WO1996006946A1 - 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 Download PDF

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WO1996006946A1
WO1996006946A1 PCT/US1995/010630 US9510630W WO9606946A1 WO 1996006946 A1 WO1996006946 A1 WO 1996006946A1 US 9510630 W US9510630 W US 9510630W WO 9606946 A1 WO9606946 A1 WO 9606946A1
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dna
nucleic acid
film
electrode
label
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PCT/US1995/010630
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English (en)
Inventor
Allen J. Bard
Xiao-Hong Xu
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Igen, Inc.
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Priority to EP95930881A priority Critical patent/EP0777741A4/fr
Priority to AU34103/95A priority patent/AU703344B2/en
Priority to JP8508822A priority patent/JPH10509025A/ja
Publication of WO1996006946A1 publication Critical patent/WO1996006946A1/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
  • 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.
  • CV and SWV electrochemical techniques
  • Suitable labels comprise
  • electrochemiluminescent compounds including organic compounds and metal chelates.
  • 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 chemiluminescence (ECL) measurements to the detection of solution phase DNA intercalated with ruthenium-containing labels has been described (Carter, M.T. et al. (1990) Bioconjugate Chem 2:257-263).
  • ECL electrogenerated chemiluminescence
  • 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.
  • chemiluminescent labels are used in immunochemical applications where the labels are excited into a luminescent state by reaction of the label with H 2 O 2 and an oxalate.
  • H 2 O 2 oxidatively converts the oxalate into a high energy derivative, which then excites the label. It is expected, that in principle, the H 2 O 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.
  • 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
  • the aluminum (III) alkanebisphosphonate can be provided as a biosensor having a coating of Al 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 Al(NO 3 ) 3 , bisphosphonic acid
  • 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
  • Figs. 3A-3C show first (A), second (B), and third
  • Figs. 4A-4C show schematic representations of
  • Figures 5A-5C show a cyclic voltammogram (Fig. 5A);
  • FIG. 5B an emission-potential transient of the Al 2 (C 4 BP) ⁇ -1 ss-DNA in the same solution
  • Figure 6 shows an emission-time transient for the
  • FIGS 7A and 7B show ECL emission-potential
  • FIGS 8A and 8B show ECL emission-potential
  • Figures 9A-9C show TEM images of Au substrate coated on a Formvar film on a #400 Cu grid (Fig. 9A); the Al 2 (C 4 BP) film on the Au substrate
  • FIG. 9B immobilized calf thymus ds-DNA on the Al 2 (C 4 BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution for - 4 h (Fig. 9C).
  • Figures 10A-10C show TEM images of the Al 2 (C 4 BP)
  • Fig. 10A immobilized calf thymus ds-DNA on the Al 2 (C 4 BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution, for -4 h (Fig. 10B);
  • FIG. 10C Figures 11A - 11B show sequencing via array
  • 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.
  • 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. In a preferred embodiment, 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 Al 2 (C 4 BP), also [Al 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. Other techniques, such as chemical vapor deposition (CVD) to apply the Au or Cr layers, can also be used.
  • CVD chemical vapor deposition
  • the gold surface supported on the silicon wafers was cleaned with hot chromic acid (saturated K 2 Cr 2 O 7 in 90% H 2 SO 4 ) for -10 s and then rinsed with copious amounts of water. This process was repeated until the surface contact angle with water was less than 15°.
  • the clean Au surface was then immediately soaked in an anchoring agent, 0.5 mM 4- mercaptobutylphosphonic acid (MBPA) solution in absolute ethanol for -24 h. See Fig. 1.
  • MBPA 4- mercaptobutylphosphonic acid
  • the "spacer” may range from 2 to 16 carbons in length.
  • the sensor described above is used by:
  • step (a) 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 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.
  • ECL active labels can also be utilized that emit at different wave lengths such as organic ECL labels, e.g. sulfonated-9,10-diphenylanthracene.
  • 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
  • the metal chelate has greater than one polydentate ligand, the polydentate ligands may be the same or 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, guanidinium, ureide, sulfur-containing groups, phosphorous containing groups, and the carboxylate ester of N- hydroxysuccinimide.
  • 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,
  • 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.
  • 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 PbO 2 or a Ce(IV) salt.
  • the electrochemiluminescent species may be measured by any suitable mechanism such as measurement of an electric current or emitted electromagnetic radiation.
  • 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
  • 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.
  • 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.
  • chemiluminescent molecules such as luminol.
  • the latter labels produce a detectable event only once per molecule (or atom) of label, thereby limiting their detectability.
  • 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 Biosystems, Model 381A) . See also L.J. McBride and M.H. Cruthers, Tetrahedron Letters, 24, 245 (1983).
  • a DNA synthesizer e.g. Applied Biosystems, 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 O, Ru(phen) 3 Cl 2 O, ethyl alcohol (200 proof), Al(NO 3 ) 3 .9H 2 O, K 2 Cr 2 O 7 , NaH 2 PO 4 and tris (hydroxymethyl) aminomethane and were used as received without purification.
  • Bisphosphonic acid H 2 O 3 P(CH 2 ) 4 PO 3 H 2 (C 4 BPA), and 4- mercaptobutylphosphonic acid (MBPA) were synthesized in accordance with the techniques taught by Mallouk et al,
  • experiments contained 0.13 M TPrA and 0.19 M phosphate buffer, prepared by dissolving TPrA into a NaH 2 PO 4 solution and
  • 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 Al 2 (C 4 BP) film on Au following the procedure described above and then immobilizing DNA on the Al 2 (C 4 BP) film.
  • a transmission electron microscope (JEOL 100CX) at 80 KV was used to image the Au substrate, the Al 2 (C 4 BP) film and the immobilized DNA.
  • Calf thymus ds-DNA was immobilized on the surface of an Al 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 (Clo 4 ) 2 solution in MeCN. Ru(phen) 3 2+ associates with ds-DNA and can be detected through its electrogenerated chemiluminescence (ECL). Alternatively, 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 electrogenerated chemiluminescence
  • ECL was produced by scanning the potential of the electrode following film formation, DNA adsorption, and
  • QCM microbalance
  • the crystal frequency decreases as the Al 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
  • the mass change, ⁇ m can be related to the frequency change, ⁇ f, by the Sauerbrey equation: where 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 ) and ⁇ q is the shear modulus of quartz (2.95 x 10 11 dyne/cm 2 ). With these constants,
  • 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 Al 2 (C 4 BP) film surface and then hybridized with complementary DNA in solution with detection of the ds-DNA produced by ECL.
  • ⁇ -1 tagged ss-DNA i.e., labeled with Ru(bpy) 3 2+
  • the amount of immobilized DNA-Ru(bpy) 3 2+ on the surface was determined by ECL resulting from the oxidation of Ru(bpy) 3 2+ and TPrA in a solution.
  • Untagged ⁇ -1c ss-DNA was immobilized on an aluminum phosphate of the present invention.
  • 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).
  • a poly(dT) solution at 70°C for 5 min and then cooled to the room temperature gradually (Fig. 4C).
  • the Al 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 2+ on the surface was determined by ECL based on the oxidation of Ru(phen) 3 2+ and TPrA in the solution.
  • 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
  • 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 Al 2 (C 4 BP) / ⁇ -1 ss-DNA- Ru(bpy) 3 2+ electrode (Fig. 5B) demonstrates that the ⁇ -1 30 bp ss-DNA-Ru(bpy) 3 2+ was immobilized on the film.
  • the decay of the light intensity (I) as a function of time (t) was also investigated with the single-photon-counting system.
  • the decay of intensity with time suggests
  • 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 2+ ) 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
  • Al 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+ .
  • the aluminum phosphonate film prepared as described above, was immersed in a 21 ⁇ M poly(dA) solution for -4 h, then in a 0.24 mM Ru(phen) 3 2+ solution for -4 h.
  • the Al 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) was employed as a working electrode in a solution of 0.19 M phosphate buffer (pH 7) containing 0.13 M TPrA.
  • FIG. 11A and 11B 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
  • 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.

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.
PCT/US1995/010630 1994-08-26 1995-08-25 Biocapteur et procede destines a la detection par luminescence electrophotochimique d'acides nucleiques adsorbes sur une surface solide WO1996006946A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP95930881A EP0777741A4 (fr) 1994-08-26 1995-08-25 Biocapteur et procede destines a la detection par luminescence electrophotochimique d'acides nucleiques adsorbes sur une surface solide
AU34103/95A AU703344B2 (en) 1994-08-26 1995-08-25 Biosensor for and method of electrogenerated chemiluminescent detection of nucleic acid adsorbed to a solid surface
JP8508822A JPH10509025A (ja) 1994-08-26 1995-08-25 固体表面に吸着された核酸の電気的に発生した化学ルミネッセンス性検出のためのバイオセンサーおよび方法

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WO2001042508A2 (fr) * 1999-12-09 2001-06-14 Motorola, Inc. Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques
US6518024B2 (en) 1999-12-13 2003-02-11 Motorola, Inc. Electrochemical detection of single base extension
EP1322787A1 (fr) * 2000-10-05 2003-07-02 Virginia Tech Intellectual Properties, Inc. Biopuce, methodes photoluminescentes permettant d'identifier un materiau biologique, et appareils utilises avec lesdites biopuces et methodes
US6673533B1 (en) 1995-03-10 2004-01-06 Meso Scale Technologies, Llc. Multi-array multi-specific electrochemiluminescence testing
US6824669B1 (en) 2000-02-17 2004-11-30 Motorola, Inc. Protein and peptide sensors using electrical detection methods
US6977151B2 (en) 1996-11-05 2005-12-20 Clinical Micro Sensors, Inc. Electrodes linked via conductive oligomers to nucleic acids
US7138121B2 (en) 2003-01-23 2006-11-21 Spangler Brenda D Biosensors utilizing dendrimer-immobilized ligands and there use thereof
US7361470B2 (en) 2003-05-13 2008-04-22 Trustees Of Boston College Electrocatalytic nucleic acid hybridization detection
WO2006076047A3 (fr) * 2004-08-06 2009-04-09 Trustees Boston College Détection d'hybridation d'acides nucléiques électrocatalytique
EP2186910A2 (fr) 1997-12-23 2010-05-19 Meso Scale Technologies LLC Procédés et compositions pour dosages a luminescence ameliorée utilisant des microparticules
US7935481B1 (en) 1999-07-26 2011-05-03 Osmetech Technology Inc. Sequence determination of nucleic acids using electronic detection
US8140148B2 (en) 1998-01-20 2012-03-20 Boston Scientific Scimed Ltd. Readable probe array for in vivo use
US8888969B2 (en) 2008-09-02 2014-11-18 The Governing Council Of The University Of Toronto Nanostructured microelectrodes and biosensing devices incorporating the same
US9151746B2 (en) 1999-04-21 2015-10-06 Osmetech Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
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WO2019201901A1 (fr) 2018-04-18 2019-10-24 F. Hoffmann-La Roche Ag Nouveaux anticorps anti-thymidine kinase
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US6066448A (en) * 1995-03-10 2000-05-23 Meso Sclae Technologies, Llc. Multi-array, multi-specific electrochemiluminescence testing
US6673533B1 (en) 1995-03-10 2004-01-06 Meso Scale Technologies, Llc. Multi-array multi-specific electrochemiluminescence testing
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US6977151B2 (en) 1996-11-05 2005-12-20 Clinical Micro Sensors, Inc. Electrodes linked via conductive oligomers to nucleic acids
US6210932B1 (en) 1997-04-09 2001-04-03 Cis Bio International System for detecting nucleic acid hybridization, preparation method and application thereof
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US8140148B2 (en) 1998-01-20 2012-03-20 Boston Scientific Scimed Ltd. Readable probe array for in vivo use
US9557295B2 (en) 1999-04-21 2017-01-31 Osmetech Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
US9151746B2 (en) 1999-04-21 2015-10-06 Osmetech Technology, Inc. Use of microfluidic systems in the electrochemical detection of target analytes
US7935481B1 (en) 1999-07-26 2011-05-03 Osmetech Technology Inc. Sequence determination of nucleic acids using electronic detection
WO2001042508A2 (fr) * 1999-12-09 2001-06-14 Motorola, Inc. Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques
WO2001042508A3 (fr) * 1999-12-09 2002-03-14 Motorola Inc Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques
US6518024B2 (en) 1999-12-13 2003-02-11 Motorola, Inc. Electrochemical detection of single base extension
US6824669B1 (en) 2000-02-17 2004-11-30 Motorola, Inc. Protein and peptide sensors using electrical detection methods
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US7138121B2 (en) 2003-01-23 2006-11-21 Spangler Brenda D Biosensors utilizing dendrimer-immobilized ligands and there use thereof
US7741033B2 (en) * 2003-05-13 2010-06-22 Trustees Of Boston College Electrocatalytic nucleic acid hybridization detection
US7361470B2 (en) 2003-05-13 2008-04-22 Trustees Of Boston College Electrocatalytic nucleic acid hybridization detection
WO2006076047A3 (fr) * 2004-08-06 2009-04-09 Trustees Boston College Détection d'hybridation d'acides nucléiques électrocatalytique
US9791402B2 (en) 2008-09-02 2017-10-17 The Governing Council Of The University Of Toronto Nanostructured microelectrodes and biosensing devices incorporating the same
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WO2018141768A1 (fr) 2017-02-02 2018-08-09 Roche Diagnostics Gmbh Dosage immunologique utilisant au moins deux agents de liaison spécifiques à un analyte pegylés
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EP0777741A1 (fr) 1997-06-11
CA2198489A1 (fr) 1996-03-07
EP0777741A4 (fr) 1999-01-13
AU3410395A (en) 1996-03-22
JPH10509025A (ja) 1998-09-08
AU703344B2 (en) 1999-03-25

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