EP0777741A1 - Biocapteur et procede destines a la detection par luminescence electrophotochimique d'acides nucleiques adsorbes sur une surface solide - Google Patents

Biocapteur et procede destines a la detection par luminescence electrophotochimique d'acides nucleiques adsorbes sur une surface solide

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Publication number
EP0777741A1
EP0777741A1 EP95930881A EP95930881A EP0777741A1 EP 0777741 A1 EP0777741 A1 EP 0777741A1 EP 95930881 A EP95930881 A EP 95930881A EP 95930881 A EP95930881 A EP 95930881A EP 0777741 A1 EP0777741 A1 EP 0777741A1
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EP
European Patent Office
Prior art keywords
dna
nucleic acid
film
electrode
biosensor
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EP95930881A
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German (de)
English (en)
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EP0777741A4 (fr
Inventor
Allen J. Bard
Xiao-Hong Xu
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IGEN International Inc
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IGEN Inc
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Publication of EP0777741A1 publication Critical patent/EP0777741A1/fr
Publication of EP0777741A4 publication Critical patent/EP0777741A4/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to the diagnostic field, and especially nucleic acid diagnostics.
  • the present invention relates to a probe or sensor * having a film containing metal centers, its preparation where a single-strand or double-strand nucleic acid sequence is immobilized thereon on, and its use in the subsequent detection of the nucleic acid by labelling with luminescent metal chelates.
  • nucleic acid diagnostics based on a surface designed modified electrical sensor, i.e., chips or electrode
  • immobilization and hybridization of nucleic acid such as DNA on a self- assembled thin film via surface reaction is also useful in studying molecular recognition of DNA.
  • Nucleic acid diagnostics has become an important area in molecular biology and biotechnology studies, with applications to the determinations of disease and food contaminating organisms and in forensic and environmental investigations.
  • the development of new DNA biosensors has led to the application of several detection techniques such as optical methods (e.g., luminescence, ellipsometry and pseudo- Brewster angle reflectometry) , piezoelectric devices (e.g., SAW, QCM) , and electrochemical techniques (e.g. , CV and SWV) .
  • optical methods e.g., luminescence, ellipsometry and pseudo- Brewster angle reflectometry
  • piezoelectric devices e.g., SAW, QCM
  • electrochemical techniques e.g. , CV and SWV
  • electrochemiluminescent methods of determining the presence of labelled materials are preferred over other methods for many reasons. They are highly diagnostic of the presence of a particular label, sensitive, nonhazardous, inexpensive and can be used in a wide variety
  • electrochemiluminescent ruthenium- and osmium-containing labels have been used in methods for detecting and quantifying analytes of interest in liquid media (U.S. Patent Nos. 5,310,687; 5,238,808; and 5,221,605).
  • electrogenerated chemi ⁇ luminescence (ECL) measurements to the detection of solution phase DNA intercalated with ruthenium-containing labels has been described (Carter, M.T. et al. (1990) Bioconiu ⁇ ate Chem 2:257-263).
  • ECL electrogenerated chemi ⁇ luminescence
  • solution phase analytes such as DNA has several drawbacks relative to detection of analytes absorbed to solid surfaces.
  • the advantages for detecting DNA via solid phase techniques as opposed to solution techniques are: (1) more sensitive (detection of monolayer quantities) ; (2) easier to separate DNA from sample (avoid interferences) ; and (3) possibility of detection of several different DNA in single analysis, with localized probes, e.g., as in sequencing studies.
  • chemilu inescent labels are used in immunochemical applications where the labels are excited into a luminescent state by reaction of the label with H 2 0 2 and an oxalate.
  • H 2 0 2 oxidatively converts the oxalate into a high energy derivative, which then excites the label.
  • the H 2 0 2 and an oxalate reaction scheme should work with any luminescent material that is stable under the oxidizing conditions of the assay, and can be excited by the high energy oxalate derivative.
  • a need exists for a solid phase system e.g., a biosensor, and method that (1) provides the necessary specificity absent systems that rely on the H 2 0 2 and oxalate reaction scheme; and (2) do not depend on solution techniques.
  • the present invention overcomes the limitations and drawbacks of the prior art.
  • the present invention provides a biosensor and its use for electrogenerated chemiluminescent detection of nucleic acid absorbed to a solid surface via the use of ruthenium- and osmium-containing chemiluminescent labels.
  • An object of the present invention is to provide a film containing an aluminum (III) alkanebisphosphonate layer having metallic aluminum centers, i.e., ionic aluminum Al(III) centers, for bonding to single-strand or double-strand DNA immobilized to said aluminum centers.
  • the aluminum (III) alkanebisphosphonate can be provided as a biosensor having a coating of A1 2 (C 4 BP) to bond to SS-DNA or ds-DNA.
  • a further object of the present invention is to provide a biosensor in the form of chips or electrodes with adsorbed DNA that is labelled with a luminescent label, such as an osmium or ruthenium moiety.
  • a still further object of the present invention is to prepare a biosensor by treating a silicon wafer to form a chromium layer and juxtaposed gold layer, then contacting the layered wafer with an anchoring agent; and subsequently immersing the product in A1(N0 3 ) 3 , bisphosphonic acid (H 2 0 3 P(CH 2 ) 4 P0 3 H 2 ) and A1(N0 3 ) 3 aqueous solutions.
  • Another object of the present invention involves the detection of a nucleic acid by labelling with luminescent metal chelates.
  • a further object of the present invention is to apply electrogenerated chemiluminescent techniques to a plurality, i.e., arrayed, oligonucleotide probes.
  • Fig. 1 shows a schematic representation of the silicon electrode of the present invention containing ionic aluminum Al(III) sites.
  • Fig. 2 shows immobilization of ds-DNA on a
  • FIG. 3A-3C show first (A), second (B) , and third (C) scans.
  • FIG. 4A-4C show schematic representations of immobilization of ss-DNA tagged with Ru(bpy) 3 2+ on the film (Fig. 4A) ; immobilization of ss-DNA on the film and hybridization of complementary strand DNA tagged with Ru(bpy) 3 2+ (Fig. 4B) ; immobilization of poly(dA) on the film, hybridization of poly(dT), and then interaction of Ru(phen) 3 2+ with the ds- DNA(poly(dA)*poly(dT) ) where the • represents an ECL active species.
  • Figures 5A-5C show a cyclic voltammogram (Fig. 5A) ; an emission-potential transient of the Al 2 (C 4 BP)/ ⁇ -l ss-DNA-Ru(bpy) 3 2+ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7
  • Figure 6 shows an emission-time transient for the
  • Figures 7A and 7B show ECL emission-potential transients at the A1 2 (C 4 BP) / ⁇ -lc ss-DNA/ ⁇ -1 ss-DNA-Ru(bpy) 3 + electrode (Fig. 7A) and at the Al 2 (C 4 BP)/ ⁇ -l ss-DNA/ ⁇ -1 ss-DNA- Ru(bpy) 3 2+ electrode (Fig. 7B) where both electrodes are immersed in 0.19 M phosphate buffer, pH 7, containing 0.13 M
  • FIGS. 8A and 8B show ECL emission-potential transients at the Al 2 (C 4 BP) /poly(dA) /poly(dT) /Ru(phen) 3 2+ electrode (Fig. 8A) and at the A1 2 (C 4 BP) /poly(dA) /Ru(phen) 3 + electrode (Fig. 8B) where both electrodes are immersed in 0.19 M phosphate buffer, pH 7, containing 0.13 M TPrA, and the potential was scanned from 0 to 1.60 at scan rate, 50 mV/s.
  • Figures 9A-9C show TEM images of Au substrate coated on a Formvar film on a #400 Cu grid (Fig. 9A) ; the A1 2 (C 4 BP) film on the Au substrate
  • FIG. 9B immobilized calf thymus ds-DNA on the A1 2 (C 4 BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution for - 4 h
  • Figures 10A-10C show TEM images of the Al 2 (C 4 BP) film on Au substrate coated on a Formvar film (Fig. 10A) ; immobilized calf thymus ds-DNA on the A1 2 (C 4 BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution, for -4 h (Fig.
  • the present invention relates to a sensor and method of detecting nucleic acids using the sensor.
  • the sensor can be a chip, electrode, or an appropriately modified surface for- adsorbing ss-DNA or ds-DNA.
  • the nucleic acids detected by the method of the present invention include DNA, cDNA or any synthetic variant thereof.
  • a nucleic acid as used throughout the specification and in the claims is meant DNA or any synthetic variant thereof.
  • Examples of DNAs detectable by the present method include chromosome DNA, plasmid DNA, viral DNA, bacterial DNA and recombinant DNA.
  • the length of nucleic acid sequence capable of detection by the present method ranges from about 2.7 nm to about 200 nm.
  • the nucleic acid sequence ranges from 8 base pair (bp) nucleotides to 3,000 base pair nucleotides. In a most preferred embodiment ranges from about 30 bp nucleotides to 1,500 bp nucleotides.
  • the nucleic acid sequence to be detected may be of purified nucleic acid or may be present in a biological sample.
  • Biological samples in which nucleic acids can be detected using the method of the present invention include but are not limited to biological fluids, e.g., serum, saliva, hair, skin, etc.
  • the nucleic acid can be purified from a sample using methods known to those skilled in the art (Current Protocols in
  • the aluminum (III) alkanebisphosphonate preferably used is a A1 2 (C 4 BP) , also [A1 2 C 4 BP] , film bearing biosensor and is prepared as follows. Silicon wafers were soaked in trichloroethylene for 30 min, rinsed twice with 2-propanol, rinsed with excess amount of deionized water, and then dried with a stream of dry nitrogen. The clean silicon wafers were primed with a 50 A chromium layer followed by deposition of a 2000 A gold layer. Chromium and gold targets (99.999%) were used to sputter the films onto the wafers in a MRC Model 8620 system at 10' 2 torr.
  • the phosphoric acid terminated surface was then thoroughly rinsed with the ethanol, dried with a stream of N 2 and then immersed alternately in 5 mM A1(N0 3 ) 3 , 5 mM bisphosphonic acid (H 2 0 3 P(CH 2 ) 4 P0 3 H 2 ) and 5 mM A1(N0 3 ) 3 aqueous solution, taking -4 h for each immersion, with washing with water between each step.
  • Al centers other metal centers, such as, lanthanum (La) and zirconium (Zr) are also contemplated.
  • the "spacer” may range from 2 to 16 carbons in length.
  • the sensor described above is used by: a) adsorbing a nucleic acid onto a film containing metal centers; b) reacting nucleic acid adsorbed to said film with a luminescent metal label; and c) detecting the nucleic acid metal label chelates formed in step b) via electrogenerated chemiluminescence of said chelates.
  • the film to which the nucleic acid is adsorbed should contain metal ions which are suitably spaced on the surface of the film to allow interaction of the metal with the phosphate backbone of the nucleic acid sequence.
  • metal centers suitable for use in binding to nucleic acid phosphate groups are aluminum, lanthanum, and zirconium.
  • the film contains an aluminum Al (III) metal center.
  • the nucleic acid adsorbed to the film in step (a) of the method of the present invention may be either double- stranded or single-stranded.
  • the adsorbed single-stranded nucleic acid is then hybridized to a complementary single- stranded nucleic acid sequence.
  • Conditions of hybridization are utilized which promote base pairing between the single- stranded DNA adsorbed to the film in its complementary sequence.
  • Factors influencing hybridization between nucleic acid sequences are known to those skilled in the art and include salt concentration of the hybridization solution, hybridization temperature and stringency of post-hybridization washes.
  • the length of hybridization may also be controlled to optimize binding.
  • Satiable buffers in which to carry out the hybridization reaction include 5mM Tris buffer, pH 7 containing 50 mM NaCl.
  • the complementary single-stranded nucleic acid sequence hybridized with the nucleic acid adsorbed to the film may be unlabeled or labeled with a luminescent metal label.
  • Suitable luminescent labels include ruthenium- and osmium- containing labels where ruthenium or osmium are bound to at least one polydentate ligand. If the metal has greater than one polydentate ligand, the polydentate ligands may be the same or different. (Other known ECL active labels can also be utilized that emit at different wave lengths such as organic ECL labels, e.g.
  • Polydentate ligands of either ruthenium or osmium include aromatic and aliphatic ligands. Suitable aromatic polydentate ligands include aromatic heterocyclic ligands. Preferred aromatic heterocyclic ligands are nitrogen-containing, such as, for example, bipyridyl, bipyrazyl, terpyridyl, and phenanthrolyl. If the metal chelate has greater than one polydentate ligand, the polydentate ligands may be the same orr different.
  • Suitable polydentate ligands may be unsubstituted, or substituted by any of a large number of substituents known to the art.
  • Suitable substituents include for example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocarbonyl, amidine, guanidiniu , ureide, sulfur-containing groups, phosphorous containing groups, and the carboxylate ester of N- hydroxysuccini ide.
  • the ruthenium or osmium may have one or more monodentate ligands, a wide variety of which are known to the art.
  • Suitable monodentate ligands include, for example, carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines.
  • a more complete list of the ligands, e.g., monodentate and polydentate ligands, that can be used in the present invention are set forth in U.S. Patent Nos.
  • one or more of the ligands of the metal to be attached to additional chemical labels, such as, for example, radioactive isotopes, fluorescent compounds, or additional luminescent ruthenium- or osmium-containing centers.
  • the complementary single-stranded nucleic acid may be tagged with the preferred luminescent metal labels of the present invention via co-valent bonding to one or more of the polydentate ligands of the metal label through one more amide linkages.
  • This linkage may be oriented so that the nucleic acid is bonded directly either to the carbinol or to the nitrogen of the amide linkage.
  • These chemical moieties may be ionized.
  • the complementary single-stranded nucleic acid is unlabeled and hence, hybridization to a single-stranded nucleic acid adsorbed to a film results in generation of unlabeled double-stranded nucleic acid adsorbed to the film.
  • double-stranded nucleic acid can be adsorbed to the film directly or can be created by first adsorbing single-stranded nucleic acid to the film and then hybridizing the adsorbed nucleic acid with its complementary sequence.
  • the film containing the adsorbed double-stranded nucleic acid is then immersed in a solution containing luminescent metal label or a solution suitable for promoting intercalation of the metal with the double-stranded nucleic acid. Examples of suitable solutions include, but are not limited to water.
  • a nucleic acid in which the luminescent metal containing labels intercalates to produce nucleic acid—metal label chelates is then detected by inducing the metal label present in the chelates to emit electromagnetic radiation by creating an excited state of the metal species that will luminesce at wave lengths from about 200 nanometers to about 900 nanometers, at ambient temperatures.
  • Intercalation (or more generally, association) of the ECL labeled species with DNA depends upon the experimental conditions in which the label is partially inserted between the base pairs of DNA. It is considered “association” because of an electrostatic interaction between a positively-charged label and the negatively-charged phosphate groups on the DNA. The exact nature of the interaction of Ru(phen) 3 2+ with DNA is uncertain, but is believed to be intercalation.
  • the temperature must be below the melting point of ds-DNA, preferably about 25-30° C.
  • the pH is typically near 7, but within a range of about 5 to about 8.
  • the intercalation or association reaction must be given sufficient time to occur; about 30 to about 60 minutes, although times as short as 10 minutes also work.
  • the metal label is excited by exposing the nucleic acid-metal label chelates to electrochemical energy. The potential at which the oxidation of the metal label will occur depends upon the exact structure of the metal label as well as factors such as the co-reactant utilized, the pH of the solution and the nature of the electrode used.
  • Suitable co- reactants which, when incubated with the nucleic acid-metal label chelates in the presence of the electrochemical energy, will result in emission of the metal label intercalated with the nucleic acid, include tripropylamine (TPrA) , oxalate or other organic acid such as pyruvate, lactate, malonate, tartrate and citrate. This oxidation can also be performed chemically, with some strong oxidants such as Pb0 2 or a Ce(IV) salt.
  • TPrA tripropylamine
  • oxalate or other organic acid such as pyruvate, lactate, malonate, tartrate and citrate.
  • This oxidation can also be performed chemically, with some strong oxidants such as Pb0 2 or a Ce(IV) salt.
  • Pb0 2 or a Ce(IV) salt Those of ordinary skill in the art recognize how to determine the optimal potential and emission wave length of an electrochemiluminescent system.
  • the rate of energy inputted into the system can provide a measure of the luminescent species.
  • Suitable measurements include, for example, measurements of electric current when the luminescent species is generated electrochemically, the rate of reductant or oxidant utilization when the luminescent species is generated chemically or the absorption of electromagnetic energy in photoluminescent techniques.
  • the luminescent species can be detected by measuring the emitted electromagnetic radiation. All of these measurements can be made either as continuous, rate-based measurements, or as cumulative methods which accumulate the signal over a long period of time.
  • An example of rate-based measurements would be by using photomultiplier tubes, photodiodes or phototransistors to produce electric currents proportional in magnitude to the incident light intensity. Examples of cumulative methods are the integration of rate-based data, and the use of photographic film to provide cumulative data directly.
  • the ECL emission was detected by a Model C123 single-photon-counting system (Hamamatsu Corp., Bridgewater, NJ) utilizing a Hamamatsu R928P PMT, cooled to -20°C in a Model TE 308 TSRF cooler controller (Products for Research Inc., Danvers, MA).
  • the meter output was fed into the y-axis of the x-y recorder, and the signal from the potentiostat was fed into the x-axis to afford ECL intensity versus bias potential display.
  • Solution analysis by ECL was carried out with a QPCR analyzer (Perkin-Elmer, Norwalk, CT) .
  • a MRC (Materials Research Corporation, Orangeburg, NY) Model 8620 sputtering system at 10 2 torr, with an RF power of 150 W and RF peak to peak voltage of 1.8 KV, was used to sputter gold (99.999%) on silicon wafers.
  • Polydeoxyadenylic acid Poly(dA)
  • polythymidylic acid poly(dT)
  • polydeoxycytidylic acid poly(dC)
  • CT calf thymus
  • ss-DNA can be carried out on a DNA synthesizer (e.g. Applied Biosyste s, Model 381A) . See also L.J. McBride and M.H. Cruthers, Tetrahedron Letters, 24, 245 (1983).
  • a DNA synthesizer e.g. Applied Biosyste s, Model 381A
  • the reagents used in the following examples include trichloroethylene (99.6%), 2-propanol (99.9%), tripropylamine (TPrA) (98%), Ru(bpy) 3 Cl 2 .6H 2 0, Ru(phen) 3 Cl 2 0, ethyl alcohol (200 proof), A1(N0 3 ) 3 .9H 2 0, K 2 Cr 2 0 7 , NaH 2 P0 4 and tris(hydroxymethyl) aminomethane and were used as received without purification.
  • TPrA tripropylamine
  • the assay buffer for ECL experiments contained 0.13 M TPrA and 0.19 M phosphate buffer, prepared by dissolving TPrA into a NaH 2 P0 4 solution and adjusting the pH to 7 with 1 M NaOH.
  • Deionized water from a Millipore Milli-Q (18 M ⁇ -cm) system was used to prepare all aqueous solutions and to rinse the electrode surface.
  • TEM samples were prepared by coating Au on a Formvar film on a #400 Cu grid with a vacum evaporator (Edwards 306) , fabricating the A1 2 (C 4 BP) film on Au following the procedure described above and then immobilizing DNA on the A1 2 (C 4 BP) film.
  • a transmission electron microscope (JEOL 100CX) at 80 KV was used to image the Au substrate, the A1 2 (C 4 BP) film and the immobilized DNA.
  • Calf thymus ds-DNA was immobilized on the surface of an A1 2 (C 4 BP) film by immersing the film in a solution of DNA (1.9 mM in nucleotide phosphate, NP) for 4 h (Figs. 1 and 2). The film was then rinsed three times with 4-mL portions of deionized water and then immersed for 4 h in either an aqueous 0.56 mM Ru(phen) 3 Cl 2 solution or a 0.12 mM Ru(phen) 3 (C10 4 ) 2 solution in MeCN. Ru(phen) 3 2+ associates with ds-DNA and can be detected through its electrogenerated chemiluminescence (ECL) .
  • ECL electrogenerated chemiluminescence
  • the film could be soaked in a mixed ds- DNA (1.9 mM NP) and Ru(phen) 3 Cl 2 (0.12mM) solution for 4 h to produce the adsorbed layer.
  • ECL was produced by scanning the potential of the electrode following film formation, DNA adsorption, and Ru(phen) 3 2+ association, from 0 to 1.6 V vs. a saturated calomel electrode (SCE) while it was immersed in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M tri-n- propylamine (TPrA) .
  • SCE saturated calomel electrode
  • TPrA tri-n- propylamine
  • the crystal frequency decreases as the A1 2 (C 4 BP) film forms and DNA and Ru(phen) 3 2+ are adsorbed on the surface, showing an increase of mass on the crystal during the different stages.
  • the mass change, ⁇ can be related to the frequency change, ⁇ f, by the Sauerbrey equation:
  • F 0 is the fundamental frequency of the unloaded crystal (6 MHz)
  • A is the electrode area (0.159 cm 2 )
  • p q is the density of quartz (2.65 g/cm 3 )
  • ⁇ q is the shear modulus of quartz (2.95 x 10 n dyne/cm 2 ) .
  • the electrode surface can be designed with immobilized DNA without adsorbing a detector molecule, Ru(phen) 3 2+ .
  • a ds-DNA on the surface can be detected by electrogenerated chemiluminescence of associated Ru(phen) 3 2+ .
  • Single-stranded DNA can also be immobilized on the A1 2 (C 4 BP) film surface and then hybridized with complementary DNA in solution with detection of the ds-DNA produced by ECL.
  • Example 2 Single-strand DNA • i-nm «hilization and hybridization.
  • ⁇ -1 tagged ss-DNA i.e., labeled with Ru(bpy) 3 2+
  • the amount of immobilized DNA-Ru(bpy) 3 + on the surface was determined by ECL resulting from the oxidation of Ru(bpy) 3 2+ and TPrA in a
  • Untagged ⁇ -lc ss-DNA was immobilized on an aluminum phosphate of the present invention.
  • the ⁇ -lc ss-DNA containing film was incubated in a complementary strand ⁇ -1 tagged ss-DNA solution at 60°C for 5 min and then cooled to room temperature gradually; during this cooling the ss-DNA hybridized with the complementary strand DNA (Fig. 4B) .
  • the hybridized DNA-Ru(bpy) 3 + on the film was detected by ECL as described above.
  • Poly(dA) was immobilized on an aluminum phosphate film of the present invention by soaking the film in a poly(dA) solution. After the immobilization, poly(dT) was hybridized with poly(dA) to produce poly(dA) «poly(dT) ds-DNA on the surface by incubating the film in a poly(dT) solution at 70°C for 5 min and then cooled to the room temperature gradually (Fig. 4C) . To intercalate Ru(phen) 3 2+ into the ds- DNA (poly(dA)-»poly(dT) ) , the A1 2 (C 4 BP)/poly(dA) »poly(dT) film was treated with a Ru(phen) 3 2+ solution. The hybridized poly(dA)*poly(dT)-Ru(phen) 3 + on the surface was determined by ECL based on the oxidation of Ru(phen) 3 2+ and TPrA in the solution.
  • Example 3 mmnfr.-. ⁇ ⁇ _*ntion and Detection of 30 bp ss-DNA.
  • the aluminum phosphate film, prepared as described above was immersed in a 1.38 ⁇ M solution of ⁇ -1 30 bp ss-DNA ⁇ tagged with Ru(bpy) 3 2+ ) for -2 h. This was employed as a working electrode for an ECL experiment in a 0.19 M phosphate buffer (pH 7) containing 0.13 M TPrA.
  • Cyclic voltammograms and emission transients were obtained by scanning the potential of the electrode from 0 to 1.6 V vs, a saturated calomel electrode (SCE) .
  • a representative voltammogram and emission detected with a single-photon-counting system are shown in Figures 5A-5C.
  • the broad oxidation was at - 1.2 V and a small reduction wave at -0.35 V observed in the voltammogram (Fig. 5A) arise from the oxidation of TPrA, Ru(bpy) 3 2+ and the Au substrate and the reduction of the oxide of Au.
  • the ECL emission from the A1 2 (CBP) / ⁇ -1 ss-DNA- Ru(bpy) 3 2+ electrode (Fig.
  • DNA on the A1 2 (C 4 BP) film the film was immersed in a 1.38 ⁇ M complementary strand ss-DNA (tagged with Ru(bpy) 3 2+ ) ( ⁇ -1 30 bp ss-DNA-Ru(bpy) 3 + ) solution.
  • the film in the solution was gradually heated to 60°C in water bath, incubated at 60°C for 5 min. and then slowly cooled to room-temperature, during which the ⁇ -1 ss-DNA-Ru(bpy) 3 2+ was hybridized with the complementary strand ⁇ -lc ss-DNA on the surface as illustrated in Fig. 4B.
  • the film with hybridized DNA was employed as the working electrode in an ECL cell as described above.
  • A1 2 (C 4 BP) / ⁇ -1 ss-DNA electrode is exposed to the ⁇ -1 ss-DNA- Ru(bpy) 3 2+ . It is preferred to cover all Al 3+ adsorption sites to avoid emission from the film/ ⁇ -1 ss-DNA/ ⁇ -1 ss-DNA- Ru(bpy) 3 2+ arising from some immobilization of ss-DNA- Ru(bpy) 3 2+ .
  • Example 5 Detection of poly.dA) Hybridized to polv(dT) using a Ruthenium I*»bei -
  • the A1 2 (C 4 BP)/poly(dA)/Ru(phen) 3 2+ film was employed as a working electrode in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M TPrA, no ECL emission was observed (Fig. 8A) . This is consistent with the lack of association of Ru(phen) 3 2+ with ss-DNA.
  • a film of poly(dA) hybridized with only poly(dT) to form ds-DNA did produce ECL.
  • This film formed by immersion in the poly(dA)solution, was incubated in a 38 ⁇ M poly(dT) solution (gradually heated to 70°C in a water bath, incubated at 70°C for 5 min and then slowly cooled to room temperature) . It was then treated with a 0.24 mM Ru(phen) 3 2+ aqueous solution and used as a working electrode in the TPrA-containing phosphate buffer for ECL investigation. ECL emission was observed as shown in a representative emission transient (Fig.
  • Example 6 TEM Images of the film and calf thymus ds-DNA. Samples, prepared by coating Au on a Formvar film on a #400 Cu grid with a vacuum evaporator, fabricating the aluminum phosphate film on the Au as described above, and then immobilizing DNA on the Al 2 (C 4 BP) film by immersing the film in a 1.65 mM [NP] of calf thymus ds-DNA for 4 h, were imaged with a transmission electron microscope (TEM) at 80 KV. As shown in Figs.
  • TEM transmission electron microscope
  • the featureless Au substrate shows formation of crystalline islands of A1 2 (C 4 BP) film (Fig. 9B) and clumps of DNA (Fig. 9C) .
  • the film (Fig. 10A) was then treated with either 1.65 mM [NP] calf thymus ds-DNA (Fig. 10B) or an identical solution of ds-DNA that had been subjected to sonication for 6 h (Fig. 10C) .
  • the results indicate that smaller clumps of DNA are adsorbed on the film after sonication.
  • FIG. 11A A further embodiment of the invention is shown in Figs. 11A and 11B.
  • a sensor surface having a multilayer film with bonding groups is provided with a complete set of oligonucleotide probes using similar techniques described above for adsorbing ds-DNA and for SS-DNA. See Fig. 11A.
  • the sensor surface of Fig. HA is contacted with DNA to be sequenced.
  • the above disclosed ECL procedures are then used to recognize zones with a complementary sequence.
  • Fig. 11B When chips are used, different types of ss-DNA that make up a test sequence are attached to the surface of a chip to make an array.
  • the chip array is then exposed to the sample solution to be sequenced, with formation of ds-DNA at the appropriate location being recognized by the ECL approach.

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Abstract

De l'ADN simple brin a été immobilisé sur une électrode recouverte d'une pellicule d'alcanebisphosphonate d'aluminium par immersion de cette électrode dans une solution d'ADN simple brin. Cet ADN simple brin immobilisé, marqué par Ru(bpy)32+, a été détecté par surveillance de la luminescence électrophotochimique (ECL) produite suite à l'oxydation dans une solution contenant de la tri-n-propylamine. Après l'immobilisation d'ADN simple brin non marqué, un brin d'ADN complémentaire marqué a été hybridé pour produire de l'ADN double brin sur la surface. L'étendue de l'hybridation d'ADN a été déterminée par la luminescence ECL de Ru(bpy)¿3?2+ marqué. L'ADN double brin immobilisé sur une surface pourrait également être détecté par observation de la luminescence ECL de Ru(phen)¿3?2+. La microscopie électronique à transmission (TEM) a été utilisée pour obtenir une image de la pellicule et de l'ADN immobilisé.
EP95930881A 1994-08-26 1995-08-25 Biocapteur et procede destines a la detection par luminescence electrophotochimique d'acides nucleiques adsorbes sur une surface solide Withdrawn EP0777741A4 (fr)

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EP0777741A4 (fr) 1999-01-13
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