreviated as hat); 9,10-phenanthrenequinone diimine (abbreviated as phi); 1,4,5,8-tetraazaphenanthrene (abbreviated as tap); 1,4 , 8,11-Tetra-azacyclotetradecane (abbreviated as cyclam), EDTA, EGTA and isocyanide. Substituted derivatives, including fused derivatives, can also be used. In some embodiments, porphyrins and substituted derivatives of the porphyrin family may be used.
For example, Comprehensive Coordination Chemistry, Ed. Wilkinson et al., Perg
ammon Press, 1987, Chapters 13.2 (pp 73-98), 21.1 (pp 813-898) and 21.3
(pp 915-957). The entirety of this document is specifically incorporated herein by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur and phosphorus are known in the art. For example, a suitable sigma carbon donor is available from Cotton and Wilkenson, Advanced
Found in Organic Chemistry, 5th Edition, John Wiley & Sons, 1988,
This document is incorporated herein by reference; see, for example, page 38. Similarly, suitable oxygen ligands include crown ethers, water, and others known in the art. Phosphines and substituted phosphines are also suitable; Cotton and Wilkenson
See page 38.

Oxygen, sulfur, phosphorus and nitrogen-donating ligands are attached in such a way that the heteroatom acts as a coordinating atom.

In a preferred embodiment, an organometallic ligand is used. Pure organic compounds used as redox moieties, and various transition metal coordination complexes with δ-linked organic ligands having donor atoms as heterocyclic or exocyclic substituents, as well as a variety of Transition metal organometallic compounds are available (Advanced
Inorganic Chemistry, 5th Ed., Cotton & Wilkinson, John Wiley & Sons, 19
88, Chapter 26; Organometallics, A Concise Introduction, Elschenbroich e
t al., 2nd Ed., 1992, VCH; and Comprehensive Organometallic Chemistry
II, A Review of the Literature 1982-1994, Abel et al. Ed., Vol. 7, Chapt
ers 7, 8, 10 & 11, Pergamon Press, specifically incorporated herein by reference). Such organometallic ligands, cyclopentadienide ion _{[C 5 H 5 (-1)} ] cyclic aromatic compounds and various ring substituted and ring fused derivatives, such as, for example, Indenirido (-1) and the like ions Produce a class of bis (cyclopentadienyl) metal compounds (ie, metallocenes); see, for example, Robins et al., J. Am.
Chem. Soc., 104: 1882-1893 (1982); and Gassman et al., J. Am. Chem. So
c., 108: 4228-4229 (1986); which are incorporated herein by reference. Of these, ferrocene [(C ₅ H ₅ ) ₂ Fe] and its derivatives have been incorporated herein by various chemicals (Connelly et al., Chem. Rev. 96: 877-910 (1996)). And electrochemical (Geiger et al., Advances in O
rganometallic Chemistry 23: 1-93; and Geiger et al., Advances in Organ
ometallic Chemistry 24:87, hereby incorporated by reference) are examples of prototypes used in electron transfer or "redox" reactions. A variety of metallocene derivatives of the first, second and third row transition metals are potential candidates as redox moieties covalently attached to either the ribose ring or the nucleoside base of nucleic acids. Other potentially suitable organometallic ligands, including cyclic allenes such as benzene, produce bis (arene) metal compounds and their ring-substituted and fused derivatives, but whose bis (benzene) chromium is a prototype It is an example. Other non-cyclic π-linked ligands such as the allyl (-1) ion or butadiene potentially produce suitable organometallic compounds, and all such ligands are free of other π
In conjunction with the-and δ-binding ligands, they constitute a general class of organometallic compounds with metal-carbon bonds. Electrochemical studies of various dimers and oligomers of such compounds, with cross-linked organic ligands and additional non-cross-linked ligands, as well as with and without metal-metal bonds, are potential candidate redox moieties in nucleic acid analysis. It is.

Where the one or more auxiliary ligands is an organometallic ligand, the ligand will generally be attached via one of the carbon atoms of the organometallic ligand, with the proviso that attachment is via another atom to the heterocyclic ligand. May be. Suitable organometallic ligands include metallocene ligands, including substituted derivatives and metallocene ophane (
See Cotton and Wilkenson, p. 1174, supra). For example, metallocene ligand derivatives such as methylcyclopentadienyl, preferably pentamethylcyclopentadienyl having a plurality of methyl groups, for example, can be used to increase the stability of the metallocene. In a preferred embodiment, only one of the two metallocene ligands of the metallocene is derivatized.

As described herein, any combination of ligands may be used. Preferred combinations are: a) all ligands are nitrogen donating ligands; b) all ligands are organometallic ligands; and c) the terminal ligand of the conductive oligomer is a metallocene ligand, and the ligand provided by the nucleic acid is nitrogen. It may be a dosing ligand, optionally with other ligands, including a nitrogen donating ligand or a metallocene ligand or a mixture thereof. These combinations are described in Representative Examples Using Conducting Oligomers of Structure 3, Structures 27 (using phenanthroline and amino as representative ligands), 28 (using ferrocene as a metal-ligand combination). ) And 29 (using cyclopentadienyl and amino as representative ligands). Structure 27

Embedded image Structure 28

Embedded image Structure 29

Embedded image

In a preferred embodiment, the ligands used in the present invention exhibit different fluorescent properties, depending on the redox state of the chelated metal ion. As described below, this therefore acts as another mode of detection of electron transfer between the ETM and the electrodes.

In addition, similar methods can be used to attach proteins to biosensor electrodes; see, eg, US Pat. No. 5,620,850, which is incorporated herein by reference.

In a preferred embodiment, as shown in more detail below, the ligand attached to the nucleic acid is an amino group attached at the 2 'or 3' position of the ribose in the ribose-phosphate backbone. The ligand may contain many amino groups to form a multidentate ligand that binds to the metal ion. Other preferred ligands include cyclopentadiene and phenanthroline.

The use of metal ions to bind nucleic acids serves as an internal control or measurement of the system, allowing the number of available nucleic acids on the surface to be determined. However, as will be apparent to those skilled in the art, when using a metal ion to attach a nucleic acid to an attachment linker, as described below, a redox potential that is different from the redox potential of the ETM used in the rest of the system is set to this metal ion. It is usually desirable that the complex has. This is commonly done so that the presence of the capture probe can be distinguished from the presence of the target sequence. This is useful for identification, measurement and / or quantification. Thus, by comparing the amount of capture probe at the electrode with the amount of hybridized double-stranded nucleic acid, the amount of target sequence in the sample can be quantified. This is very important to function as a sensor or internal control of the system. This allows for the measurement of the same molecule used for detection, either before or after adding the target, rather than relying on a similar but different control system. Thus, the actual molecule used for detection can be quantified prior to any experiment. This is a significant advantage over previous methods.

In a preferred embodiment, the capture probe nucleic acid (or other binding ligand) is covalently attached to the electrode via an insulator. It is well known that nucleic acids (and other binding ligands) attach to insulators, such as alkyl groups, and their attachment can be to a base, or to a backbone containing ribose or phosphate moieties, or to nucleic acid analogs. It can be implemented for an alternative backbone.

[0196] In a preferred embodiment, there may be one or more different capture probe species on the surface. In some embodiments, there may be one type of capture probe or one type of capture probe extender, as described in more detail below. Alternatively, different capture probes, or a single capture probe with a variety of different capture extension probes, can be used. Similarly, it is desirable to use an auxiliary capture probe that includes a relatively short probe sequence (particularly for nucleic acid analytes and binding ligands in a mechanism-2 system), which "takes away" components of the system, such as the recruitment linker. ETM on the surface used to "press"
The concentration can be increased.

Thus, the present invention is directed to a tissue collection device comprising a biosensor having a substrate comprising at least one detection electrode, comprising a monolayer and a capture binding ligand, useful for a system for detecting a target analyte of a collected sample. I will provide a.

In a preferred embodiment, the composition further comprises a solution or a soluble binding ligand, but as described in detail below for the Mechanism-1 system, the ETM is attached to a non-covalently attached hybridization indicator. It may be added in the form. Solution-bound ligands preferably resemble capture-bound ligands in that they bind specifically to a target analyte. The solution binding ligand may be the same or different from the capture binding ligand. In general, solution-bound ligands do not bind directly to the surface (although in some embodiments they do). The solution binding ligand has at least one ET
Either directly comprises a recruiting linker comprising M or the recruiting linker binds directly or indirectly to the solution binding ligand.

Thus, a “solution binding ligand” or “soluble binding ligand” or “signal carrier” or “labeled probe” or “labeled binding ligand” having a recruiting linker comprising a covalently attached ETM. Is provided. That is, some portion of the labeled probe or solution-bound ligand, directly or indirectly,
It includes a recruiting linker that includes an ETM that is bound to a target analyte and that has a covalently attached moiety. In some systems, such as the mechanism-1 nucleic acid system, they may be identical. Similarly, in mechanism-1, the recruitment linker comprises a nucleic acid that can hybridize to the detection probe. “
The terms "electron donor moiety", "electron acceptor moiety", and "ETM" (ETM) or grammatical equivalents refer to molecules capable of electron transfer under certain conditions. It should be understood that tolerance is relative; that is, a molecule that loses electrons under certain experimental conditions can accept electrons under different experimental conditions. It should also be understood that the number of moieties and electron acceptor moieties is very large, and that those skilled in the art of electron transfer compounds will be able to utilize many compounds in the present invention. ETMs include, but are not limited to, transition metal complexes, organic ETMs, and electrodes.

[0200] In a preferred embodiment, the ETM is a transition metal complex. Transition metals are those whose atoms have a partial or complete d-shell of electrons. Suitable transition metals for use in the present invention are listed above. The transition metal forms a complex with the various ligands L defined above, resulting in a suitable transition metal complex, as is well known in the art.

In addition to transition metal complexes, other organic electron donors and acceptors may be used in the present invention.
May be covalently attached to the nucleic acid. These organic molecules are
Bottle, xanthene dye, azine dye, acridine orange, N, N'-dimethyl
-2,7-diazapyrenium dichloride (DAP²⁺), Methyl viologen,
Ethidium bromide, quinones such as N, N'-dimethylanthra (2,1,9
-Def. 6,5,10-d'e'f ') diisoquinoline dichloride (ADIQ ²⁺ ); Porphyrin ([meso-tetrakis (N-methyl-x-pyridinium)
Luffyrin tetrachloride]), berramine blue B hydrochloride, bindshed
Bindschedler green; 2,6-dichloroindophenol, 2,6-
Dibromophenol indophenol; Brilliant Crest Blue (chloride 3
-Amino-9-dimethylamino-10-methylphenoxyazine), methylene butyl
Lou: Nile Blue A (Aminoaphthodiethylaminophenoxazine sulfate)
, Indigo-5,5 ', 7,7'-tetrasulfonic acid, indigo-5,5', 7-
Trisulfonic acid; phenosafuranine, indigo-5-monosulfonic acid; safrani
Bis (dimethylglyoximato) iron (II) chloride; indulin scare
, Neutral red, anthracene, coronene, pyrene, 9-phenyla
Anthracene, rubrene, binaphthyl, DPA, phenothiazine, fluoranthene
, Phenanthrene, chrysene, 1,8-diphenyl-1,3,5,7-octate
Toracene, naphthalene, acenaphthalene, perylene, TMPD and their compounds
Analogs and substituted derivatives, but are not limited to.

In one embodiment, the electron donor and acceptor are redox proteins known in the art. However, redox proteins are not preferred in many embodiments.

The selection of a specific ETM is influenced by the type of electron transfer detection used, as generally outlined below. Preferred ETMs are metallocenes, particularly preferred are ferrocene and its derivatives.

[0204] In a preferred embodiment, multiple ETMs are used. The use of various ETMs leads to signal amplification, which allows for more sensitive detection limits. The use of multiple ETM on nucleic acids that hybridize to complementary strands, the number, but to cause a decrease in T _m s of depending on the spatial relationship between the site of attachment and the plurality ETM hybridization complex, whereas it is The ETM is on the recruiting linker (ie,
In the case of "Mechanism-2"), it is no longer a factor because it does not hybridize to the complementary sequence. Thus, multiple ETMs are preferred, at least about 2 ETMs per recruiting linker are preferred, and at least 10 are particularly preferred, and at least about 20-50 are particularly preferred. In some cases, a very large number of ETMs (50-1000) are available.

[0205] Thus, a solution-bound ligand or labeled probe having a covalently attached ETM is provided. The method of attaching the ETM to the solution-bound ligand is based on the detection mode (
That is, it changes depending on the mechanism-1 or 2) and the composition of the solution-bound ligand. As described in more detail below, in the mechanism-2 system, the portion of the solution-bound ligand (or labeled probe) comprising the ETM is called the "recruitment linker" and may be composed of nucleic acids or non-nucleic acids. . For the mechanism-1 system, the recruitment linker must be a nucleic acid.

Thus, as the skilled artisan will appreciate, there may be various configurations that can be used. In a preferred embodiment, the recruitment linker is a nucleic acid (including analogs) and ET
The attachment of M can be accomplished by (1) a base; (2) the corresponding structure of a backbone, eg, a ribose, phosphate ester, or nucleic acid analog; (3) the following nucleoside substitution; or (4) A metallocene polymer as described below; In a preferred embodiment, the recruitment linker is non-nucleic acid and may be a metallocene polymer or an alkyl-type polymer containing ETM substituents (including heteroalkyl, as described in more detail below). These options are depicted generally in FIG.

[0207] In a preferred embodiment, the recruitment linker is a nucleic acid, comprising a covalently attached ETM. The ETM may be attached to the nucleoside in the nucleic acid at various positions. Preferred embodiments include (1) attachment to nucleoside bases, (2) attachment of ETMs as base substitutes, (3) analogous structures to the ribose or phosphate moieties of the ribose-phosphate backbone, or nucleic acid analogs. And (4) attachment via a metallocene polymer, but are not limited thereto.

Further, when the recruiting linker is a nucleic acid, as described below, it is desirable to use a second label probe, which probe hybridizes to a portion of the first label probe as defined herein. And a second portion comprising a recruitment linker. This is similar to the use of an amplifier probe, except where both the first and second label probes comprise an ETM.

In a preferred embodiment, for the attachment of the conductive oligomer, it is attached to the base of the nucleoside as generally outlined above. Attachment is to an internal or terminal nucleoside.

[0210] Covalent attachment to a base depends on the moiety on the ETM chosen, but generally resembles attaching a conductive oligomer to a base, as described above.
Attachment may generally be at any site on the base. In a preferred embodiment, E
TM is a transition metal complex, and thus attachment of the appropriate metal ligand to the base leads to covalent attachment of the ETM. Alternatively, as the skilled artisan will appreciate, a similar type of linkage may be used for attaching the organic ETM.

In one embodiment, 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.

A ligand containing an aromatic group can be attached via an acetylene bond as is known in the art (Comprehensive Organic Synthesis, Trost et al., Ed., Perg.
amon Press, Chapter 2.4; Coupling Reactions Between sp ² and sp Carbon Ce
nters, Sonogashira, pp 521-549, and pp 950-953; incorporated herein by reference). Structure 30 illustrates a representative structure in the presence of metal ions and other necessary ligands; although structure 30 illustrates uridine, other bases can be used throughout this specification. is there.

[0213]

Embedded image La is _a ligand and includes organometallic ligands such as nitrogen, oxygen, sulfur or phosphorus donating ligands or metallocene ligands. Suitable ligand L _a is phenanthroline, imidazole, etc. bpy and terpy, but not limited thereto. L _r and M are as defined above. Again, as the skilled artisan will appreciate, a linker ("Z") is included between the nucleoside and the ETM.

Similarly, as an attachment linker, the bond is made using a linker, but the linker can utilize an amide bond (generally Telser et al., J. Am.
Chem. Soc. 111: 7221-7226 (1989); Telser et al., J. Am. Chem. Soc. 111.
: 7226-7232 (1989); both references are specifically incorporated herein by reference). These structures are illustrated generally in Structure 31 below. Again, uridine is used here as a base, but other bases can be used, as described above.

Embedded image

In this embodiment, L is a ligand as defined above, and Lr and M are also as defined above. Preferably, L is amino, phen, byp and terpy
It is.

In a preferred embodiment, the ETM attached to the nucleoside is a metallocene; that is, L and Lr of Structure 31 are both metallocene ligands, and are L _{m '} as described above. Structure 32 illustrates a preferred embodiment, where the metallocene is ferrocene and the base is uridine, although other bases can be used.

Embedded image

Preliminary data suggests that Structure 32 is cyclizable, with the secondary acetylene carbon atom attacking the carbonyl oxygen to form a furan-like structure. Suitable metallocenes include ferrocene, cobaltene and osmium oxene.

In a preferred embodiment, the ETM is attached to ribose at any position on the ribose-phosphate backbone of the nucleic acid, ie, at the 5 ′ or 3 ′ end or at any internal nucleoside. Ribose in this case includes a ribose analog. As is known in the art, nucleosides modified at either the 2 'or 3' position of ribose may be made possible by nitrogen, oxygen, sulfur and phosphorus-containing modifications. Amino-modified and oxygen-modified ribose are preferred. Generally, PC
See T Publication WO 95/15971, which is incorporated herein by reference. These modifying groups can be used as transition metal ligands or as chemically functional moieties for attachment of other transition metal and organometallic ligands, or as organic electron donor moieties recognized by those skilled in the art. In this aspect,
Linkers as illustrated herein as "Z" can be used as well, or a conductive oligomer between ribose and ETM. Preferred embodiments utilize attachment at the 2 'or 3' position of ribose, with the 2 'position being preferred. Thus, for example, the conductive oligomer illustrated in Structures 13, 14 and 15 can be replaced by an ETM; alternatively, the ETM may be added to the free end of the conductive oligomer.

[0219] In a preferred embodiment, the metallocene acts as an ETM and is attached via an amide bond as illustrated in Structure 33 below. The illustration outlines the synthesis of suitable compounds when the metallocene is ferrocene.

Embedded image

In a preferred embodiment, an amine linkage is used as shown generally in Structure 34.

Embedded image Z is a linker as defined herein, preferably having 1 to 16 atoms,
Two to four atoms are particularly preferred. t is 1 or 0.

In a preferred embodiment, an oxo bond is used as shown generally in Structure 35.

Embedded image In structure 35, Z is a linker as defined herein and t is 1 or 0. Suitable Z linkers include alkyl groups, including heteroalkyl groups such as (CH ₂ ) _n and (CH ₂ CH ₂ O) _n , where n is preferably 1 to 10,
n = 1 to 4 are particularly preferred, and n = 4 is particularly preferred.

[0222] Bonds utilizing other heteroatoms are also possible.

In a preferred embodiment, the ETM is attached to the phosphate at any position on the ribose-phosphate backbone of the nucleic acid. This can be done in various ways. In one embodiment, a phosphodiester bond analog, such as a phosphoramide or phosphoramidite bond, may be incorporated into the nucleic acid, where the heteroatom (ie, nitrogen) acts as a transition metal ligand (PCT Publication WO95 / 15971
See; incorporated herein by reference). Alternatively, the conductive oligomer illustrated in structures 23 and 24 may be replaced by ETM. In a preferred embodiment, the composition has the structure shown in Structure 36.

Embedded image

In structure 36, the ETM is attached via a phosphate ester bond, generally by using a linker Z. Suitable Z linkers are (CH ₂ ) _n , (CH ₂
CH ₂ O) _n , including an alkyl group including a heteroalkyl group, wherein n is 1 to 1
0 is preferred, n = 1 to 4 is particularly preferred, and n = 4 is particularly preferred.

For the Mechanism-2 system, if the ETM is attached to the base or backbone of the nucleoside, it is possible to attach the ETM via a “dendritic” structure, as outlined in more detail below. Using an alkyl-based linker, a number of branched structures comprising one or more ETMs at the end of each branch can be created. Generally, this is accomplished by creating a branch point that contains a large number of hydroxy groups, which can be used to add additional branch points. The terminal hydroxy group is then used for the phosphoramidite reaction, generally as described below for nucleoside substitution and metallocene polymer reactions, as described below.
M can be added.

In a preferred embodiment, an ETM such as a metallocene is used as a “nucleoside substitution” to operate as an ETM. For example, the distance between two cyclopentadiene rings of ferrocene is similar to the orthogonal distance between two bases in a double-stranded nucleic acid. In addition to ferrocene, other metallocenes can be used, for example, air-stable metallocenes such as those containing cobalt or ruthenium.
Thus, the metallocene moiety may be incorporated into the nucleic acid backbone, as generally illustrated in structure 37 (nucleic acid with a ribose-phosphate backbone) and structure 38 (peptide nucleic acid backbone). While structures 37 and 38 illustrate ferrocene, other metallocenes may be used as well, as those skilled in the art will recognize. Generally, air stable metallocenes are preferred, including metallocenes utilizing ruthenium and cobalt as the metal.

[0227]

Embedded image In Structure 37, Z is a linker as defined above, generally having a short alkyl group and preferably containing a heteroatom such as oxygen. In general, what is important is the length of the linker, as described in more detail below, so as to provide minimal perturbation of the double-stranded nucleic acid. Thus, methylene, ethylene,
Ethylene glycol, propylene and butylene are all suitable, with ethylene and ethylene glycol being particularly preferred. Further, each Z linker may be the same or different. Although structure 37 represents a ribose-phosphate backbone, as those skilled in the art will recognize, nucleic acid analogs such as ribose analogs and phosphate ester bond analogs may be used.

[0228]

Embedded image In Structure 38, suitable Z groups are as described above, but again, each Z linker may be the same or different. As mentioned above, other nucleic acid analogs may be used as well.

Further, while the above structures and discussions depict metallocenes, especially ferrocenes, using the same general idea, as described below, as a substituent of a nucleoside in addition to a metallocene, or in a polymer embodiment, ETM can be added.
Thus, for example, in the case of a transition metal complex other than a metallocene comprising one, two or three (or more) ligands, the ligand is functionalized as described for ferrocene and the phosphoramidite group Additions can be made possible. In particular, preferred in this embodiment are at least two rings (eg,
Aryl and substituted aryl) ligands, wherein:
Each ligand comprises a functional group for attachment by phosphoramidite chemistry. As the skilled artisan will appreciate, this type of reaction creates a polymer of the ETM as part of the nucleic acid backbone or as a "side group" of the nucleic acid to allow amplification of the signal generated here, It can be performed with virtually any ETM that can be functionalized to include the correct chemical groups.

Thus, nucleic acid analogs are prepared by inserting a metallocene, such as ferrocene (or other ETM), into a nucleic acid backbone; that is, the present invention provides a backbone comprising at least one metallocene. A nucleic acid having the formula:
This is distinguished from metallocene-bearing nucleic acids by attachment to the backbone, ie, via ribose, phosphate esters, and the like. That is, two nucleic acids made from a traditional nucleic acid or analog, in which case the nucleic acid comprises a single nucleoside, can each be covalently attached to each other via a metallocene. In a different aspect, there is provided a metallocene derivative or substituted metallocene, wherein each of the two aromatic rings of the metallocene has a nucleic acid substituent.

Further, as described in more detail below, it is possible to incorporate more than one metallocene into the backbone with nucleotides in between and / or with adjacent metallocenes. When an adjacent metallocene is added to the backbone, this is the same as the process described below as a "metallocene polymer"; that is, there are regions of the metallocene polymer within the backbone.

In addition to nucleic acid substituents, in some cases, it may be desirable to add additional substituents to one or both of the aromatic rings of the metallocene (or ETM). For example, these nucleoside substitutions are generally substantially complementary nucleic acids, e.g., those portions of the probe sequence to be hybridized to the target sequence or another probe sequence, so that a substituent can be added to the metallocene ring to form the opposite strand. It is possible to facilitate hydrogen bond formation with one or more bases above. These may be added at any position on the metallocene ring. Suitable substituents include, but are not limited to, amide groups, amine groups, carboxylic acids, and alcohols, including substituted alcohols.
In addition, these substituents can be attached via a linker as well,
Generally not preferred.

Further, a substituent may be added to the ETM, particularly a metallocene such as ferrocene to form an ET.
The redox properties of M may be changed. Thus, for example, in certain embodiments, as described in more detail below, different attached differently (ie, base or ribose attached), on different probes, or for different purposes (eg, as a calibration or internal reference). It is desirable to have an ETM. Thus, adding a substituent on the metallocene allows the distinction of two different ETMs.

An intermediate component is also provided to generate these metallocene-backbone nucleic acid analogs. Thus, in a preferred embodiment, the present invention provides a phosphoramidite metallocene, as generally depicted in Structure 39.

Embedded image

In Structure 39, PG is a protecting group, which is generally a group suitable for nucleic acid synthesis, with DMT, MMT, TMT, and the like all preferred. The aromatic ring can be a metallocene ring or an aromatic ring of a transition metal complex or other ligand for an organic ETM. The aromatic rings can be the same or different and can be substituted as discussed herein.

Structure 40 illustrates a ferrocene derivative:

Embedded image

[0237] These phosphoramidite analogs can be added to standard oligonucleotide synthesis known in the art.

Structure 41 illustrates a ferrocene peptide nucleic acid (PNA) monomer and can be added to the synthesis of PNAs known in the art.

Embedded image In structure 41, the PG protecting group is suitable for use in peptide nucleic acid synthesis, with MMT, boc, Fmoc and the like being preferred.

[0239] These same intermediate compounds can be used to form ETM or metallocene polymers, which are added to nucleic acids rather than as backbone substitutes, as described in more detail below.

In a preferred embodiment, especially for the use of a mechanism-2 system,
TM is described herein as a polymer, for example, as a metallocene polymer,
As described in S Patent No. 5,124,246, modified functionalized nucleotides are attached in a "branched" configuration similar to the "branched DNA" embodiment. The general idea is as follows. A modified phosphoramidite nucleotide is produced, which can ultimately contain a free hydroxy group that can be used for attachment of a phosphoramidite ETM such as a metallocene. The free hydroxy group can be on a base or backbone, such as ribose or phosphate esters (although those skilled in the art will recognize, nucleic acid analogs containing other structures can also be used). The modified nucleotide is incorporated into the nucleic acid and the hydroxy protecting group is removed, resulting in free hydroxy. As described above in structures 39 and 40, based on the addition of a phosphoramidite ETM such as a metallocene, an ET such as a metallocene ETM
M is added. Additional phosphoramidite ETMs, such as metallocenes, were added and included "ETMs" including "metallocene polymers" as depicted for ferrocene in FIG.
In addition, in some embodiments, increasing the solubility of the polymer by adding a "capping" group to a terminal ETM in the polymer, for example, adding a final phosphate group to the metallocene. It will be appreciated by those skilled in the art that other suitable solubility-enhancing "capping" groups will be appreciated. Can be added to the polymer at other locations.

In a preferred embodiment, the ribose 2 ′ position of the phosphoramidite nucleotide is first functionalized to include a hydroxy group protected in this case via an oxo bond, but with respect to the number of linkers, Any number may be used as generally described herein for the linker. The protected modified nucleotide is then incorporated into the growing nucleic acid by standard phosphoramidite chemistry. The protecting group was removed, the free hydroxy group was used, again using standard phosphoramidite chemistry,
Add a phosphoramidite metallocene such as ferrocene. Similar reactions are possible with nucleic acid analogs. For example, a peptide nucleic acid structure containing a metallocene polymer could be generated using the peptide nucleic acid shown in Structure 41 and a metallocene monomer.

Thus, the present invention provides recruitment linkers for nucleic acids that include “branches” of a metallocene polymer. In a preferred embodiment, the length of metallocene is from 1 to about 50,
Preferably from about 5 to about 20, particularly preferably from about 5 to about 10, metallocene polymers are utilized.

Further, when the recruitment linker is a nucleic acid, the ETM is attached in any combination. In general, when using the mechanism-1 system, as outlined herein, clusters of nucleosides containing ETMs can reduce the Tm of hybridization of a probe to a target sequence; In general, in the mechanism-1 system, the ETMs are intervened over the entire length of the sequence, or a small number of them are used.

In Mechanism-1, non-covalently attached ETMs can be used. In some embodiments, the ETM is a hybridization indicator. The hybridization indicator acts as an ETM (which, in addition, selectively and usually reversibly associates with double-stranded nucleic acid), which is described in Millan et al., Anal. Chem. 65: 2.
317-2323 (1993); similar to Millan et al., Anal. Chem. 662943-2948 (1994), both of which are expressly incorporated by reference. In this embodiment, an increase in the local concentration of ETM (due to the association of the ETM hybridization indicator with the duplex nucleic acid at the surface) can be monitored using a monolayer containing conductive oligomers. Hybridization indicators include intercalators and minor and / or major groove binding molecules. In a preferred embodiment, an intercalator may be used; since intercalation generally occurs only in the presence of double-stranded nucleic acid, the ETM is only enriched in the presence of double-stranded nucleic acid. Intercalation of the transition metal complex ETM is known to those skilled in the art. Similarly, major or minor groove binding molecules such as methylene blue may be used in this implementation.

In addition, the biosensors of the present invention may be used substantially in any method that relies on electrochemical detection of a target analyte, and are particularly useful for nucleic acid detection. For example,
The methods and compositions of the present invention can be used in nucleic acid detection methods that rely on the detection of an ETM specific to the target analyte. For example, Napier et al., Bioconj. Chem. 8:
As outlined in 906 (1997) (explicitly incorporated herein by reference), the guanine base of a nucleic acid is converted by a change in the redox state (i.e., guanine oxidation by a ruthenium complex). Can be detected. Similarly, the method of the present invention comprises:
Use in a detection system for oxidizing ribose of nucleic acids using a copper surface as a catalyst electrode is also useful.

In a preferred embodiment, the recruitment linker is not a nucleic acid, but may instead be any type of linker or polymer. As the skilled artisan will appreciate, generally any linker or polymer that may be modified and include ETM can be used. In general, these polymers or linkers should be reasonably soluble and contain functional groups suitable for ETM attachment.

In a preferred embodiment, the recruitment linker is not a nucleic acid, but instead may be any type of linker or polymer. As those skilled in the art will recognize, ETM
General linkers or polymers that can be modified to include Generally, the polymer or linker should be reasonably soluble and should contain the appropriate functional groups for ETM addition.

As used herein, a “recruiting polymer” is at least 2 or 3
Subunits and are covalently attached. At least some of the monomer subunits contain functional groups for covalent attachment of ETMs. In some embodiments, the coupling moiety is used to covalently link the subunit and the ETM. Suitable functional groups for attachment are amino, carboxy,
Amino groups are particularly preferred, such as oxo and thiol groups. As the skilled artisan will appreciate, a variety of recruiting polymers are possible.

Suitable linkers include, but are not limited to, alkyl linkers (including heteroalkyl (including (poly) ethylene glycol type structures), substituted alkyl, arylalkyl linkers). For polymers, as described above, the linker comprises one or more functional groups for ETM attachment, and attachment is accomplished using art-recognized, e.g., well-known homo- or hetero-bifunctional linkers. (See Pierce Chemical Company's catalog, 1994, section on techniques for crosslinkers, pages 155 to 200; incorporated herein by reference).

Suitable recruiting polymers include, but are not limited to, functionalized styrenes, such as aminostyrene, functionalized dextran, and polyamino acids. Suitable polymers include polyamino acids such as polylysine (both poly-D-amino acids and poly-L-amino acids), and polymers containing lysine and other amino acids that are particularly preferred. As outlined above, in some embodiments, a charged recruitment linker is preferred (eg, when detecting an uncharged target analyte). Other suitable polyamino acids include polyglutamic acid, polyaspartic acid, copolymers of lysine with glutamic acid or aspartic acid, copolymers of lysine with alanine, tyrosine, phenylalanine, serine, tryptophan, and / or proline. is there.

In a preferred embodiment, the recruitment linker comprises a metallocene polymer as described above.

The attachment of the recruitment linker to the first portion of the label probe (ie, the moiety that binds either directly or indirectly to the target analyte) depends on the composition of the recruitment linker, as will be appreciated by those skilled in the art. . When the recruitment linker is a nucleic acid, it is generally formed upon synthesis of the first part of the labeled probe, with the incorporation of the required ETM-containing nucleoside. Alternatively, the first portion of the label probe and the recruitment linker may be made separately and then attached. For example, there may be overlapping overlapping sections, for example, using psoralens known in the art, to form sections of double-stranded nucleic acid that can be chemically crosslinked.

When a non-nucleic acid recruiting linker is used, the linker /
Polymer deposition is generally performed using standard chemical techniques as will be recognized by those skilled in the art. For example, when using an alkyl-based linker, attachment is similar to insulator attachment to nucleic acids.

In addition, the recruitment linker, which is a mixture of nucleic acids and non-nucleic acids, may be in a linear form (ie, where the nucleic acid segments are joined together with an alkyl linker) or a branched form (including ETMs and further branched). ("Nucleic acids with alkyl" branches ").

In a preferred embodiment, for example, where the target analyte is a nucleic acid, it is the target sequence itself that carries the ETM rather than the recruiting linker of the labeled probe. For example, as described in more detail below, nucleotide triphosphates containing each ETM of the present invention can be enzymatically added to growing nucleic acids, for example, during the polymerase chain reaction (PCR). As the skilled artisan will appreciate, some enzymes have been shown to be generally tolerant to modified nucleotides, but some of the modified nucleotides of the invention, for example, "nucleoside substitutions" And possibly some phosphate ester attachment may or may not be recognized by enzymes that allow incorporation into growing nucleic acids. Thus, the preferred attachment in this embodiment is to a nucleotide base or ribose.

Thus, for example, as is well known to those of skill in the art, PCR amplification of a target sequence will result in a target sequence comprising an ETM that is generally randomly incorporated into the sequence. The system of the present invention can be configured to be detectable using these ETMs.

Alternatively, as described in more detail below, a nucleotide comprising the ETM can be enzymatically added to the end of the nucleic acid, eg, to the target nucleic acid. In this embodiment, an effective "recruiting linker" can be added to the end of the target sequence and used for detection. Thus, the present invention provides a composition utilizing an electrode comprising a monolayer having a capture probe, and a first portion capable of hybridizing to a component of an assay complex, and a hybridizable component of the assay complex. But not a second moiety comprising at least one covalently attached electron transfer moiety. Also provided are methods of utilizing these compositions.

Linking the ETM to a probe sequence, ie, a sequence designed to hybridize to a complementary sequence (ie, a mechanism-1 sequence, but which can also be used in a mechanism-2 system) Is also possible. Thus, ETMs can be added to non-recruiting linkers as well. For example, a label probe fragment that hybridizes to a component of the assay complex, for example,
An ETM may be present in the first part or in addition to the target sequence described above.
These ETMs may be used in some embodiments for electronic movement detection, or cannot be used depending on their location and system. For example, in some embodiments, for example, where a target sequence containing a randomly incorporated ETM hybridizes directly to a capture probe, the ETM is present in the portion that hybridizes to the capture probe. If a capture probe attaches to an electrode using a conductive oligomer, these ETMs can be used to detect electron transfer as previously described. Alternatively, these ETMs may not be specifically detectable.

Similarly, in some embodiments, when the recruitment linker is a nucleic acid, it may be desirable in some instances (eg, the mechanism-2 system) that some or all of the recruitment linker is double-stranded. In one embodiment, there may be a second recruitment linker, which is substantially complementary to the first recruitment linker and capable of hybridizing to the first recruitment linker. In a preferred embodiment,
The first recruitment linker comprises an ETM attached by a covalent bond. In another embodiment, the second recruitment linker comprises an ETM and no first recruitment linker, and the ETM is collected on its surface by the second recruitment linker hybridizing to the first. In yet another embodiment, both the first and second recruitment linkers comprise an ETM. It should be noted that, as noted above, nucleic acids comprising a large number of ETMs do not hybridize as well, ie, the _Tm may be reduced depending on the attachment site and properties of the ETM. Thus, in general, when multiple ETMs are used for strand hybridization,
That is, in a mechanism-1 system, it is generally less than about 5, preferably less than about 3. Alternatively, the ETM should have a sufficient spatial distance so that the intervening nucleotides hybridize sufficiently to allow good reaction rates.

For nucleic acid systems, the probes of the present invention are designed to be complementary to a target sequence (to a target sequence or other probe sequence in a sample, as described below);
The hybridization between the target sequence and the probe of the present invention takes place. As outlined below, this complementarity need not be perfect; there may be a significant number of base pair mismatches that would inhibit hybridization between the target sequence and the single stranded nucleic acid of the invention. However, if the number of mutations is so large that no hybridization occurs under minimal stringent hybridization conditions, the sequence is not a complementary target sequence. Thus, "substantially complementary" as used herein means that the probe is sufficiently complementary to the target sequence to hybridize under normal reaction conditions.

Generally, the nucleic acid compositions of the present invention are useful as oligonucleotide probes. As will be appreciated by those skilled in the art, the length of the probe will vary with the length of the target sequence and the hybridization and washing conditions. Generally, oligonucleotide probes range from about 8 to about 50 nucleotides, preferably from about 10 to about 3 nucleotides.
0 and particularly preferred is about 12 to about 25. In some cases, very long probes may be used, for example, 50 to 200 to 300 nucleotides in length. Thus, in the structures depicted herein, nucleosides can replace nucleic acids.

A wide variety of hybridization conditions can be used in the present invention, including high, moderate and low stringency conditions; for example, Maniatis et al., Molecular C
loning: A Laboratory Manual, 2nd Edition, 1989, and Short Protocols in M
See olecular Biology, ed. Ausubel, et al. (herein incorporated by reference). Stringency conditions are sequence-dependent and will be different in different circumstances. The longer the sequence, the higher the temperature and specifically hybridized. A detailed description of nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Mo
lecular Biology--Hybridization with Nucleic Acid Probes, "Overview of p
rinciples of hybridization and the strategy of nucleic acid assays "(1993
)It is in. Generally, stringent conditions are selected to be about 5-10 ° C. below the thermal melting point (Tm) of the specific sequence at a defined ionic strength pH. Tm (at a defined ionic strength, pH and nucleic acid concentration) is the temperature at which 50% of the probes complementary to the target will hybridize to the target sequence at equilibrium (at Tm, the target sequence is present in excess, Occupy equilibrium). Stringent conditions are pH 7.0-8.0.
3, the salt concentration is about 1.0 sodium ion or less, typically about 0.01 to 1.0 M sodium ion concentration (or other salt), and the temperature is for short probes (eg, 10 to 50 nucleotides) at least about 30 ° C.
For long probes (eg, 50 nucleotides or more), the conditions should be at least about 60 ° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

In another embodiment, less stringent hybridization conditions are used;
For example, mild or low stringency conditions known to those skilled in the art may be used; see Maniatis and Ausubel, supra, and Tijssen, supra.

Hybridization conditions can also be varied using a non-ionic backbone, ie, PNA, as is known in the art. In addition, a cross-linking agent may be added to cross-link, ie, covalently attach, the double-stranded hybridization complex following target binding.

As will be apparent to those skilled in the art, the system of the present invention may be mounted on a number of different shapes, some of which are depicted in FIGS. In general, there are three types of systems that can be used: (1) systems in which the target sequence itself is labeled with an ETM (see FIG. 6A; generally useful for nucleic acid systems); A system that binds to the sequence (see FIG. 6C); and (3) a system where the labeled probe binds indirectly to the target sequence, for example, through the use of an amplification probe (
See FIG. 6E).

In all three systems, it is preferred, but not required, to immobilize the target sequence on the electrode surface. This is preferably performed using a capture binding ligand and, optionally, one or more capture extension probes; see FIG. 3 for a representative example of a nucleic acid. If only capture probes are used, it is necessary to have a unique capture probe for each target sequence; that is, the surface must be designed to contain the unique capture probe. Alternatively, a capture extension probe can be used, which is a "universal" surface, ie,
Enables a surface containing a single type of capture probe that can be used to detect all target sequences. A "capture extension" probe is generally depicted, such as in FIG. 6C, having a first portion that hybridizes to all or a portion of the capture probe and a second portion that hybridizes to a portion of the target sequence. This then allows for the production of designed designed soluble probes, but, as the skilled artisan will appreciate, is generally simpler and less costly. As shown herein, two capture extension probes may be used. This is generally done to stabilize the assay complex (eg, if the target sequence is large, or if a large amplification probe is used, especially a branched or dendritic amplification probe).

If the capture binding ligand is not a nucleic acid, a capture extension component may still be used. In one embodiment, as depicted in FIG. 2C, the capture binding ligand has an associated capture extender of nucleic acid (as will be appreciated by those skilled in the art, but may also be part of a binding pair) and the electrode surface Can be used to target Alternatively, an additional capture extension component is used to enable a “generic” surface (see FIG. 1).

Although the discussion and figures herein generally depict nucleic acid embodiments,
The same concept can be used for non-nucleic acid target analytes. For example, capture extension ligands may be prepared as needed by those skilled in the art. For example, a nucleic acid "tail" may be added to the binding ligand.

In a preferred embodiment, the binding ligand is added after the formation of the SAMs discussed below ((4) above). This can be implemented in various ways, as will be appreciated by those skilled in the art. In one embodiment, the conductive oligomer having terminal functional groups is prepared by a preferred embodiment utilizing an activated carboxylic acid ester and an isothiocyanate ester, which comprises an activated carboxylic acid ester. Or with a primary amine attached to a binding ligand such as a nucleic acid. These two reagents have the advantage of being stable in aqueous solution and further react with primary alkylamines. However, of the bases, the primary aromatic amines and the secondary and tertiary amines must not react, and thus must allow for the site-specific addition of nucleic acids to the surface. Similar techniques can be used for non-nucleic acid components; for example, SAMs containing metal chelates of proteins, as outlined above.
Attachment to is known; see US Patent No. 5,620,850. This allows spotting of probes (either capture probes or detection probes, or both) on the surface by known methods (inkjet, spotting, etc.).

In addition, there are many non-nucleic acid methods that can be used to immobilize nucleic acids on a surface.
For example, a pair of binding partners may be utilized; one binding partner is attached to the end of the conductive oligomer and the other is attached to the end of the nucleic acid. This can also be performed without the use of a nucleic acid capture probe; that is, one binding partner acts as a capture probe and the other attaches to either the target sequence or the capture extension probe. That is, either the target sequence contains a binding partner or the capture extension probe that hybridizes to the target sequence contains a binding partner. Suitable binding partner pairs include, but are not limited to, hapten pairs such as biotin / streptavidin, antigens / antibodies, NTA / histidine tags, and the like. In general, smaller binding partners are preferred, so that the electrons move from the nucleic acid into the conductive oligomer, allowing detection.

In a preferred embodiment, when the target sequence itself is modified to include a binding partner, the binding partner is linked via a modified nucleotide that can be enzymatically attached to the target sequence.
For example, it attaches during the PCR target amplification step. Alternatively, the binding partner readily attaches to the target sequence.

Alternatively, a capture extension probe having a nucleic acid moiety for hybridizing to a target and a binding partner may be utilized (eg, the capture extension probe may be a non-binding linker such as an alkyl linker used to attach to the binding partner). A nucleic acid moiety). In this embodiment, it is desirable to crosslink the target double-stranded nucleic acid and the capture extension probe using, for example, a psoralen known in the art for stability.

In a preferred embodiment, the target sequence itself comprises an ETM. As discussed above, this can be done with a target sequence having an ETM incorporated at a significant number of positions, as outlined above. In this embodiment, for the rest of the system, the 3′-5 ′ orientation of the probe and target is such that the ETM-containing structure (ie, the recruitment linker or target sequence) is as close as possible to the surface of the monolayer, and , Are selected to have the correct orientation. This can be accomplished by deposition via an insulator or a conductive oligomer, as shown generally in the figures. Further, as one of skill in the art will appreciate, the multiple capture probes were in the shape as depicted in FIG. 5D, where the 5′-3 ′ orientation of the capture probes was different, or used multiple capture probes. It can be used where the target "loop" creates.

In a preferred embodiment, the labeled probe hybridizes directly to the target sequence, as depicted generally in the figure. In these embodiments, the target sequence is preferably immobilized on the surface by a capture probe, including a capture extension probe. Next, an ETM is brought near the surface of the monolayer using a labeled probe. In a preferred embodiment, multiple labeled probes are used; that is, the labeled probes are designed such that the portion that hybridizes to the target sequence can be different for a number of different labeled probes;
The result is amplification of the signal because multiple labeled probes can bind to each labeled sequence. Thus, as depicted in the figure, n is an integer of at least one. Depending on the desired sensitivity, the length of the target sequence and the number of ETMs per labeled probe, a suitable range for n is from 1 to 50, particularly preferably from about 1 to about 2
0, and especially preferred is about 2 to about 5. In addition, if "generic" labeled probes are desired, labeled extension probes can be used, as described generally below for the use of amplification probes.

As described above, in this embodiment, generally, the shape of the system and the arrangement of the labeled probes are designed so that the ETMs gather as close as possible to the monolayer surface. In a preferred embodiment, the labeled probe hybridizes indirectly to the target sequence. That is, the present invention finds use in a novel combination of signal amplification techniques and electron transfer detection on electrodes, which, as generally depicted in the figures (see below) for nucleic acid embodiments, Particularly useful for sandwich hybridization assays; similar systems can be developed for non-nucleic acid target analytes. In these embodiments, the amplification probes of the invention bind directly or indirectly to a target sequence in a sample. Since the amplification probe preferably contains a relatively large number of amplification sequences available for binding of the labeled probe, the detectable signal can be significantly increased and the detection limit of the target can be significantly improved. These labels and amplification probes, and the detection methods described herein can be used in any manner by essentially known nucleic acid hybridization, for example, by directly binding the target to a solid phase, or by using one target. It can be used in sandwich hybridization assays that bind to the above nucleic acids, which in turn bind to a solid phase.

In general, these embodiments may be described as follows, with particular reference to nucleic acids. Amplification probes hybridize directly to the target sequence or through the use of labeled extension probes, which allows for the preparation of "generic" amplification probes. The target sequence is
Preferably, it is immobilized on the electrode using a capture probe, but this is not necessary. Preferably, the amplification probe comprises a variety of amplification sequences, but in some embodiments,
As described below, the amplification probe contains only one amplification sequence. Amplification probes can take many different forms; for example, branched conformations, dendritic conformations,
Or a linear "string" of the amplified sequence. Using these amplified sequences, a hybridization complex with a labeled probe can be formed, and ETM can be detected using an electrode.

Accordingly, the present invention provides an assay complex consisting of at least one amplification probe. As used herein, "amplification probe" or "nucleic acid multimer" or "
By "amplification multimer" or grammatical equivalent is meant a nucleic acid probe used to facilitate signal amplification. The amplification probe may have at least one as defined below.
It contains one single-stranded nucleic acid probe sequence and at least one single-stranded nucleic acid amplification sequence, preferably a variety of amplification sequences.

An amplification probe contains a first probe sequence that is used, directly or indirectly, to hybridize to a labeled sequence. That is, the amplification probe itself may have a first probe sequence that is substantially complementary to a target sequence, or it may have a first probe sequence that is substantially complementary to a portion of an additional probe, and The additional probe, in this case called a labeled extension probe, has a first portion that is substantially complementary to the target sequence. In a preferred embodiment, the first probe sequence of the amplification probe is substantially complementary to the target sequence.

In general, for all probes herein, the first probe sequence is of sufficient length to provide specificity and stability. Thus, in general, a probe sequence of the invention designed to hybridize to another nucleic acid (ie, a probe sequence, an amplification sequence, a portion or domain of a larger probe) will have a length of at least about 5 nucleosides. , Preferably at least about 10, and particularly preferably at least about 15.

In a preferred embodiment, either the amplification probe or other probes of the invention may form a hairpin base-loop structure in the absence of their target. The length of the base duplex sequence is chosen such that the hairpin structure is not advantageous in the presence of the target. The use of these types of probes in the system of the present invention, or in any nucleic acid detection system, significantly reduces non-specific binding, resulting in an increased signal-to-noise ratio.

In general, these hairpin structures contain four components. The first component is a target binding sequence, ie, a region complementary to the target (which may be a sample target sequence or other probe sequence for which binding is desired) and comprises about 10 nucleotides , Preferably about 15. The second component is a loop sequence, which can facilitate the formation of a nucleic acid loop. Particularly preferred in this regard are GTC repeats, which have been identified as forming bends in Fragile X Syndrome. (If a PNA analog is used, the bend preferably contains a proline residue). Generally, three to five repetitions 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 includes a first portion of the labeled probe binding sequence. The fourth component is substantially complementary to a labeled probe (or, optionally, another probe). The fourth component further includes "sticky ends", ie, portions that do not hybridize to other portions of the probe, and preferably include most, but not all, of the ETM. As the skilled artisan will appreciate, any or all of the probes described herein may include amplification, capture, capture extension, label, and labeled extension probes, including:
They can be arranged to form a hairpin in the absence of their target.

In a preferred embodiment, several different growth probes are used, each with a first probe sequence that hybridizes to a different portion of the target sequence. That is, there is more than one amplification level; the amplification probes amplify the signal by multiple labeling events, and several different amplification probes, each with a different label, are used for each target sequence. Thus, the preferred embodiment utilizes a pool of at least two different amplification probes, each pool having a different probe sequence for hybridizing to a different portion of the target sequence; The only practical limitation would be the length of the target sequence initially. Furthermore, it is possible, but not generally preferred, that different amplification probes include different amplification sequences.

In a preferred embodiment, the amplification probe does not hybridize directly to the target sequence of the sample, but instead hybridizes to the first portion of the labeled extension probe. This is particularly useful to allow the use of "generic" amplification probes, ie, amplification probes that can be used with a variety of different targets. This is desirable because some of the amplification probes require special synthesis techniques. Thus, it is preferable to add a relatively short probe as the label extension probe.
Thus, the first probe sequence of the amplification probe is substantially complementary to the first portion or domain of the first labeled extended single-stranded nucleic acid probe. The label extension probe also includes a second portion or domain that is substantially complementary to a portion of the label sequence.
Both of these moieties are preferably at least about 10 to about 50 nucleosides in length, and preferably range from about 15 to about 30. The terms "first" and "second" are not meant to confer sequence orientation with respect to the 5'-3 'orientation of the target or probe sequence. For example, 5'-3 'of the complementary target sequence
Assuming orientation, the first portion may be located 5 'to the second portion, or may be located 3' to the second portion. In this specification, for convenience, the order of the probe sequences is generally shown from left to right.

In a preferred embodiment, one or more labeled extension probe amplification probe pairs are used, ie, n is 1 or more. That is, using a plurality of labeled extension probes,
Each has a portion that is substantially complementary to a different portion of the target sequence; this can serve as another level of amplification. Thus, the preferred embodiment utilizes a pool of at least two labeled extension probes, the upper limit being determined by the length of the target sequence.

In a preferred embodiment, one or more labels are depicted in FIG. 5G and as generally outlined in US Pat. No. 5,681,697 (hereby incorporated by reference). An extension probe is used with a single amplification probe to reduce non-specific binding. In this embodiment, a first portion of the first labeled extension probe hybridizes to a first portion of the target sequence, and a second portion of the first labeled extension probe hybridizes to a first probe sequence of the amplification probe. A first portion of the second labeled extension probe hybridizes to a second portion of the target sequence, and a second portion of the second labeled extension probe hybridizes to a second probe sequence of the amplification probe. In some cases, these features
This is referred to as a "cross-shaped" structure or shape, and is generally performed to provide stability when using large branched or dendritic amplification probes.

Further, as will be appreciated by those skilled in the art, the labeled extension probe is as follows:
It may interact with the pre-amplification probe rather than the direct amplification probe. Similarly, as outlined above, the preferred embodiment utilizes several different amplification probes, each with a first probe sequence that hybridizes to a different portion of the labeled extension probe. Further, as outlined above, although this is not generally preferred, it is possible that different amplification probes include different amplification sequences.

[0287] In addition to the first probe sequence, the amplification probe also contains at least one amplification sequence. As used herein, "amplified sequence" or "amplified segment" or grammatical equivalent means a sequence used directly or indirectly to bind to a first portion of a labeled probe, as described in more detail below. I do. Preferably, the amplification probe contains a variety of amplification sequences, preferably from about 3 to about 100
0, particularly preferably about 10 to about 100, and especially preferred is about 50. In some cases, for example, when a linear amplification probe is used, 1 to about 20 is preferable, and about 5 to about 10 is particularly preferable.

[0288] Amplification sequences can be linked together in various ways, as will be appreciated by those skilled in the art. These may be directly linked to each other by covalent bonds, or via a nucleic acid bond such as a phosphodiester bond, a PNA bond, or via an insert binder such as an amino acid, carbohydrate or polyol bridge, or other crosslinker or bond Via a partner, it may be attached to an intervening sequence or chemical moiety. The binding site may be at the end of the segment and / or at an internal nucleotide in one or more chains. In a preferred embodiment, the amplification sequence is attached via a nucleic acid bond.

In a preferred embodiment, branched amplification sequences are used, as generally described in US Pat. No. 5,124,246 (hereby incorporated by reference).
Branched amplification sequences can adopt a "fork-like" or "comb-like" conformation. "Fork-like" branched amplification sequences generally have three or more oligonucleotide segments emanating from an origin forming a branched structure. An origin is another nucleotide segment or a multifunctional molecule to which at least three segments can be covalently or tightly linked. A "comb-like" branched amplification sequence has a linear spine from which a number of side-chain oligonucleotides extend. In either conformation, the overhanging segment usually depends on the modified nucleotide or other organic moiety with appropriate functionalities for oligonucleotide attachment. In addition, in any conformation, a large number of amplification sequences may be available for direct or indirect binding to a detection probe. In general, these structures use modified multifunctional nucleotides, particularly those described in US Pat.
Prepared as known in the art, as described in 5,352 and 5,124,246.

In a preferred embodiment, dendritic amplification probes are used as described in US Pat. No. 5,175,270 (hereby incorporated by reference). Dendritic amplification probes have amplification sequences that attach via hybridization and, as a result, have a portion of double-stranded nucleic acid as a component of their structure. The outer surface of the dendritic amplification probe has various amplification sequences.

In a preferred embodiment, a linear amplification probe is used, which has sequences where the individual amplification sequences are linked end-to-end directly or with short intervening sequences to form a polymer. As in other amplification configurations, there may be additional sequences or portions between the amplification sequences.

In one embodiment, the linear amplification probe has a single amplification sequence. This may be the case if a hybridization / dissociation cycle occurs to form a pool of amplified probes, which hybridizes to the target, and is then removed to allow more probes to bind, or a large amount of ETMs It is useful when used for each probe. However, in a preferred embodiment, the linear amplification probe contains various amplification sequences.

Furthermore, the amplification probe may be wholly linear, wholly branched, wholly dendritic, or a combination thereof. The amplification sequence of the amplification probe is used, directly or indirectly, to bind to a labeled probe that allows detection. In a preferred embodiment, the amplification sequence of the amplification probe is substantially complementary to the first portion of the labeled probe. Alternatively, an amplification extension probe is used, which has a first portion that binds to the amplification sequence and a second portion that binds to the first portion of the labeled probe.

Further, the compositions of the present invention may include a “pre-amplification” molecule, which serves as a bridge between the label extension molecule and the amplification probe. In this way, more amplification factors, and thus more ETMs, are ultimately bound to the detection probe. The preamplified molecule may be linear or branched, typically about 3
It contains nucleotides in the range of 0-3000.

[0295] The tissue collection device of the present invention can be used in various ways. The reactants may be added simultaneously or in any order, but the preferred embodiments are as outlined below.

The invention thus generally relates to target sequences, capture probes, and labeled probes.
(Or, if the target analyte is labeled with an ETM, the assay complex includes a target sequence and a capture probe).
As used herein, "assay complex" refers to a collection of attachment or hybridization complexes that include an analyte and includes a binding ligand and target that allow for detection, and at least one ETM. The composition of the assay complex will depend on the use of the different probe components outlined herein. Thus, in FIG. 6A, the assay complex includes the capture probe and the target sequence. A preferred embodiment utilizes a capture probe, a target sequence, and a labeled probe. Assay complexes also include capture extension probes, labeled extension probes, and amplification probes, depending on the shape used, as outlined herein.

[0297] This assay is generally performed under stringent conditions that allow the formation of labeled probe attachment complexes only in the presence of the target. Stringency can be controlled by changing process parameters, which are thermodynamic variations. The parameters include, but are not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, and the like. Stringing, including the use of an electrophoresis step, may also drive non-specific (ie, low stringency) substances away from the detection electrode.

[0298] These parameters can also be used to control non-specific binding on nucleic acids, as generally reviewed in US Pat. No. 5,681,697. Thus, it is desirable that certain steps be performed under high stringency conditions; for example, when the first hybridization step is performed between the target sequence and the labeled extender and capture extension probe. Performing this step under conditions that prioritize particular binding allows for a reduction in non-specific binding.

In a preferred nucleic acid embodiment, when using all of the components outlined herein,
The preferred method is as follows. The single-stranded target sequence is incubated with the capture extension probe and the labeled extension probe under hybridization conditions. In a preferred embodiment, the reaction is performed with an immobilized capture probe in the presence of an electrode, but the reaction may be performed in two steps by initial incubation and subsequent application to the electrode. Excess reagent is washed off and then the amplification probe is added. If preamplification probes are used, they may be added before or simultaneously with the amplification probes. Excess reagent is washed off and the labeled probe is then added. Excess reagent is washed off and detection is started as outlined below.

In one embodiment, a number of capture probes (or capture and capture extension probes) are used, each of which is substantially complementary to a different portion of the target sequence. Again, when using an amplification probe, as outlined herein, the system generally involves the recruitment linker containing the ETM upon binding of the labeled probe to the monolayer surface containing the conductive oligomer (mechanism-2). Or in the vicinity of the detection probe. Thus, for example, for the mechanism-2 system, as outlined herein, when the ETM is attached via a "dendritic" type structure, the length of the linker from the point of attachment of the nucleic acid to the ETM is It can vary with the length of the capture probe, especially when using a capture extension probe. That is, a longer capture probe has a capture extender, resulting in a target sequence being "retained" further away from the surface than in a shorter capture probe.
Adding an extra linking sequence between the probe nucleic acid and the ETM will bring the ETM into spatial proximity to the surface and give better results. Similarly, for the mechanism-1 system, the length of the recruitment linker, the length of the detection probe, and their distance can be optimized.

In addition, if desired, the nucleic acids utilized in the present invention can be coupled prior to detection, if applicable, using standard molecular biology techniques, such as using ligase. Similarly, if stability is desired, it is possible to add a crosslinking agent to stabilize the structure. As will be appreciated by those skilled in the art, although described for nucleic acids, the systems outlined herein can be used for other target non-analytes as well.

The compositions of the present invention are generally described below and in US Patent Nos. 5,591,578; 5,824,473.
5,770,369; 5,705,348; 5,780,234 and 5,952,171; WO98 / 20162; WO99 /
37819; PCT / US98 / 12430, PCT / US99 / 01703; PCT / US98 / 12082; PCT / US99 / 14191; P
CT / US99 / 21683; PCT / US99 / 25464; PCT / US99 / 10104; and USSNs 09 / 135,18
3, 09 / 295,691 and 09 / 306,653, all of which are incorporated herein by reference, and are synthesized using methods generally known in the art. As will be appreciated by those skilled in the art, many of the methods outlined below involve nucleic acids that include a ribose-phosphate backbone. However, as outlined above, many other nucleic acid analogs may be used, some of which do not contain ribose or phosphate in the backbone. In these embodiments, for conjugation at positions other than the base, conjugation is performed as recognized by those skilled in the art, depending on the backbone. Thus, for example, the binding is PNA
This can be done at the backbone carbon atom, as described below, or at either end of the PNA. In addition, systems can be created which utilize non-nucleic acid components, as generally outlined in the above references.

The composition may be manufactured in a number of ways, depending on the mechanism and components used. A preferred method is to first synthesize an attached linker attached to the nucleoside, add another nucleoside to form a capture probe, and then attach to the electrode.
Combine. Alternatively, a full capture probe is made, then the complete attachment linker is added, followed by binding to the electrode. Alternatively, monolayers of the attached linker, some of which have functional groups for attachment of the capture probe, are first attached to the electrode, followed by attachment of the capture probe. In the latter two methods, the attachment linker used is
It may be preferred when it is not stable in solvents and under conditions of conventional nucleic acid synthesis.

In a preferred embodiment, the compositions of the present invention first form an attachment linker covalently linked to a nucleoside, followed by the addition of additional nucleosides to form a capture probe nucleic acid, and the attachment linker to the electrode. Manufactured by a final step including adding.

The attachment of the attachment linker to the nucleoside can be accomplished in several ways. In a preferred embodiment, all or some of the attached linkers are first synthesized (generally,
It has a functional group at the end for binding to the electrode), which is then bound to the nucleoside. Then, further nucleosides are added as needed, and in the last step generally coupled to the electrode. Alternatively, for example, where the attachment linker is a conductive oligomer, the oligomer units are added to the nucleoside at once, and the nucleoside is added and bound to the electrode. Many representative syntheses are described in WO98 / 20162, PCT US98 / 12430, PCT US98 / 12082, PCT US99 / 0.
1705, PCT US99 / 01703 and USSN 09/135.
183, 60/105, 875, and 09 / 295,691 are shown in the drawings, all of which are incorporated herein by reference.

The attachment linker is then attached to a nucleoside, which may comprise one (or more) oligomer units, attached as described herein.

In a preferred embodiment, the linkage is to the ribose of the ribose-phosphate backbone, including amide and amine linkages. In a preferred embodiment, there is at least one methylene group or other short aliphatic alkyl group (as a Z group) between the nitrogen attached to the ribose and the aromatic ring of the conductive oligomer.

Alternatively, the linkage is through a phosphate of the ribose-phosphate backbone, as generally outlined in PCT US97 / 20014.

In a preferred embodiment, the linkage is through a base. In a preferred embodiment, a protecting group may be added to the base prior to the addition of the conductive oligomer, as is generally known to those skilled in the art. In addition, the palladium cross-linking reaction can be altered to prevent the dimerization problem; that is, the two conductive oligomers do not bind to the base but rather dimerize.

Alternatively, conjugation to a base can be achieved by making a nucleoside having one unit of oligomer, followed by the addition of another.

Once the modified nucleosides have been prepared, protected and activated, prior to coupling to the electrode, standard synthetic methods (Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Pres
s, Oxford, UK 1984; Eckstein)
They can be included in several ways.

In one embodiment, one or more modified nucleosides are transformed into a triphosphate form, and the growing oligonucleotide strand is added to the enzyme DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptase. And using standard molecular biology techniques, such as using RNA polymerase. For inclusion of the 3 'modified nucleoside in the nucleic acid, a terminal deoxynucleotidyl transferase may be used. (Ratliff, Terminal deoxy
nucleotidyltransferase.In The Enzymes, Vol. 14A.PD Boyer ed.pp 105-
118. Academic Press, San Diego, CA 1981). Accordingly, the present invention provides a deoxyribonucleoside triphosphate comprising a covalently linked ETM. Preferred embodiments utilize ETM binding to a base or backbone, such as ribose (preferably at the 2 'position), as generally shown in structures 42 and 43 below.

[0313]

Embedded image

[0314]

Embedded image

Thus, in some embodiments, nucleic acids comprising ETMs can be produced in situ. For example, a target sequence can hybridize to a capture probe (eg, on a surface) such that the ends of the target sequence are exposed, ie, non-hybridized. The addition of the enzyme and ETM-labeled triphosphate nucleotides allows for the production of the label in situ. Similarly, with labeled nucleotides recognized by the polymerase, PCR and detection can occur simultaneously; that is, the target sequence is generated in situ.

In a preferred embodiment, the modified nucleoside is phosphoramidate or H
-Converted to the phosphonate form, which is then used for solid-phase or solution synthesis of oligonucleotide synthesis. In this way, the modified nucleotide is included in the oligonucleotide at an internal position or at the 5 'end for attachment at the ribose (ie, amino- or thiol-modified nucleoside) or at the base. This is generally done in one of two ways. First, the 5'-position of ribose is 4 ', 4-dimethoxytrityl (
DMT), followed by reaction with 2-cyanoethoxy-bis-diisopropylaminophosphine in the presence of diisopropylammonium tetrazolide or with 2'-cyanoethoxyphosphine chlorodiisopropylamino,
Obtain phosphoramidates as known in the art; however, other methods may be used as will be appreciated by those skilled in the art. See Gait supra; Caruthers, Science 230: 281 (1985), both of which are incorporated herein by reference.

For attachment of the group to the 3 'end, a preferred method uses the attachment of a modified nucleoside (or nucleoside substitute) to a controlled pore glass (CPG) or other oligomeric support. In this embodiment, 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 another oligomer support known in the art. In addition, phosphoramidate nucleosides, with or without modification, are attached to the 5 'end after deprotection. Accordingly, the present invention provides conductive oligomers or insulators covalently linked to nucleosides attached to a solid oligomer support such as CPG, and phosphoramidate derivatives of the nucleosides of the present invention.

The present invention further provides a method for producing a labeled probe having a recruitment linker containing ETM. As will be apparent to those skilled in the art, these synthetic reactions depend on the characteristics of the recruitment linker and the method of coupling the ETM. For nucleic acid recruitment linkers, the labeled probe is generally made to include an ETM at one or more positions, as outlined herein. When a transition metal complex is used as the ETM, the synthesis is performed in several ways. In a preferred embodiment, the ligand, followed by the transition metal ion, is added to the nucleoside, and then the nucleoside to which the transition metal complex is attached is added to the oligonucleotide, ie, to the nucleic acid synthesizer. Alternatively, the ligand is bound and subsequently incorporated into the growing oligonucleotide chain, and a metal ion is added.

In a preferred embodiment, the ETM is attached to ribose in the ribose-phosphate backbone. This is usually done as outlined herein for conductive oligomers, and as described herein and in PCT Publication WO 95/15971, using amino-modified or oxo-modified nucleosides to form the 2 Perform 'or 3' position. It is then recognized in the art as a ligand, for example as a transition metal ligand for the binding of a metal ion, or as a chemical functional group that can be used for the binding of other ligands or organic ETMs, for example via an amide bond. In addition, an amino group may be used. For example, as an example, the synthesis of nucleosides with various ETMs linked via ribose is described.

[0320] In a preferred embodiment, the ETM binds to the phosphate of the ribose-phosphate backbone. As outlined herein, this may be performed using a phosphodiester analog such as a phosphoramidate linkage (see generally PCT Publication WO 95/15971) or as described in PCT US 97/20014. Methods similar to those described are used, except that the conductive oligomer is replaced with a transition metal ligand or complex or an organic ETM.

The attachment to another backbone, such as a peptide nucleic acid or another phosphate bond, is performed as will be appreciated by those skilled in the art.

In a preferred embodiment, the ETM binds to a nucleoside base. This can be done in various ways. In one embodiment, the amino group of a base that is naturally occurring or added as described herein (e.g., see the figure) can be used as a ligand for a transition metal complex or, for example, via another amide bond. Use the ligand or as a chemical functional group that can be used to add an organic ETM. This is done as will be appreciated by those skilled in the art. Alternatively, nucleosides containing a halogen atom attached to the heterocyclic ring are commercially available. Acetylene binding ligands can be added using a halogenated base, as is generally known; see, for example, Tzalis et.
al., Tetrahedron Lett. 36 (34): 6017-6020 (1995); Tzalis et al., Tetrahed.
ron Lett. 36 (2): 3489-3490 (1995); and Tzalis et al., Chem. Communicat.
ions (submitted) See 1996, all incorporated herein by reference. See also the Figures and Examples which describe the synthesis of metallocenes (in this case ferrocenes) linked via an acetylene bond to the base.

In one embodiment, a nucleoside is prepared with a transition metal ligand incorporated into a nucleic acid, and the transition metal ion and the remaining required ligand are then added as is known in the art. In another embodiment, the transition metal ion and additional ligand are added prior to inclusion in the nucleic acid.

The nucleic acids of the invention are prepared with a covalently attached linker (ie, either an insulator or a conductive oligomer), and the linker is attached to an electrode. The method depends on the type of electrode used. As described herein, binding linkers are generally made with a terminal "A" linker to facilitate binding to an electrode. For the purposes of this application, a sulfur-gold bond is considered a covalent bond.

In a preferred embodiment, the conductive oligomer, insulator and binding linker are covalently attached to the electrode via a sulfur bond. However, surprisingly, conventional protecting groups used to attach molecules to gold electrodes are generally ideal for use in both the synthesis of the compositions described herein and their inclusion in oligonucleotide synthesis reactions. Not a target. Thus, the present invention provides a novel method of coupling conductive oligomers to gold electrodes using unusual protective protecting groups, including ethylpyridine and trimethylsilylethyl as illustrated. However, as will be appreciated by those skilled in the art, when the conductive oligomer is free of nucleic acids, conventional protecting groups such as acetyl groups may be used. See Greene et al., Supra.

This can be done in several ways. In a preferred embodiment, the conductive oligomer subunit containing a sulfur atom is protected with an ethyl-pyridine or trimethylsilylethyl group for attachment to an electrode. With respect to the former, this generally involves substituting a sulfur atom (preferably in the form of sulfhydryl) with a vinylpyridine or vinyltrimethylsilylethyl group and under conditions such that an ethylpyridine or trimethylsilylethyl group is added to the sulfur atom. This is done by contact.

The subunit also generally contains a functional moiety for attachment of the additional subunit, so that the additional subunits combine to form a conductive oligomer.
The conductive oligomer is then attached to the nucleoside, and additional nucleosides are attached. The protecting group is then removed and a covalent sulfur-gold bond is performed. Alternatively, all or part of the conductive oligomer is prepared and then protected by adding a subunit containing a protected sulfur atom or by adding a sulfur atom. The conductive oligomer is then attached to the nucleoside and additional nucleotides are attached. Alternatively, a conductive oligomer attached to the nucleic acid is prepared and then protected by adding a subunit containing a protected sulfur atom or by adding a sulfur atom. Alternatively, the ethylpyridine protecting group may be used as described above, but is removed after one or more steps and replaced with a standard protecting group such as disulfide. Thus, an ethylpyridine or trimethylsilylethyl group can act as a protecting group in some of the synthetic reactions, and is then removed and replaced with a conventional protecting group.

A “subunit” of a conductive polymer herein refers to at least a portion of a conductive oligomer to which a sulfur atom is attached, but a functional group that allows for the addition of additional components of the conductive oligomer, Alternatively, additional atoms may be present, including additional components of the conductive oligomer. Thus, for example, when using an oligomer of structure 1, the subunit contains at least a first Y group.

A preferred method is 1) the addition of an ethylpyridine or trimethylsilylethyl protecting group to the sulfur atom attached to the first subunit of the conductive oligomer, generally by adding a vinylpyridine or trimethylsilylethyl group to the sulfhydryl 2) addition of additional subunits for the formation of conductive oligomers; 3) addition of at least the first nucleoside to the conductive oligomer; 4) addition of additional nucleosides to the first nucleoside to form nucleic acids. 4) including binding of the conductive oligomer to the gold electrode. This can also be done in the absence of nucleosides, as described in the examples.

The above method can also be used to attach insulating molecules to gold electrodes.

In a preferred embodiment, a monolayer containing surface species (including conductive oligomers and insulators) is added to the electrode. Generally, the chemistry of the addition is similar or the same as the addition of the conductive oligomer to the electrode, ie, using a sulfur atom for attachment to the gold electrode. A composition comprising a monolayer, in addition to a conductive oligomer covalently linked to a nucleic acid,
It can be done in one of at least five ways: (1) the addition of a monolayer followed by a binding linker
(2) Addition of a linked linker-nucleic acid complex, followed by addition of a monolayer;
(3) Simultaneous addition of monolayer and binding linker-nucleic acid complex; (4) Formation of monolayer containing binding linker terminated with a functional moiety suitable for complete nucleic acid binding (any of 1, 2, or 3) Or (5) formation of a monolayer containing a binding linker terminated with a functional moiety suitable for nucleic acid synthesis, ie, nucleic acids are synthesized on a monolayer surface as is known in the art. Such suitable functionalities include, but are not limited to, nucleosides for phosphoramidate addition, amino groups, carboxyl groups, protected sulfur moieties, or hydroxyl groups. As an example, the formation of a single layer on a gold electrode using the preferred method (1) is described.

[0332] In a preferred embodiment, the nucleic acid is a peptide nucleic acid or analog. In this embodiment, the invention provides a peptide nucleic acid having at least one covalently linked ETM or linking linker. In a preferred embodiment, these moieties are covalently linked to the monomeric subunit of PNA. "Monomeric subunit of PNA" _{herein, -NH-CH 2 CH 2 -N} (COCH 2 - base) -CH ₂ -CO
-Monomers or derivatives of PNA (herein included within the definition of "nucleoside")
Means For example, the number of carbon atoms in the PNA backbone can be varied; generally, see Nielsen et al., Chem. Soc. Rev. 1997, page 73, which describes the number of PNA derivatives, incorporated herein by reference. I do. Similarly, the amide bond attaching the base to the backbone can be altered; phosphoramide and sulfamide linkages can be used. Alternatively, the moiety is linked to an internal monomer subunit. As used herein, "internal" means that the monomer subunit is not an N-terminal monomer subunit or a C-terminal monomer subunit. In this embodiment, the moiety can be attached to the base or backbone of the monomer subunit. The coupling of the bases is performed as outlined herein or as known from the literature. Generally, a moiety is added to the base, which is then included in the PNA as outlined herein. The base is added to the P before or after the addition of the chemical substituent.
Protected as necessary for inclusion in the NA synthesis reaction or derivatized to be included. Base protection and derivatization is shown in PCT US97 / 20014. The base can then be included in the monomer subunit.

In a preferred embodiment, the moiety is covalently linked to the backbone of the PNA monomer. The bond is generally at one of the unsubstituted carbon atoms of the monomer subunit, preferably at the α of the backbone.
-At the carbon, but also at the α-carbon of the amide bond connecting the 1- or 2-position carbon, or the base to the main chain. In the case of PNA analogs, other carbons or atoms may be similarly substituted. In a preferred embodiment, a moiety is added to the terminal monomeric subunit at the α-carbon atom or to an internal terminal monomeric subunit.

In this embodiment, the modified monomer subunit is synthesized with an ETM, a linking linker or a functional group for its attachment, and then a base is added to attach the modified monomer to the growing PNA chain. May be included.

The prepared monomeric subunit having a covalent moiety was prepared according to Will et al., Tetrahe
dron 51 (44): 12069-12082 (1995) and Vanderlaan et al., Tett. Let.38: 2
249-2252 (1987) (both are hereby incorporated by reference in their entirety)
The PNA is incorporated using methods as outlined in These methods allow the addition of chemical substituents to peptide nucleic acids without destroying the chemical substituents.

As will be appreciated by those skilled in the art, electrodes can be manufactured to have any combination of nucleic acids, conductive oligomers and insulators.

The compositions of the present invention may further comprise, in addition to the ETM, one or more other labels at any position. As used herein, “label” refers to an element (eg, an isotope) or a chemical compound that has been attached to allow detection of the compound. Preferred labels are radioactive isotope labels, colored or fluorescent dyes, and electrochemical labels such as ETM. The label can be included in the compound at any position. In addition, the compositions of the present invention may also include other moieties, such as a cross-linking agent, to promote cross-linking of the target-probe complex. For example, Lukhtanov et al., Nucl. Acids. Res. 24 (4): 683 (1996) and Tabone et a.
l., Biochem. 33: 375 (1994), both of which are incorporated herein by reference.

Once manufactured, the compositions are used in many applications, as described herein. In particular, the compositions of the invention are used in binding assays for target analyte detection, especially nucleic acid target sequences. As will be apparent to those skilled in the art, electrodes can be made to have a single species of binding ligand, or multiple species of binding ligand (ie, an array format).

In addition, the use of solid supports, such as electrodes, as outlined herein, allows the use of these genetic assays in an array.

In general, once an assay complex of the invention comprising at least one capture probe, one target analyte and one labeled probe is formed, detection proceeds by electronic initiation. Without being limited to a mechanism or theory, detection is based on electron transfer from the ETM to the electrode and involves the interposition of π orbitals.

Detection of electron transfer, ie, the presence of the ETM, is generally initiated electronically by a suitable voltage. The potential is applied to the assay complex. Precise control and variation of the applied potential can be achieved with a potentiostat and a three-electrode system (one for reference, one for the sample (or working), one for the counter electrode) or a two-electrode system (one For the sample and one for the counter electrode). This depends, in part, on the peak potential of the system, which depends on the choice of ETM, and also on the conductive oligomer used, the composition and consistency of the monolayer, and the type of reference electrode used. It allows the applied potentials to match.

In a preferred embodiment, the co-reductant or co-oxidant (integrally, the co-redox product)
Is used as an additional source or sink. In general, Sato et al., Bull. C
hem. Soc. Jpn 66: 1032 (1993); Uosaki et al., Electrochimica Acta 36: 17
99 (1991); and Alleman et al., J. Phys. Chem. 100: 17050 (1996), all of which are incorporated herein by reference.

In a preferred embodiment, an input source in solution is used to initiate electron transfer.
Preferably when initiation and detection is carried out using a DC current or at an AC frequency where diffusion is not limited. In general, as will be appreciated by those skilled in the art, short circuits in the system can be avoided by using a "hole" containing monolayer in a preferred embodiment. This can be done in several general ways. In a preferred embodiment, the input electrode source has a redox potential that is less than or equal to the ETM of the labeled probe.
Thus, at voltages above the redox potential of the input electron source, the ETM and the input electron source can be oxidized to provide electrons. The ETM provides electrons to the electrodes and the input source is E
Give to TM. For example, as an ETM bound to the compositions of the invention described in the Examples, ferrocene has a redox potential of approximately 200 mV in aqueous solution (depending on the method of binding and the presence of any substituents, even when ferrocene is bound). Very variable). The electron source ferrocyanide also has a redox potential of about 200 mV. Thus, at voltages of about 200 mV or more, ferrocene replaces ferricenium and transfers electrons to the electrodes. The ferricyanide can be oxidized to transfer electrons to the ETM. In this method, the electron source (or co-reducing agent) serves to amplify the signal generated in the system, and the electron source molecules rapidly and repeatedly transfer electrons to the ETM bound to the nucleic acid. The rate at which electrons are donated and accepted is limited to the rate of diffusion of the co-reducing agent, ie, electron transfer between the co-reducing agent and the ETM. Electron transfer is affected by density and size.

On the other hand, an input electron source having a redox potential lower than ETM is used. E
At a voltage lower than the redox potential of the TM, but higher than the redox of the electron source, an input source such as ferrocyanide cannot be oxidized and cannot provide electrons to the ETM.
That is, electron transfer does not occur. When ferrocene is oxidized, an electron transfer pathway is created.

In another preferred embodiment, the input electron source has a higher redox potential than the labeled probe ETM. For example, luminol, an electron source, has a redox potential of approximately 720 mV. A voltage lower than the redox potential of the ETM, ie, 20
At 0-720 mV, the ETM cannot accept electrons from the luminol electron source because the voltage is lower than the luminol redox potential. However, at or above the luminol redox potential, luminol transfers electrons to the ETM,
Enables quick and repetitive electron transfer. In this method, the electron source (or co-reducing agent) serves to amplify the signal generated in the system, and the electron source molecules donate electrons quickly and repeatedly to the ETM of the labeled probe.

Luminol also has the additional advantage of becoming a chemiluminescent species upon oxidation (see Ji.
rka et al., Analytica Chemica Acta 284: 345 (1993)), which allows optical detection of electron transfer from the ETM to the electrode. As long as luminol does not directly contact the electrode, ie, in the presence of an ETM where there is no efficient electron transfer path to the electrode, luminol is oxidized only by transferring electrons to the ETM on the assay complex.
In the absence of ETM (ie, when the target sequence does not hybridize the composition of the present invention), luminal is not significantly oxidized, resulting in low photon emission and low (if any) signal emission from luminol. . Very large signals occur in the presence of the target. Thus, measuring luminol oxidation by photon emission is an indirect measure of the ETM's ability to donate electrons to the electrodes. In addition, photon detection is generally more sensitive than electronic detection, thus increasing the sensitivity of the system. Initial results suggest that luminescence is dependent on hydrogen peroxide concentration, pH and luminol concentration, which is not linear.

Suitable electron source molecules are well known and include ferricyanide and luminol,
It is not limited to these.

On the other hand, an output electron acceptor can be used. That is, the above reaction is performed in reverse.
ETM such as metallocene which receives electrons from the electrodes is used. An output electron acceptor that rapidly and repeatedly receives electrons converts metallocene to metallisenium. In this embodiment, cobalt isenium is the preferred ETM.

The presence of ETM on the surface of the monolayer can be detected by various methods. Optical detection includes, but is not limited to, for example, fluorescein,
There are phosphorescens, luminescens, chemirminsens, electroluminescens and refractive indexes. Electronic detection includes, but is not limited to, amperometry, voltammetry, capacitance, and impedance. These methods include time- and frequency-dependent methods based on AC or DC current, pulse methods, lock-in methods, filter methods (high-pass, low-pass, bandpass) and time-resolved methods such as time-resolved fluoresence. is there.

In one embodiment, the efficient transfer of electrons from the ETM to the electrode is based on the ETM
In the redox state. With many ETMs, including ruthenium complexes containing bipyridine, pyridine, and imidazole rings, these changes in the redox state are related to changes in the spectrum. Significant differences in absorption are seen between the reduced and oxidized states for these molecules. For example, reference, Fabbri
zzi et al., Chem. Soc. Rev. 1995 pp192-202. These differences can be monitored using a molecular photometer or a photomultiplier.

In this embodiment, the electron donor and acceptor include all derivatives described above for optical activation or initiation. Preferred electron donors and acceptors are characterized by a large spectral change in redox that allows sensitive monitoring of electron transfer. Preferred examples include Ru (NH ₃ ) ₄ py and Ru (bpy) zim. It should be understood that only the donor or receiver monitored by absorption has ideal spectral properties.

In a preferred embodiment, electron transfer is detected by fluorimetry. Many transition metal complexes, such as ruthenium, have apparent fluorescence. Therefore, the redox state of the electron donor bound to the nucleic acid and the receptor charge, Ru (4,7-biphenyl ₂ -
Phenanthroline) ₃ ²⁺ using fluorescence due, can be monitored sensitively. The formation of this compound can be easily measured by standard fluorescence detection methods. For example, laser-induced fluorescence is recorded on a standard cell fluorimeter, on-line fluorimeter flow (eg, coupled to chromatography), or a multi-sample “plate reader” similar to that commercially available for 96-well immunoassays. can do.

On the other hand, fluorescence can be measured using an optical binding sensor with a binding ligand in solution or a nucleic acid probe bound to an optical fiber. Fluorescence can be monitored using an optical intensifier or other photodetector coupled to the optical fiber. The advantage of this is that the amount of sample used for detection can be very small.

In addition, scanning fluorescence detectors such as Fluorlmager sold by Molecular Dynamic are well suited for monitoring the fluorescence of modified nucleic acid molecules lined up on solid surfaces.
The advantage of this system is that multiple electron transfer probes can be scanned at once using a chip covered with thousands of different nucleic acid probes.

Many transition metal complexes fluoresce with large Stokes shifts. Suitable examples include bis and trisphenanthroline complexes of transition metals such as ruthenium,
And bis and tribipyridine complexes (see Juris, A., Balzani, V.
, et al. Coord. Chem. Rev., V.84, p.85-277, 1988). Preferred examples show efficient fluorescence (reasonably high quantum yield) and low reconstruction energy. These include, Ru (4,7-biphenyl ₂ - phenanthroline) ₃ ^2+, Ru (4,4'-
There are diphenyl 2,2'-bipyridine) ₃ ²⁺ and platinum complexes (see, Cumming
s et al., J. Am. Chem. Soc. 118: 1949-1960 (1996), which is hereby incorporated by reference). On the other hand, the decrease in fluorescence associated with hybridization can be measured using these systems.

In another embodiment, electrochemiluminescence is used as the basis for electron transfer detection. Some ETMs, such as Ru ²⁺ (bpy) ₃ , have reduced excitation states in direct luminescence.
This change in property is related to nucleic acid hybridization and can be monitored with a simple optical intensifier. (See, Blackburn, GF Clin. Chem. 37: 1534-
1539 (1991); and Juris et al., Supra).

In a preferred embodiment, amperometry, voltammetry, capacitance and impedance are used for electron detection. Preferred techniques include, but are not limited to, electrogravimetric analysis, coulometry (including control potential coulometry and constant current coulometry), voltammetry (cycle voltammetry, pulse voltammetry (normal pulse voltammetry, square wave voltametry). Measurement, differential pulse voltammetry, osteo wing
Square wave voltammetry, electrostatic pulse method), stripping analysis (anion stripping, cation stripping, square wave stripping voltammetry), conduction analysis (electronic conduction, direct analysis), time-dependent electrochemical analysis (chronoan (Perometry, chronopotentiometry, cycle chronoamperometry, cycle chronopotentiometry, ac porography, chronogalvometry, chronochromometry), ac impedance method, capacitance method, ac voltammetry, opto-electrochemical method.

In a preferred embodiment, monitoring of electron transfer is performed with amperometric detection. In this detection method, the potential (as compared to an isolated control electrode) between a nucleic acid binding electrode and a control (reverse) electrode in a sample containing the desired target gene is utilized. Different efficiencies of electron transfer result from the presence or absence of the target nucleic acid. That is, the presence or absence of the target nucleic acid, and thus the labeled probe, produces different currents.

Instruments for measuring electron transfer with amperometry include sensitive current detection,
Means for controlling the voltage potential, always including a potentiostat. This voltage is optimized by reference to the potential of the electron donor complex on the label probe. Electron-donating complexes include those described above for complexes of iron, osmium, platinum, cobalt, rhenium, and ruthenium, with iron complexes being most preferred.

In a preferred embodiment, other electron detection methods are used. For example, potentiometry (ie, voltammetry) requires the non-Faraday method (no net current).
And are commonly used in pH and other ion detectors. Similar sensors are used to monitor the electron transfer between the ETM and the electrodes. In addition, insulators (
Other properties such as resistance (such as resistance) and electrical conductors (conductivity, impedance, capacitance) are used to monitor electron transfer between the ETM and the electrodes. Also, any system that produces a current (such as electron transfer) produces a small magnetic field and can be monitored in certain embodiments.

One advantage provided by the fast rate of electron transfer found in the compositions of the present invention is that time resolution can generally enhance signal to noise results in monitors such as absorption, fluorescence or electron flow. The high speed of electron transfer of the present invention results in a high signal and a typical delay between electron transfer onset and completion. By amplifying the signal of a particular delay, the pulse onset of electron transfer and "lock-in" amplification detection and Fourier transformation occur.

In a preferred embodiment, electron transfer is initiated using an alternating current (AC) method. Without being bound by theory, an ETM coupled to an electrode responds similarly to an alternating voltage through a connected resistor and capacitor. Basically, any method by which the properties of the complex acting as these resistors and capacitors can be measured can be the basis of detection. Surprisingly, conventional electrochemical theory, such as Laviron et a
l., J. Electroanal. Chem. 97: 135 (1979) and Laviron et al., J. Electro.
.. anal Chem 105: 35 ( 1979) ( which is hereby incorporated by reference) is (hereinafter 10 mV) very small E _AC and a relatively large number of molecules, except, the system described herein Does not become a model. That is, the alternating current (I) is not accurately described in Laviron's equation. This stems in part from this theory assuming an unlimited source and sink of electrons, which is not the case for the system of the present invention.

The AC voltage theory, which is a good model for these systems, is described by O 'Connor et al., J.
Electroanal. Chem. 466 (2): 197-202 (1999), which is hereby incorporated by reference. The equation for predicting these systems is shown below as Equation 1:

(Equation 1) In Formula 1, n is the number of oxidation or reduction electrons for each redox molecule, f is the applied frequency,
F is the total number of Faraday constant, N _total is the redox molecules, E ₀ is the formal potential, R represents gas constant, T is the temperature in Kelvin degrees and E _Dc is the electrode potential, the redox molecules. This model fits very well with the experimental data. In some cases, the current is lower than expected, but this is due to a decrease in ferrocene and can be restored in many ways.

In addition, the Faraday current can also be expressed as a function of time, as shown in Equation 2:

(Equation 2) _IF is the Faraday current and q _e is the elementary charge.

[0365] However, neither the effect of the electron transfer speed nor the instrumental factor is incorporated into Equation 1. The speed of electron transfer is important if it is near or below the applied frequency. Thus, the true i _AC is a function of three factors as shown in Equation 12 below. Equation 3 i _AC = f (Nernst factor) f (k _ET ) f (instrument factor)

[0366] These equations can model and predict the expected AC current in systems utilizing input signals including AC and DC elements. As noted above, surprisingly, conventional theory does not model these systems at all except at very low voltages.

In general, non-specifically bound labeled probes / ETMs show a difference in impedance (ie, higher impedance) than when the labeled probe containing the ETMs specifically bound in the correct direction. In a preferred embodiment, washing off non-specific binding substances results in an infinite effective impedance. Thus, alternating current detection has several advantages, generally described below, including increased sensitivity and the ability to eliminate background noise. In particular, changes in impedance (including, for example, bulk impedance) can be monitored as the difference between non-specific binding of the ETM-containing probe and target-specific assay complex formation.

Therefore, using the AC initiation and detection method, the frequency response of the system is ETM
It changes as a result of being. "Frequency response" means a signal modification as a result of electron transfer between the electrode and the ETM. This modification differs according to the signal frequency. The frequency response includes AC current, phase shift, DC offset voltage, Faraday impedance, etc. at one or more frequencies.

Once an assay complex comprising the target sequence and the labeled probe is created, the first input electronic signal is used in the system, preferably via at least the sample electrode (including the complex of the invention) and the counter electrode. , And electron transfer between the electrode and the ETM is started. Electrode systems are also used at the voltages applied to the control and working electrodes. The first input signal includes at least one AC element. AC elements are of variable amplitude and frequency. Generally, for use in the method of the invention, the AC amplitude is about 1 mV-1.1 V, preferably about 10 mV-800 mV, particularly preferably about 10-500 mV. The AC frequency is about 0.01 Hz to 100 KHz, preferably about 10 Hz to 10 MHz, and particularly preferably about 100 Hz to 20 MHz.

The combined use of AC and DC signals has various advantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises an AC element and a DC element. That is, the DC offset voltage of the sample and the reverse electrode is the ETM (eg, with ferrocene, the sweep is typically 0 to 500 mV) (or, when the working electrode is grounded, the control electrode is swept from 0 to -500 mV). Swept through the chemical potential. This sweep is used to identify the DC voltage at which the maximum response of the system is seen. This is generally at or near the electrochemical potential of the ETM.
Once this voltage is measured, a sweep or one or more uniform DC onset voltages are used. The DC onset voltage is about -1V to + 1.1V, and about -500V.
mV to +800 mV is desirable, and about -300 to 500 mV is particularly desirable.
In a preferred embodiment, the DC onset voltage is not zero. At the top of the DC onset voltage, a signal AC element of variable amplitude and frequency is applied. If the ETM is present and can respond to an AC perturbation, the transfer of electrons between the electrode and the ETM will produce an AC current.

In established systems, a single input signal is sufficient to identify the presence or absence of an ETM (ie, the presence of a target sequence) nucleic acid. On the other hand, multiple input signals are also adapted. There are many types, multiple frequencies, multiple AC amplitudes, or combinations thereof.

Thus, in a preferred embodiment, using multiple DC onset voltages,
DC voltage sweeps are preferred. This can be done at a single frequency or at two or more frequencies.

In a preferred embodiment, the AC amplitude can be varied. Without being bound by theory, increasing the amplitude seems to increase propulsion. Higher amplitudes result in higher overpotentials and give faster speeds of electron transfer. Generally, the same system improves response at a single frequency through the use of a high overpotential at that frequency (ie, a faster output signal). As the amplitude increases at higher frequencies, the speed of electron transfer through the system increases and sensitivity increases. In addition, for example, it can be used to provoke a response in a slow system that does not have a suitable spatial arrangement.

In a preferred embodiment, measurements of the system are made at at least two isolated amplitudes or overpotentials. Multiple amplitudes are preferred. As mentioned above, changes in response as a result of amplitude changes form the basis for system identification, calibration and quantification. Furthermore, one or more AC frequencies can be used as well.

In a preferred embodiment, the alternating frequency is varied. At different frequencies, different molecules respond in different ways. As those skilled in the art will appreciate, the output current generally increases with increasing frequency. However, when the frequency is higher than the speed at which electrons travel to and from the electrode and the ETM, the higher frequency results in a loss or reduction of the output signal. At some point, the frequency will be greater than the speed of electron transfer between the ETM and the electrode, and the output signal will also decrease.

In some embodiments, a single measurement of the output signal at a single frequency is used for detection. That is, the frequency response of the system in the absence of the target sequence and thus the absence of the labeled probe containing the ETM can be pre-measured and is very low at certain high frequencies. With this information, a particular frequency response indicates the presence of the assay complex. That is, all responses at a particular frequency characterize the assay complex. It is only necessary to use a single input high frequency, and any change in all frequency responses is indicative of the presence of the analyte, the presence of the target sequence.

Further, the use of alternating current techniques results in a significant reduction in background signal at all single frequencies due to substances other than ETMs. That is, "closing out" or "filtration" of unwanted signals. The frequency response of a charge carrier or redox active molecule in solution is limited by its diffusion coefficient and charge transfer coefficient. Thus, at high frequencies, the charge carrier cannot spread its charge fast enough to transfer it to the electrodes and / or the charge transfer rate is not fast enough. This is especially true if no suitable monolayer is used or if a partial or incomplete monolayer is used, ie no solvent can reach the electrode. As already outlined,
In direct current techniques, the presence of "holes" through which the solvent can reach the electrodes depends on the "
Short-circuiting "may result in solvent charge carriers: reaching the electrodes and generating a background signal. However, using current alternating current techniques, one or more frequencies may be chosen to prevent the presence of a monolayer. Prevents the frequency response of one or more charge carriers in solution, whether in the absence or the presence of many biological fluids, such as blood, that contain significant amounts of redox-active molecules that can interfere with amperometric detection. So it's especially meaningful.

In a preferred embodiment, measurements of the system are made at at least two isolated frequencies, with measurements at multiple frequencies being preferred. There are scans at multiple frequencies.
For example, the alternating current is 10-100 kHz at a low input frequency such as 1-20 Hz.
When compared with the response to an output signal at a higher frequency, such as the presence or absence of the ETM, the difference in the frequency response is shown. In a preferred embodiment, the frequency is measured at at least 2, preferably about 5, and more preferably at least about 10 frequencies.

After conducting the input signal to initiate electron transfer, the output signal is received, ie, detected. The presence and increase of the output signal depends on many factors. Input signal overpotential / amplitude; input AC signal frequency; intervening medium composition; DC onset; system environment; ETM properties; For a given input signal, the presence and enhancement of the output signal generally depends on the presence or absence of the ETM, the location and distance of the ETM from the monolayer surface, and the nature of the input signal. In some embodiments, it is possible to discriminate, based on impedance, the difference between the non-specific binding of the labeled probe and the formation of a target-specific assay complex containing the labeled probe.

In a preferred embodiment, the output signal comprises an alternating current. As described above, the magnitude of the output current depends on the number of parameters. Changing these parameters will optimize the system in number.

The alternating current produced by the present invention is generally at about 1 femptoamp to about 1 milliamp,
About 50 femptoamps to about 100 microamps are preferred, and about 1 picoamp to about 1 microamp are particularly preferred.

[0383] In a preferred embodiment, the output signal is a phase shifted by the AC element as compared to the input signal. Without being bound by theory, it appears that the uniformity of the system of the present invention allows for the detection of phase shifts. That is, the biocomposite in which electron transfer of the present invention occurs uniformly reacts with an AC input, which is the same as a standard electronic device, and the phase shift can be measured. This means that ETM
Serve as the basis for the detection of the presence or absence of, and / or the difference between the presence of a target-specific assay complex containing a labeled probe and the non-specific binding of a substance to a system component.

The output signal is characterized by the presence of ETM. That is, the output signal is characterized by the presence of a target-specific assay complex comprising the labeled probe and the ETM. In a preferred embodiment, the basis for detection is the difference in the Faraday impedance of the system as a result of the formation of the assay complex. Faraday impedance is the impedance of the electrode and ETM system. Faraday impedance is quite different from bulk or two-electron impedance, and bulk impedance is the impedance of the bulk solution between the electrodes. Many factors can change the Faraday impedance, which does not affect the bulk impedance, and vice versa.
Thus, the assay complex of the present system containing nucleic acids has a certain Faraday impedance, which depends on the distance between the ETM and the electrode, its electronic properties, the composition of the intervening medium, and the like. What is important in the method of the present invention is that the Faraday impedance between the ETM and the electrode is very different depending on whether the labeled probe containing the ETM binds specifically or non-specifically to the electrode.

Accordingly, the present invention further provides an electronic device or device for detecting a subject using the composition of the present invention. The apparatus includes a test chamber for receiving a sample solution having at least a first measurement or sample electrode and a second measurement or counter electrode. A three electrode system is also used. The first and second measurement electrodes contact the test sample receiving area, and in the presence of a liquid test sample, the two electrophoresis electrodes may be in electronic contact.

In a preferred embodiment, the first measurement electrode comprises a mounting linker and a single-stranded nucleic acid capture probe covalently linked via a monolayer containing the conductive oligomer described above.

The device includes an AC voltage source electrically connected to the test room or measurement electrode. Preferably, the AC voltage source may emit the offset voltage as well.

[0388] In a preferred embodiment, the device further comprises a processor capable of comparing the input signal and the output signal. A processor is coupled to the electrode and positioned to receive the output signal and detects the presence of a surface nucleoside. Accordingly, the compositions of the present invention may be used in various studies, clinical, quality control, field tests, etc., as abbreviated herein.

All references cited herein are hereby incorporated by reference in their entirety.

[Brief description of the drawings]

FIG. 1A depicts a tissue collection device with a container positioned.

FIG. 1B depicts the tissue collection device with the container positioned.

FIG. 1C depicts the tissue collection device with the container positioned.

FIG. 1D depicts the tissue collection device with the container positioned.

FIG. 2A depicts a tissue collection device having a container for testing a portion of a specimen.

FIG. 2B depicts a tissue collection device having a container for testing a portion of a specimen.

FIG. 3A depicts the electrodes of the biosensor 80 in the cap 20 with the interconnect device 90.

FIG. 3B depicts the electrodes of the biosensor 80 as components of the tube 10.

FIG. 3C depicts the electrodes of the biosensor 80 as components of the tube 10.

FIG. 3D depicts the biosensor 80 in the container 30 of the device 10.

FIG. 4A. Capture probe 100 linked via an attachment linker 106.
Depict the target sequence 120 hybridized to.

FIG. 4B depicts the use of a single capture extension probe 110.

FIG. 4C depicts the use of two capture extension probes 110 and 130.

FIG. 5A depicts different possible structures of binding of the labeled probe to the ETM.

FIG. 5B depicts different possible structures of binding of the labeled probe to the ETM.

FIG. 5C depicts different possible structures of binding of the labeled probe to the ETM.

FIG. 5D depicts different possible structures of binding of the labeled probe to the ETM.

FIG. 5E depicts different possible structures of binding between the labeled probe and the ETM.

FIG. 6A depicts one embodiment of the present invention.

FIG. 6B depicts one embodiment of the present invention.

FIG. 6C: Labeled probe 1 that hybridizes directly to labeling sequence 120
FIG. 45 depicts the use of 45.

FIG. 6D depicts one embodiment of the present invention.

FIG. 6E does not hybridize directly to the target, but rather directly (FIG.
E) depicts a system utilizing a labeled probe 145 that hybridizes to an amplification probe that hybridizes to the target sequence, either indirectly (FIGS. 6F and 6G).

FIG. 6F does not hybridize directly to the target, but rather directly (FIG. 6).
E) depicts a system utilizing a labeled probe 145 that hybridizes to an amplification probe that hybridizes to the target sequence, either indirectly (FIGS. 6F and 6G).

FIG. 6G does not hybridize directly to the target, but rather directly (FIG.
E) depicts a system utilizing a labeled probe 145 that hybridizes to an amplification probe that hybridizes to the target sequence, either indirectly (FIGS. 6F and 6G).

FIG. 6H depicts a system utilizing multiple labeled probes.

FIG. 7A depicts a “Mechanism-1” detection system.

FIG. 7B depicts a “mechanism-1” detection system.

FIG. 7C depicts a “Mechanism-1” detection system.

FIG. 7D depicts a “Mechanism-1” detection system.

FIG. 7E depicts a “Mechanism-1” detection system.

FIG. 7F depicts the same utilizing a linear amplification probe 150.

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Claims (21)

[Claims] 1. A tissue collection device having an electrode. 2. The tissue collection device of claim 1, wherein said electrode comprises a capture binding ligand. 3. The tissue collection device according to claim 1, wherein the electrode comprises: a) a self-assembled monolayer; and b) a capture binding ligand. 4. The tissue collection device according to claim 2, wherein the capture binding ligand is a nucleic acid capture probe. 5. The tissue collection device according to claim 3, wherein the self-assembled monolayer includes an insulator. 6. The tissue collection device according to claim 3, wherein the self-assembled monolayer contains an electrical path forming molecular species (EFS). 7. The tissue collection device according to claim 6, wherein the EFS is a conductive oligomer. 8. The method according to claim 1, further comprising at least one reagent.
The tissue collection device according to 5, 6, or 7. 9. The tissue collection device according to claim 8, wherein said reagent is selected from the group consisting of an anticoagulant, a probe nucleic acid and a lysing agent. 10. The tissue collection device according to claim 4, wherein the probe nucleic acid includes an electron transfer unit. 11. The tissue collection device according to claim 10, wherein said electron transfer unit is ferrocene. 12. The method according to claim 1, wherein the tissue collection device is a blood collection device.
The apparatus according to 4, 5, 6, 7, 8, 9, 10, or 11. 13. The method of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, wherein the electrodes are on a substrate comprising printed circuit board (PCB) material. Tissue collection device. 14. A tissue collection device having an electrode comprising: a) an i) self-assembled monolayer; and ii) an assay complex comprising: 1) a capture binding ligand; 2) a target analyte; and 3) an electron transporter. And b) detecting electron transfer between the electrode and the electron transfer unit. 15. The method according to claim 14, wherein said sample is blood. 16. The method of claim 14, wherein said self-assembled monolayer comprises an insulator. 17. The method of claim 14, 15, or 16, wherein said self-assembled monolayer comprises EFS. 18. The method of claim 14, 15, 16 or 17, wherein said target analyte is a nucleic acid. 19. The method of claim 14, wherein said capture binding ligand is a capture probe.
The method according to 15, 16, 17 or 18. 20. The method of claim 14, 15, 16, 17, 18, or 19, wherein said assay complex comprises a labeled probe comprising said electron transfer moiety. 21. The method according to claim 20, wherein said electron transfer unit is ferrocene.