WO1996006946A1

WO1996006946A1 - Biosensor for and method of electrogenerated chemiluminescent detection of nucleic acid adsorbed to a solid surface

Info

Publication number: WO1996006946A1
Application number: PCT/US1995/010630
Authority: WO
Inventors: Allen J. Bard; Xiao-Hong Xu
Original assignee: Igen, Inc.
Priority date: 1994-08-26
Filing date: 1995-08-25
Publication date: 1996-03-07
Also published as: JPH10509025A; AU3410395A; AU703344B2; EP0777741A1; CA2198489A1; EP0777741A4

Abstract

Single-strand DNA was immobilized on an electrode covered with an aluminum alkanebisphosphonate film by immersing it in an ss-DNA solution. The immobilized ss-DNA labeled with Ru(bpy)32+ was detected by monitoring the electrogenerated chemiluminescence (ECL) produced upon oxidation in a solution containing tri-n-propylamine. After immobilization of unlabeled ss-DNA, a complementary labeled strand DNA was hybridized to produce ds-DNA on the surface. The extent of DNA hybridization was determined by ECL of the labeled Ru(bpy)¿3?2+. Surface immobilized ds-DNA could also be detected by observing the ECL of intercalated Ru(phen)¿3?2+. Transmission electron microscopy (TEM) was employed to image the film and the immobilized DNA.

Description

Title of the Invention

BIOSENSOR FOR AND METHOD OF ELECTROGENERATED CHEMILUMINESCENT DETECTION OF NUCLEIC ACID ADSORBED TO A SOLID SURFACE Field Of Invention

The present invention relates to the diagnostic field, and especially nucleic acid diagnostics. In

particular, 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.

In addition to using the subject biosensor for 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. Background Of Invention

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). Of particular interest are labels which can be made to

luminesce through photochemical, chemical, and electrochemical reaction schemes. In particular, 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 of applications. Suitable labels comprise

electrochemiluminescent compounds, including organic compounds and metal chelates.

For example, 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). In addition, the application of 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). However, reaction schemes that are viable in the solution phase are often not applicable in the solid phase. More importantly, the detection of 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.

These luminescent systems are of increasing importance in diagnostics. For example, in U.S. Pat. No.

4,372,745, chemiluminescent labels are used in immunochemical applications where the labels are excited into a luminescent state by reaction of the label with H₂O₂ and an oxalate. In these systems, H₂O₂ oxidatively converts the oxalate into a high energy derivative, which then excites the label. It is expected, that in principle, the H₂O₂ 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. Unfortunately, this very versatility is a major drawback of the 4,372,745 patent: lack of selectivity or specificity, i.e., typical biological fluids containing the analyte of interest also contain a large number of potentially luminescent substances that can cause high background levels of luminescence.

Thus 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 H₂O₂ and oxalate reaction scheme; and (2) do not depend on solution techniques. The present invention overcomes the limitations and drawbacks of the prior art.

Summary Of Invention

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 Al₂(C₄BP) 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(NO₃)₃, bisphosphonic acid

(H₂O₃P(CH₂)₄PO₃H₂) and Al(NO₃)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.

These and other objects will become more apparent from the following detailed description and drawings.

Brief Description Of The Drawinσs

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

Al₂(C₄BP) film and interaction of

Ru(phen)₃ ²⁺ with the ds-DNA.

Figs. 3A-3C show first (A), second (B), and third

(C) scans. ECL emission-potential transients at the Al₂(C₄BP) /DNA-Ru(phen)₃ ²⁺ electrode in 0.19 M phosphate buffer, pH 7, containing 0.13 M TPrA. (Scanning was halted and the solution stirred after each scan.) Scans were initiated at 0 V and were directed toward more positive potentials. Scan rate, 50 mV/s.

Figs. 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 Al₂(C₄BP)/λ-1 ss-DNA-Ru(bpy)₃ ²⁺ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7

(Fig. 5B); and an emission-potential transient of the Al₂(C₄BP)λ-1 ss-DNA in the same solution (Fig. 5C). The electrode used in the experiments shown in Figures 5A-5C was prepared as described in the specification with immersion in a 1.38 μM λ-1 ss-DNA-Ru(bpy)₃ ²⁺ or λ-1 ss-DNA solution for -4 h, respectively. In each case, the potential was scanned from 0 to 1.60 V at v = 50 mV/s.

Figure 6 shows an emission-time transient for the

Al₂(C₄BP)/λ-1 ss-DNA-Ru(bpy)₃ ²⁺ electrode in 0.19 M phosphate buffer/0.13 M TPrA, pH 7, when the potential was stepped from 0 to 1.5 V.

Figures 7A and 7B show ECL emission-potential

transients at the Al₂(C₄BP)/λ-1c ss-DNA/λ-1 ss-DNA-Ru(bpy)₃ ²⁺ electrode (Fig. 7A) and at the Al₂(C₄BP)/λ-l ss-DNA/λ-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

TPrA and the potential was scanned from 0 to 1.60 at scan rate, 50 Mv/s.

Figures 8A and 8B show ECL emission-potential

transients at the

Al₂(C₄BP)/poly(dA)/poly(dT)/Ru (phen)₃ ²⁺ electrode (Fig. 8A) and at the

Al₂(C₄BP)/poly(dA)/Ru(phen)₃ ²⁺ 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 Al₂(C₄BP) film on the Au substrate

(Fig. 9B); immobilized calf thymus ds-DNA on the Al₂(C₄BP) 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 Al₂(C₄BP)

film on Au substrate coated on a Formvar film (Fig. 10A); immobilized calf thymus ds-DNA on the Al₂(C₄BP) film, prepared by immersing the film in a 1.65 mM [NP] of ds-DNA solution, for -4 h (Fig. 10B);

immobilized sonicated calf thymus ds-DNA on the Al₂(C₄BP) film, prepared by

immersing the film in a 1.65 mM [NP] of ds-DNA solution, sonicated for -6 h at room-temperature, for -4 h. (Fig. 10C) Figures 11A - 11B show sequencing via array

hybridization.

Detailed Description Of Invention

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. 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.

In the present invention, 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. Alternatively, the nucleic acid can be purified from a sample using methods known to those skilled in the art (Current Protocols in

Electrobiology (1994 ed. Ausubel, F.M. et al. John Wiley & Sons, Inc.)).

The aluminum (III) alkanebisphosphonate preferably used is a Al₂(C₄BP), also [Al₂C₄BP], 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.

The gold surface supported on the silicon wafers was cleaned with hot chromic acid (saturated K₂Cr₂O₇ in 90% H₂SO₄) 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. The phosphoric acid

terminated surface was then thoroughly rinsed with the

ethanol, dried with a stream of N₂ and then immersed

alternately in 5 mM A1(NO₃)₃, 5 mM bisphosphonic acid

(H₂0₃P(CH₂)₄P0₃H₂) and 5 mM A1(NO₃)₃ aqueous solution, taking -4 h for each immersion, with washing with water between each step. Although the preferred embodiment contemplates the use of Al centers, other metal centers, such as, lanthanum (La) and zirconium (Zr) are also contemplated. In addition, although a C₄ specie was utilized in producing the alkanebisphosphonate film, other chain links, C₂-C₁₆ may be utilized, which result, in adequate spacing of the aluminum ions on the surface of the film to permit contact with the phosphates of the DNA

backbone. In other words, 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. In step (a), 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.

Examples of metal centers suitable for use in binding to nucleic acid phosphate groups are aluminum, lanthanum, and zirconium. In a preferred embodiment, 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. When a single-stranded nucleic acid is adsorbed to the film, 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. In addition, 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. 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

phenanthrolyl. If 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.

5,310,687, 5,238,808 and 5,221,605, the subject matter of which are incorporated herein by reference.

It is also within the scope of this invention for 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. A more elaborate description of methods for

attaching a luminescent ruthenium-containing or osmium- containing label to amino groups of biological substances such as nucleic acid is provided in U.S. Patent No. 5,221,605 incorporated by reference.

In an alternative embodiment, 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. Thus, 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. In either case, 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.

In one embodiment of the present invention, 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. Examples of 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 PbO₂ 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 electrochemiluminescent species may be measured by any suitable mechanism such as measurement of an electric current or emitted electromagnetic radiation. For example, 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. In addition, of course, 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.

All of these luminescence-based methods entail repeated luminescence by the ruthenium-containing compound. The repetitive nature of the detectable event distinguishes these labels from radioactive isotopes or bound

chemiluminescent molecules such as luminol. The latter labels produce a detectable event only once per molecule (or atom) of label, thereby limiting their detectability.

The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof. Analysis was performed in a Plexiglas cell designed for ECL and electrochemical studies using the film of the present invention, a gold surface supported on the silicon wafer containing metallic sites, e.g., Al. A saturated calomel reference electrode (SCE) and a platinum wire counter electrode were used for all measurements. The electrochemical measurements coupled with ECL experiments were carried out with a Model 175 universal programmer, a Model 173

potentiostat (Princeton Applied Research, PAR, Princeton, NJ), and an Ominigraphic 2000 X-Y recorder (Houston Instruments, Houston, TX). 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 10² 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)) and calf thymus (CT) ss-DNA and ds-DNA were obtained from Sigma

Chemical Co. (St. Louis, MO) and were used without additional purification. The ss-DNA samples, λ-1 DNA

(5'GAAAATGTGCTGACCGGACATGAAAATGAG3'), (Seq. ID. No. 1) λ DNA tagged with Ru(bpy)₃ ²⁺(5'Ru(bpy)₃ ²⁺-

GAAAATGTGCTGACCGGACATGAAAATGAG3'), (Seq. ID. No. 2) and a complementary strand λ-lc DNA

(5'CTCATTTTCATGTCCGGTCAGCACATTTTC3') , were obtained from

Perkin-Elmer and diluted with a 5 mM tris buffer containing 50 mM NaCl(pH 7). Synthesis of 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).

The reagents used in the following examples include trichloroethylene (99.6%), 2-propanol (99.9%), tripropylamine (TPrA) (98%), Ru(bpy)₃Cl₂.6H₂O, Ru(phen)₃Cl₂O, ethyl alcohol (200 proof), Al(NO₃)₃.9H₂O, K₂Cr₂O₇, NaH₂PO₄ and tris (hydroxymethyl) aminomethane and were used as received without purification. Bisphosphonic acid H₂O₃P(CH₂)₄PO₃H₂ (C₄BPA), and 4- mercaptobutylphosphonic acid (MBPA) were synthesized in accordance with the techniques taught by Mallouk et al,

J.A.C.S., 115, 11855 (1993). The assay buffer for ECL

experiments contained 0.13 M TPrA and 0.19 M phosphate buffer, prepared by dissolving TPrA into a NaH₂PO₄ solution and

adjusting the pH to 7 with 1 M NaOH.

Deionized water from a Millipore Milli-Q (18 MΩ-cm) system was used to prepare all aqueous solutions and to rinse the electrode surface.

TEM Sample Preparation.

TEM samples were prepared by coating Au on a Formvar film on a #400 Cu grid with a vacum evaporator (Edwards 306), fabricating the Al₂(C₄BP) film on Au following the procedure described above and then immobilizing DNA on the Al₂(C₄BP) film. A transmission electron microscope (JEOL 100CX) at 80 KV was used to image the Au substrate, the Al₂(C₄BP) film and the immobilized DNA.

Example 1

Electrogenerated Chemiluminescent Detection of lmmobilized ds-DNA using A Ruthenium Label.

Calf thymus ds-DNA was immobilized on the surface of an Al₂(C₄BP) 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)₃Cl₂ solution or a 0.12 mM Ru(phen)₃(Clo₄)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)₃Cl₂ (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). Typical ECL transients, detected with a single-photon-counting apparatus, are shown in Figures 3A-3C. Emission arises from the energetic electron-transfer reaction between electrogenerated Ru(phen)₃ ³⁺ and an intermediate in the oxidation of TPrA:

DNA-Ru(phen)₃ ²⁺ (ads) - e → DNA-Ru(phen)₃ ³⁺ [1]

CH₃CH₂CH₂-N-Pr₂ - e - CH₃CH*,CH_:NPr-,-^f [2]

CH₃CH₂CH₂NPr₂ ⁺ → CH₃CH₂CHNPr₂ + H⁺ [3] DNA-Ru(phen)₃ ³⁺ + CH₃CH₂CHNPr₂→DNA-Ru(phen)₃ ^2+●+CH₃CH₂CHNPr₂ ⁺ [4] DNA-Ru(phen)₃ ^2+●→ DNA-Ru(phen)₃ ²⁺ + hυ [5]

The emission intensity decreased on a second scan and none was seen on a third, suggesting loss of Ru(phen)₃ ³⁺ from the film and diffusion into the bulk solution.

In a control experiment, an electrode with a film of Al₂(C₄BP) that had not been treated with DNA was soaked for 4 h in either an aqueous 0.56 mM Ru(phen)₃ ²⁺ or a 0.12 mM

Ru(phen)₃ ²⁺ in MeCN solution and then rinsed with MeCN and water. This electrode showed no ECL emission upon potential sweep in the same TPrA solution described above, demonstrating the adsorption of Ru(phen)₃ ²⁺ on the Al₂(C₄BP) film does not occur. The ability to generate Ru(phen)₃ ³⁺ electrochemically in this ECL experiment demonstrates that the Al₂(C₄BP) film and the DNA layer do not prevent heterogeneous electron-transfer reactions.

To obtain further evidence for Al, (C₄BP) film formation and DNA immobilization, experiments were undertaken with a gold-coated quartz crystal and a quartz crystal

microbalance (QCM). The gold was repeatedly treated with hot chromic acid and then rinsed with water and EtOH until the surface was hydrophilic, as indicated by contact angle

measurements. The crystal frequency was then measured in air during different stages of Al₂(C₄BP) film formation, after DNA adsorption, and after Ru(phen)₃ ²⁺ association. The film was rinsed after each step with deionized water and dried in a stream of N₂ before measurement of the frequency. Results are shown in Table 1.

a Immersions were sequential in order given from top to bottom; bare surface was gold; bare crystal frequency, 6.011329 MHz. ^b 0.5 mM MBPA in EtOH for - 24 h.

c 5 mM aq. Al(NO₃)₃ for - 4 h.

d 5 mM phosphoric acid for 4 h.

e Calf thymus DNA (3.8 mM NP) for 4 h.

f 0.24 mM Ru(phen)₃ ²⁺ in MeCN for 4 h.

g The variation and drift of the signal over the series of measurements was about ± 1 Hz.

h Assuming roughness factor for gold of 2 (total surface area, 0.6 cm²). For comparison, for a close-packed monolayer of

MBPA, T = 0.6 x 10-⁹ mol/cm².

i Assuming an Al(H₂O)₃ ³⁺ until adsorbed.

j Corresponding to moles of nucleotide phosphate (NP) per cm².

Clearly, the crystal frequency decreases as the Al₂(C₄BP) 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, Δm, can be related to the frequency change, Δf, by the Sauerbrey equation:

where F₀ is the fundamental frequency of the unloaded crystal (6 MHz), A is the electrode area (0.159 cm²), p_q is the density of quartz (2.65 g/cm³) and μ_q is the shear modulus of quartz (2.95 x 10¹¹ dyne/cm²). With these constants,

Δm (ng) = -1.95 Δf (Hz)

The mass changes calculated in this way are also given in Table l. These can be converted to approximate surface concentrations, r, assuming a roughness factor of 2 (i.e., total surface area of both sides of the quartz crystal of 0.6 cm²) . ECL could also be detected from both gold surfaces of the quartz crystal, when used as a substrate for Al₂(C₄BP) film formation, DNA adsorption and Ru(phen)₃ ²⁺ association and then scanned in the TPrA solution.

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 Al₂(C₄BP) film surface and then hybridized with complementary DNA in solution with detection of the ds-DNA produced by ECL. Example 2

Single-strand DNA immobilization and hybridization. λ-1 tagged ss-DNA (i.e., labeled with Ru(bpy)₃ ²⁺) was immobilized on an aluminum phosphate film of the present invention by immersing the film in the DNA solution (Fig. 4A). 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 λ-1c ss-DNA was immobilized on an aluminum phosphate of the present invention. The λ-1c ss-DNA

containing film was incubated in a complementary strand λ-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 ²⁺ 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 Al₂(C₄BP) /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.

Example 3

lmmobilization and Detection of 30 bp ss-DNA.

lmmobilized On Aluminum Phosphate Film.

The aluminum phosphate film, prepared as described above was immersed in a 1.38 μM solution of λ-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 Al₂(C₄BP) /λ-1 ss-DNA- Ru(bpy)₃ ²⁺ electrode (Fig. 5B) demonstrates that the λ-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 light intensity detected by the photomultiplier tube, when the potential was stepped from 0 to 1.50 V, decreased with time (Fig. 6). The decay of intensity with time suggests

desorption of ss-DNA-Ru(bpy)₃ ²⁺ from the electrode surface or irreversible decomposition of the emitter. Emission results from the energetic electron-transfer reaction between

electrogenerated Ru(bpy)₃ ³⁺ and an intermediate in the

oxidation of TPrA: DNA-Ru(bpy)₃ ²⁺ (ads) → DNA-Ru(bpy)₃ ³⁺ + e' [6]

CH₃CH₂CH₂NPr₂ - e → CH₃CH₂CH₂NPr₂ ⁺ [7]

CH₃CH₂CH₂NPr₂ ⁺ → CH₃CH₂CHNPr₂ + H⁺ [8]

DNA-Ru(bpy)₃ ³⁺+CH₃CH₂CHNPr₂ → DNA-Ru(bpy) ₃ ^2+*+CH₃CH₂CHNPr₂ ⁺ [9]

DNA-Ru(bpy)₃ ^2+*→ DNA-Ru(bpy)₃ ²⁺ + hυ [10] No ECL emission was observed from a film prepared by immersing it in solution of unlabeled ss-DNA: 1.38 μM λ-1 30 bp ss-DNA or a 0.37 μM λ-1c 30 bp ss-DNA solution (Fig. 5C).

A control experiment with the aluminum phosphate film immersed in a 304 nM Ru(bpy)₃ ²⁺ solution for - 4 h shows a negligible ECL signal, indicating that Ru(bpy)₃ ²⁺ does not adsorb on the film and oxidize.

Example 4

Detection of 30 bp ss-DNA Hybridized to

Complementary SS-DNA Tagged With A Ruthewium Label.

After the immobilization of unlabeled λ-1c 30 bp ss-

DNA on the Al₂(C₄BP) film, the film was immersed in a 1.38 μM complementary strand ss-DNA (tagged with Ru(bpy)₃ ²⁺) (λ-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 λ-1 ss-DNA-Ru(bpy)₃ ²⁺ was hybridized with the

complementary strand λ-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. The ECL emission from the film was observed as shown in Fig. 7A.

However, when λ-1 ss-DNA, rather than λ-1c ss-DNA, was immobilized on the film and followed by the same hybridization procedure as described above by incubating the film in the λ-1 ss-DNA-Ru(bpy)₃ ²⁺ solution, heating to 60°C, and then cooling to room-temperature, no obvious ECL emission was evident as shown in Fig. 7B. This demonstrates that the ECL emission in Fig. 7A arises from the hybridization of immobilized λ-lc ss- DNA with λ-1 ss-DNA-Ru(bpy)₃ ²⁺ in the solution. The emission again results from the oxidation of Ru(bpy)₃ ²⁺-tagged

hybridized ss-DNA and TPrA in the solution as described by Eqs. [6] - [10]. In this experiment the Al₂(C₄BP) film should be incubated in the λ-1 ss-DNA solution for a sufficiently long time (at least 4 h) to cover all possible Al (III) binding sites on the film with the λ-1 ss-DNA before the

Al₂(C₄BP) /λ-1 ss-DNA electrode is exposed to the λ-1 ss-DNA- Ru(bpy)₃ ²⁺. It is preferred to cover all Al³⁺ adsorption sites to avoid emission from the film/λ-1 ss-DNA/λ-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 poly(dT) using a Ruthenium Label.

The aluminum phosphonate film, prepared as described above, was immersed in a 21 μM poly(dA) solution for -4 h, then in a 0.24 mM Ru(phen)₃ ²⁺ solution for -4 h. When the Al₂(C₄BP) /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. However, 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 μM 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. 8B), demonstrating that poly(dT) in the solution hybridized with the poly(dA) on the surface to produce a ds-DNA [poly(dA)●poly(dT] and

Ru(phen)₃ ²⁺ intercalated with the ds-DNA as shown in Fig. 4c.

These examples show that Ru(phen)₃ ²⁺ only

intercalates with ds-DNA [poly (dA)●poly (dT], but not with ss- DNA (poly(dA)). Intercalation of Ru(phen)₃ ²⁺ with ss-DNA and ds-DNA, ECL emission was measured from a 43 nM Ru(phen)₃ ²⁺ solution, a 43 nM Ru(phen)₃ ²⁺ containing 57 μM nucleotide phosphate ([NP]) of calf thymus ss-DNA and a 43 nM Ru(phen)₃ ²⁺ containing 33 μM [NP] of calf thymus ds-DNA with a QPCR analyzer. Results are given in Table 2,

indicating that the ECL emission decreased after ds-DNA was added to the Ru(phen)₃ ²⁺ solution. A negligible change, however, in the ECL signal was shown when ss-DNA was added, further demonstrating that Ru(phen)₃ ²⁺ only intercalates with ds-DNA, but not with ss-DNA.

A control experiment in which a film, after the immobilization of poly(dA), was incubated in a 21 μM poly(dA) or a 46 μM poly(dC) solution instead of poly(dT) (70°C for 5 min then cooled to room-temperature) and then treated with Ru(phen)₃ ²⁺ for -2 h produces no ECL emission. This indicates that the Al₂(C₄BP) /poly(dA) electrode can distinguish a complementary strand of poly(dT) DNA from non-complementary ones. 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 Al₂(C₄BP) 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. 9A-9C the featureless Au substrate (Fig. 9A) shows formation of crystalline islands of A1₂(C₄BP) 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.

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.

Although the invention has been described in

conjunction with the specific embodiments, it is evident that many alternatives and variations will be apparent to those skilled in the art in light of the foregoing description.

Accordingly, the invention is intended to embrace all of the alternatives and variations that fall within the spirit and scope of the appended claims.

Claims

1. A method of detecting nucleic acids comprising:

a. forming a sensor surface bearing a film having metallic centers;

b. adsorbing at least one nucleic acid onto to said metal centers;

c. reacting nucleic acid adsorbed to said film with a luminescent metal label; and

d. detecting the nucleic acid metal label chelates formed in step (c) via electrogenerated chemiluminescence of said chelates.

2. The method of claim 1, wherein the nucleic acid of step (b) is double-stranded.

3. The method of claim 1, wherein the nucleic acid of step (b) is single-stranded.

4. The method of claim 3, wherein step (c) further comprises binding the single-stranded nucleic acid adsorbed to the film, to a complementary single-stranded nucleic acid sequence.

5. The method of claim 1, wherein said electrogenerated chemiluminescence arises from a reaction of an

electrogenerated metal label in the nucleic acid -metal label chelate and a suitable coreactant.

6. The method of claim 5, wherein said electrogenerated metal label is produced via emission from the sensor which is an electrode.

7. A method of detecting nucleic acids comprising:

a. forming an electrode by adsorbing at least one nucleic acid onto said electrode;

b. reacting nucleic acid adsorbed to said electrode with a luminescent label; and

c. detecting the nucleic acid label formed in step (b) via electrogenerated chemiluminescence.

8. The method of claim 7, wherein the nucleic acid of step (b) is double-stranded.

9. The method of claim 7, wherein the nucleic acid of step (b) is single-stranded.

10. The method of claim 8, wherein step (c) further comprises binding the single-stranded nucleic acid adsorbed to the electrode, to a complementary single-stranded nucleic acid sequence.

11. The method of claim 7, wherein said electrogenerated chemiluminescence arises from a reaction of an electrogenerated metal label in the nucleic acid - metal label chelate and a suitable coreactant.

12. The method of claim 11, wherein said

electrogenerated metal label is produced via emission from the electrode.

13. An aluminum alkanebisphosphonate biosensor

comprising:

a substrate and a first metallic layer; and an aluminum alkanebisphosphonate layer forming (Al) metallic centers.

14. A biosensor according to claim 13 , wherein said biosensor further includes single-strand DNA immobilized to said aluminum centers.

15. A biosensor according to claim 14, wherein said DNA is labelled with and an osmium or ruthenium moiety.

16. A biosensor according to claim 13, wherein said biosensor further includes double-strand DNA immobilized to said aluminum centers.

17. A biosensor according to claim 16, wherein said DNA is labelled with an osmium or ruthenium moiety.

18. A method of preparing a biosensor for nucleic acid, comprising:

a) treating a silicon wafer to form a chromium layer and juxtaposed gold layer;

b) contacting said layered wafer with an anchoring agent; and

c) immersing the product of step b) alternately in Al(NO₃)₃, bisphosphonic (H₂O₃P(CH₂)₄PO₃H₂) and Al(NO₃)₃ aqueous solutions.

19. A biosensor comprising:

an electrode substrate, adsorbed ss-DNA or ds-DNA immobilized on said electrode, and a detectable label on said ss-DNA or ds-DNA.

20. A biosensor according to claim 19, wherein said DNA is labelled with an osmium or ruthenium moiety or a

luminescent label.