MXPA99011881A - Nucleic acid biosensor diagnostics - Google Patents

Abstract

Se describe un biosensor para análisis directo de hibridación deácido nucleico por medio del uso de una fibraóptica funcionalizada con moléculas deácido nucleico y transducción de fluorescencia. Se inmovilizan sondas deácidos nucleicos sobre la superficie de fibrasópticas y sufren hibridación conácidos nucleicos complementarios introducidos en el ambiente local del detector. Se detectan eventos de hibridación por el uso de compuestos fluorescentes, los cuales se enlazan en los híbridos deácidos nucleicos. La invención encuentra uso en la detección y tamizado de trastornos genéticos, virus y microorganismos patogénicos. Las aplicaciones en biotecnología incluyen monitoreo de cultivos de genes y expresión de genes y la eficacia (por ejemplo dosis-respuesta) de farmacéuticos para terapia de genes. La invención incluye sistemas biosensores en los cuales se conectan moléculas fluorescentes a las moléculas deácidos nucleicos inmovilizadas. El método preferido para inmovilización deácidos nucleicos es por síntesis deácido nucleico de fase sólida in situ. Se alcanza el control delíndice de refracción delácido nucleico inmovilizado, por la química de derivación de soporte y la síntesis deácido nucleico. La derivación preferida de fibraóptica produce un recubrimiento de ADN de mayoríndice de refracción que el núcleo de fibra sobre la superficie de la fibra.

Description

- DIAGNOSTICS WITH NUCLEIC ACID BIOSENSOR DESCRIPTION OF THE INVENTION The present invention is generally directed to biosensors that are useful in the identification and analysis of biologically important nucleic acids. The biosensors of the present invention and their applied methods provide a means for the direct analysis of nucleic acid hybridization, and, therefore, have application to a myriad of biological fields including clinical diagnosis. 10 The detection and identification of microorganisms is a common problem for many areas of human and veterinary health. For example, the detection of pathogenic species such as Salmonella typhimurium, Listeria monocytogenes, and Escherichia coli that are causative agents of major food-borne epidemics is a major concern within the food industry with respect to the quality and safety of food supply. In other areas of human and veterinary health care, the detection and identification of infectious diseases caused by microorganisms and pathogenic viruses is a first step in the diagnosis and treatment. For example, it is estimated that 10-15 million visits to clinics per year are for the detection and treatment of three main pathogens - Clamydia ssp. , Trichomonas vaginalis and Gradenerella vaginitis. The infections of these organisms annually affect 3.75 million, 0.75 million and 1.5 million patients, respectively. The classical techniques routinely used for the detection and identification of microorganisms are often labor-intensive, involving plating procedures that require long analysis times. To illustrate, the method currently used for the detection of Listeria monocytogenes in foods and food facilities involves a three-stage analysis.The analysis begins with the enrichment of the sample that will be analyzed in a nutrient broth for 2 to 4 days. After the enrichment period, the plating of the sample in selective agar medium is carried out and the sample is allowed to incubate for 2 days in order to obtain colonies for biotyping and serotyping, which can take 20 days to finish (T cLauchlin et al., 1988, Microbiology Review, 5_5: 578) Crop-based detection processes require analysis times that are too long for effective monitoring and timely intervention in order to avoid the spread of biologically hazardous materials. or threat diseases.In addition, although these methods have been improved in the last decade, the opportunity to obtain false negative results It is still important and it is difficult to grow many microorganisms. Therefore, plating / culture methods are limited with respect to their sensitivity, specificity and longevity of analysis times that are required. In order to shorten the time required to detect and identify bacteria, viruses and pathogenic genetic diseases, rapid tests such as enzyme immunoassay (EIA) have been developed (Olapedo et al., 1992). Although immunoassay techniques can be very sensitive and effective, they are practical drawbacks that have restricted the use of these methods. These drawbacks include the need for highly experienced personnel, long analysis and preparation times, as well as the large quantities of expensive reagents that are required to perform such analyzes. With the advent of nucleic acid amplification techniques (the polymerase chain reaction), in vitro amplification of specific sequences of a portion of DNA or RNA is now possible. The detection of very low numbers of microorganisms has been demonstrated (Roseen et al., 1991; Golsteyn et al., 1991, Ernas, K., et al., 1991). The technique of polymerase chain reaction is sensitive and specific but involves complex manipulations to carry out the tests and particularly is not suitable for large numbers of samples. Due to the sensitivity of the polymerase chain reaction (PCR) technology, rooms or special areas for the preparation of samples and analysis are required to avoid contamination. In many tests, PCR results must be confirmed by additional hybridization assays. RNAs are difficult to analyze by PCR, but they are very important for human viral detection. In general, CPR needs to be automatic to be accepted as a practical diagnostic tool. Hybridization methods require three to four days to complete the results. Although the current hybridization step can be as short as 18 hours, the entire detection process of a DNA / DNA hybrid can take up to three days with a radioisotope marker. Therefore, there is a great need for simpler, faster and more cost-effective means to detect biologically important RNA and specific DNA sequences in the fields of human and veterinary diagnosis, food microbiology and applications. forensic The biosensors developed to date begin to overcome the drawbacks associated with the current state of the art to detect and identify microorganisms. A biosensor is a device that consists of a biologically active material connected to a transducer that converts a selective chemical reaction into a measurable analytical signal (Thompson et al., 1984. Trends in Analytical Chemistry, 3_: 173; Guilbault, 1991, Current Opinion in Biotechnology, 2: 3). The advantages offered by biosensors over other forms of analysis include ease of use (by non-expert personnel), low cost, ease of manufacture, small size, roughness, easy interfacing with computers, low detection limits, high sensitivity, high selectivity, rapid response and reusability of the devices. Biosensors have been used to selectively detect cells, viruses and other biologically important materials, biochemical reactions and inmonulogical reactions using detection strategies involving the immobilization of enzymes, antibodies or other selective proteins on solid substrates such as quartz and fused silica (for sensors piezoelectric and optical) or metals (for electrochemical sensors) (Andrade et al., 1990, Biosensor Technology: Fundamentals and Applications, RP Buck, E. Hatfield, M. Umana, EF Bowden, Eds., Marcel Dekker Inc., NY , pp. 219; ise, 1990, Bioinstrumentation: Research, Developments and Applications, B-utter orth Publishers, Stoneham, MA). However, such sensors are not widely available from commercial sources because of the problems associated with the long-term stability of selective recognition elements when immobilized on solid surfaces (Kallury et al, 1992, Analytical Chemistry, 64: 1062; Krull et al, 1991, Journal of Electron Microscopy Techniques, 18: 212). _ An alternative approach is to create biosensors with long-term chemical stability. One such approach takes advantage of DNA stability. With the recent advent of DNA probe technology, a number of selective oligomers have been identified that interact with the DNA of important biological species, for example salmonella, (Symons, 1989, Nucleic Acid Probes, CRC Press, Boca Raton, FL; Bock et al., 1992, Nature, 355: 564; Tay et al., 1992 Oral Microbiology and Immunolsgy,! _ • '34; Sherman et al., 1993, Bioorganic & Medicinal Chemistry Letters, 3_: 469). These have been used to provide a new type of biorecognition elements which is highly selective, stable, and can be easily synthesized in the laboratory (Letsinger et al., 1976, Journal of the American Chemical Society, J38_: 3655; Beaucage et al. ., 1981, Tetrahedron Letters, 22: 1859, Alvarado-Urbina et al., 1981, Science, 214: 270). Until recently, the only research group in existence that has published the work done in fluorimetric detection of nucleic acid hybridization and immobilized on optical substrates is by Squirrell et al. (C.

R. Graham, D. Leslie, and D. J. Squirrell, B? Osensors and Bioelectronics 7 (1992) 487-493). In this work, single-stranded nucleic acid sequences ranging in length from 16 mer oligonucleotides to oligomers of 204 bases functionalized with an aminohexyl interlayer at the 5'-terminus conveniently bound to functionalized fiber sections with 3- aminopropyltriethoxysilane via a glutaraldehyde ligation. All nucleic acid hybridization investigations were performed by monitoring fluorescence intensity in an intrinsically modeled configuration using complementary strands that were previously labeled with a portion of fluorescein. This gave a reusable analysis system in which signal generation was observed within minutes and nanomolar detection was achieved. However, this optical sensor technology developed by Squirrel et al does not contain an element of transduction that can transduce the binding event in a non-reactive form. For this analysis to work, the target strands must be marked before performing the analysis in order to detect, making this technique inappropriate for practice applications. Abel and co-workers (Abel, AP, Weller, MG, Duveneck, GL, Ehrat, M. and Widmer, HM Anal, Chem. 1996 68, 2905-2912) of Novartis Ltd. (formerly Ciba-Geigy Ltd.) have recently reported an automatic optical biosensor system. Their device uses 5 '-biotinylated 16 mer oligonucleotide probes attached to an avidin-functionalized optical fiber to detect complementary oligonucleotides pre-labeled with fluorescein portions in an evanescent wavelength of total internal reflection (TIRF) waveform. Squirrell. Each analysis consisted of a pre-equilibrium of 3 minutes, a hybridisation time of 15 minutes, a high procedure of 10 minutes followed by a regeneration cycle of 5 minutes (chemical or thermal). A chemical denaturation scheme was observed as the preferred modality for sensor regeneration as exposure of the functionalized optical sensor of oligonucleotides at temperatures exceeding 52 ° C causing irreversible damage to the device, resulting in the denaturation of the avidin used for immobilization. This limitation renders labile the function of the device against sterilization techniques, such as by autoclaving, and also indicates the rigorous cleaning of the sensor surface, such as by sound treatment, which also compromises sensor integrity via denaturation of the torque. of affinity used to anchor the probe oligonucleotide. In order to detect nucleic acids not previously labeled with fluorescein, and to overcome the Squirrell limitation, a competitive binding analysis was employed by Abel and co-workers. Detection of the unlabeled analyte was performed by sensor pre-treatment with a fluorescein-labeled "DNA tracker" followed by monitoring that decreases a fluorescence intensity of the sensor by exposure and subsequent displacement of the tracer DNA by the acid nucleic of complementary analyte. The dose-response curves reported by Abel et al. they show a limit of detection of 132 pmoles (8 x 1013 molecules) for this detection strategy. Nevertheless, in addition to the high detection limits and the inability of the device to withstand sterilization, this device can not be classified as a biosensor technology due to the need for external treatment with DNA tracker in order to achieve transduction. The prior art with respect to the patent literature contains many examples of "sensor" devices that are based on nucleic acid molecules immobilized on waveguide carriers and transduction strategies based on evanescent excitation. The technology of Gerdt and Herr (David W. Gerdt, John C. Herr "Fiber Optic Evanescent Wave Sensor for Immunoassay", US Patent No. 5,494,798) describes the detection of nucleic acid hybridization based on alterations in the amount of light transmitted from an optical fiber in a coupled fiber system (similar to Mach-Zehnder interferometer) to the second fiber of the waveguide system. The amount of light transferred is a function of the refractive index of the medium in or around the waveguides. The alterations of the refractive index affect a penetration depth of the evanescent wave emitted from a first waveguide in which the optical radiation is released. This vertical wave of electromagnetic radiation is subsequently propagated in (and therefore transfers the optical radiation to) the second waveguide. Therefore, the device is sensitive to refractive index alterations that occur within a volume surrounding the first waveguide with a thickness of approximately one wavelength of light propagating within the waveguide. One of the extensions of the waveguide can be functionalized with immobilized nucleic acid molecules that serve to provide selective binding portions. The change in the refractive index of the nucleic acid thin film is the first waveguide to occur when hybridization with the target nucleic acid sequences alters the amount of light transferred to the second waveguide, thus providing a means of transducing signal. Hybridization events can be identified based on changes in the output ratios of the two waveguide extensions in the coupled fiber system. A limitation of this technology lies in the fact that any alterations in the refractive index near the surfaces of the waveguides will provide alterations in output ratios of the two fibers. Therefore, non-specific binding events (such as protein adsorption) will provide false positive results. In order to avoid the problem of interference that provides false positive results, a transduction strategy is required that is sensitive to the structure of the binding pair (ie, the recognition and objective element). The technologies of Fodor, Squirrell (David s Squirrell "Gene Probé Biosensor Method" International Application Number PCT / GB92 / 01698, International Publication Number WO 93/06241, International Publication Dated April 1, 1993), Sutherland et al. (Ranald Macdonald Sutherland, Peter Bromley and Bernanrd Gentile "Analytical Method for Detecting and Measuring Specifically Sequenced Nucleic Acid." European Patent Application Number 87810274.8, Publication Number 0 245 206 A1, filing date: April 30, 1987). Hirschfeld (Tomas B. Hirschfeld, "Nucleic Acid Assay Method", US Patent Number 5,242,797, patent date: September 7, 1993.), and Abel et al. (Andreas P. Abel, Michael G. Weller, Gert L. Deveneck, Markus Ehrat, and H. Michael Widmer, Analytical Chemistry, 1996, 68, 2905-2912.) Overcomes this limitation by using fluorescent probes that are associated with the pair of or join the selective portions capable of joining a portion of binding pair. These inventions provide methods for measuring nucleic acid hybridization on waveguide surfaces based on evanescent excitation and TIRF. In each embodiment an oligonucleotide probe capable of selective binding to a target sequence is covalently immobilized on a waveguide surface. For the cases of Squirrell and Abel et al., each one defines two preferred modalities for the detection of hybridization events. The first modality of Squirrell and Abel et al. they are essentially identical in which the target nucleic acid is functionalized with a fluorescently detectable agent (by chemical or enzymatic methods) as a first step before detection. By hybridizing between the labeled target and the immobilized nucleic acid, the fluorescent agent binds in close proximity to the surface of the waveguide where it can be excited by the formation of an evanescent wave and emission of fluorophore collected and measured quantitatively. In the second preferred Squirrell embodiment, hybridization between the immobilized oligonucleotide and the target sequence is performed first. After the first hybridization event, a fluorescently labeled oligonucleotide present in the system may undergo hybridization with all or a portion of the remainder of the target sequence not hybridized to the immobilized sequence. The binding of the third (labeled) oligonucleotide provides a fluorescent species bound in close proximity to the waveguide that can provide transduction via evanescent excitation and the collection of emitted radiation. In the second modality of Abel et al. , a method for the detection of nucleic acids not previously labeled with a fluorescent portion via competitive binding analysis is described. Detection of the unlabeled analyte was performed by first pretreating the optical sensor with the immobilized probe nucleic acid with "tracer DNA" labeled with fluorescein. The amount of DNA tracked can be monitored via evanescent excitement and the reason for collection. Analyte binding could be followed by monitoring decreases in the fluorescence intensity of the sensor as a function of displacement of the tracer DNA via competitive binding to non-fluorescent analyte nucleic acid in a dose-response manner. In the methods of Sutherland et al. and Hirschfeld, the transduction of hybridization events are provided with fluorescent intercalation dyes (e.g., ethidium bromide). After hybridization between the single-stranded target and the immobilizing nucleic acids, the intercalating fluorescent dye molecules of the solution are inserted into the base stacking regions of the immobilized double-stranded nucleic acid. An increase in quantum efficiency of fluorescence, fluorescence lifespan, stoichiof fluorescent intercalary probes frequently occurs when associating with double-stranded nucleic acid. The inventors claim that these improved aspects can be monitored by evanescent excitation and fluorescence emission compilation. Fodor et al. have used light-directed chemical synthesis to generate miniaturized high-density arrays of oligonucleotide probes. The DNA oligonucleotide arrays have been manufactured using high resolution photolithography in combination with solid phase oligonucleotide synthesis. This form of DNA microcircuit technology can be used for DNA hybridization analysis in parallel, directly giving the sequence information of genomic DNA segments. Prior to sequence identification, the nucleic acid targets must be fluorescently labeled, either before or after hybridization to the oligonucleotide array via direct chemical modification of the target strand or by the use of a subsequent intercalation dye for hybridization in the DNA microcircuit. The hybridization pattern determined by fluorescence microscopy is then deconvolved by appropriate chemometric processing to reveal the target nucleic acid sequence. Instead of focusing on the selective detection of trace amounts of a particular nucleic acid sequence, this technology focuses on the analysis of nucleic acid sequences in adequately high number of copies so that it sufficiently occupies the oligonucleotide array. Regardless of the undoubted achievements of the prior art mentioned above, there are still limitations in these technologies for which additional advantages are desired. Although the strategies employed by Sutherland et al. , and Hirschfeld overcomes the limitations of Gertd and Herr regarding the origin of the signal - and the generation of false positive results, these methods of analysis are limited by the amount of signal that can be generated by the evanescent excitation. For multi-mode waveguides, less than 0.01% of the optical radiation carried within the waveguide is exposed to the external environment in the form of an evanescent wave (RB Thompson and FS Ligler, "Chemistry and Technology of Evanescent Wave Biosensor" , in Biosensors with Fiberoptics, Eds .: Wise and Wingard, Human Press Inc., New Jersey, 1991, pp. 111-138.). In the case where monomodal waveguides are used, approximately 10% of the radiation carried by the waveguide is exposed in the external environment in the form of an evanescent wave (David W. Gerdt, John C. Herr "Fiber Optic Evanescent Wave Sensor for Immunoassay ", US Patent No. 5,494,798). In the classical total internal reflection fluorescence evanescent wave (TIRF) configuration the critical angle (? C) for the waveguide / solution guide interface (? C / s) is larger than? C for the waveguide / biological (Tc / B), only the evanescent component of the propagated radiation will enter the biological film. The principle of these optical reciprocals that couple the light back into a waveguide as a plane wave will be in the same way as the primary process when a plane wave generates an evanescent wave (Ranald Macdonald Sutherland, Peter Bromley and Bernanrd Gentile "Analytical Method for Detecting and Measuring Specifically Sequenced Nucleic Acid" European Patent Application Number 87810274.8 *, Publication Number 0 245 206 A1, filing date: April 30, 1987, p.13.). Thus, for fluorophores excited by evanescent waves created from modes propagate at or near? Cw s, no fluorescence emission can be coupled back into the waveguide in its propagation mode, as? C / s that would be >; 90 ° (U. J. Krull, R. S. Brown, and E. T. Vandenberg, "Fiber Optic Chemoreception" in Fiber Optic Chemical Sensors and Biosensors, vol.2, Ed. O. S. Wolfbeis, CRC Press, Boca Raton, 1991, pp. 315-340). Therefore a large portion of the signal could be lost in the vicinity for systems in which fluorescence emission originates from thin films of a lower refractive index than from the waveguide in which they are immobilized. It has been shown by Love et al. that under optimal conditions, only 2% of the light emitted by the fluorophore in the lower refractive index medium can be captured and guided by the fiber (WF Love, LJ Button and RE Slovacek, "Optical Characteristics of Fiberoptic Evanescent Wave Sensors: Theory and Experiment "" in Biosensors with Fiberoptics Eds .: Wise and Wingard, Human Press Inc., New Jersey, 1991, pp. 139-180.) The present invention relates to biosensors for the direct detection of nucleic acids and acid analogs. The device comprises a light source, a detector and an optical element for receiving light from the source and transporting it to an interaction surface of the optical element, a nucleic acid or nucleic acid analogue for a particular sequence or structure of nucleic acid ( that is, which is complementary to the target nucleic acids) is immobilized on the optical surface of the optical element. in which they will bind in, or over the hybridized nucleic acid complex and fluoresce when stimulated by the light source. After excitation by electromagnetic radiation of the appropriate wavelength bound to the optical element, the resulting fluorescence is collected within the optical element and the detector is guided to signal that the target nucleic acid has complexed with the immobilized probe and therefore both indicates the presence of the target in the sample. An interaction surface is defined to mean a surface of the optical element on which the nucleic acid is immobilized and in which the fluorescent molecules interact with the light. This invention provides biosensors in which the interaction surface is functionalized with sequences of nucleic acid probes such as the refractive index of the immobilized layer (Substrate Interleaver / Acid / Fluorescent Ligand) is equal to or greater than the refractive index to the surface of the waveguide so that the organic coating becomes an extension of the waveguide. The refractive index of the immobilized layer depends at least in part on the charge of the immobilized and interlacing molecules on the surface and the chemical nature of the immobilized molecules and linkers. Preferred biosensors offer high sensitivity and low detection limits, can be made by activating the interaction surface of an optical element with interlaced substrate molecules of at least about 25 A (Angstrom) in length followed by the binding of a nucleic acid sequence of probe selected to the interleaver. (A probe nucleic acid is, at least in part, complementary to a target nucleic acid). The preferred method for binding a probe nucleic acid to the substrate interleaver is by in situ synthesis of the nucleic acid sequence at the interlayer terminus using solid phase nucleic acid synthesis methods or routine modifications thereof. Such in situ synthesis methods are particularly useful for the immobilization of nucleic acids of 50 or less bases and more particularly they are useful for nucleic acids of 30 or less bases. The fluorophore can be trapped for immobilized DNA, for example, by the use of hydrocarbon in captivity. The use of captive probes can significantly reduce the response time of the biosensor since the response mechanism is not diffusionally controlled. The associated fluorophore provides internal calibration for the intensity of the optical source and the derivation to the detector. It also provides photobleaching calibration and provides internal calibration by monitoring the binding against the free colorant by the use of, for example, time resolution fluorescence measurements. The optical element preferably comprises an optical waveguide that also transports the fluorescent light to the detector. The optical waveguide preferably transports the light emitted by the total internal reflection to the detector. The optical waveguide can comprise an optical fiber, a channel waveguide or a substrate that confines the light by total internal reflection. The fluorescent molecules preferably provide sufficient change of Stochies so that the wavelength of the light source and the wavelength of the fluorescent light are easily separated. Fluorescent molecules can be provided in a solution in which the optical element is immersed, or by capturing the nucleic acid that is immobilized in the interlayer. In the practice of the present invention, the light source can be any suitable source such as a gas laser, a solid state laser, a semiconductor laser, a light emitting diode or a white light source. The detector can be any suitable detector such as a photomultiplier tube, an avalanche photodiode, an image intensifier, a multi-channel plate, a semiconductor detector. The biosensor system can be a system of multiple wavelengths and multiple fluorescence. The luminous coupling of the system can also be modified to allow a multitude of disposable biosensors that are analyzed sequentially or in parallel.

The biosensor system of the present invention can be constructed and used to detect each of a target nucleic acid mixture (e.g., Chlamydia and Gonorrhea in urogenital infections or E. coli and Salmonella during food processing). This can be done using a plurality of fluorophores (which, for example, fluoresce at different wavelengths) each of which is placed in captivity to an immobilized nucleic acid probe that is characteristic of * or specific for the detection of a given species or strain. In this example, the observed wavelength of fluorescence emission will be specific for the hybridization of a target nucleic acid given to its complementary immobilized probe. The biosensors of the present invention have an improved detection limit and sensitivity with respect to the prior art and are shown to be stable to prolonged storage and severe washing and sterilization conditions. The sensors stored for 1 year in vacuum, in solutions of 1: 1 ethanol / water, absolute ethanol or dried at -20 ° C provided response characteristics identical to those newly prepared. The adsorbed fluorescent contaminants accumulated through storage can be removed (as confirmed through 'fluorescence microscopy' research) by sound treatment of the biosensors in ethanol / water 1: 1 where the sensitivity of the device was consistently observed to be increases by a factor of approximately 2.5 from this previous treatment with respect to the freshly prepared biosensors not cleaned before use. Unlike those of the prior art (for example, Abel et al.) The optical biosensors of the present invention also show to be thermally stable wherein the function of the device is maintained after sterilization by the autoclave treatment (20 minutes, 120 ° C, over pressure of 4 atmospheres). The ability to clean and sterilize a biosensor device so that it can be used in an online configuration and / or in clinical applications is an important advantage that is only realized by the technology reported herein. The biosensors of this invention also allow faster sample analysis with improved response time for signal generation. The present invention also provides a recyclable or disposable biosensor for detecting an objective nucleic acid whose biosensor includes an optical element for receiving and transporting light to an interaction surface of the optical element and nucleic acid for a particular nucleic acid sequence that is complementary to acid. nucleic target, immobilized on the interaction surface of the optical element. The recycled or disposable biosensor preferably comprises an optical waveguide, which preferably conveys light by total internal reflection to the interaction surface of the optical waveguide when the organic coating has a refractive index equal to or greater than the surface of the optical waveguide. the waveguide. The optical waveguide preferably comprises an optical fiber. Fluorescent molecules are poured into a solution in which the recyclable or disposable biosensor is immersed which will bind when the immobilized nucleic acid is hybridized with the complementary target nucleic acid and becomes fluorescent when stimulated by light. Alternatively, the fluorescent molecules are provided bound by a binding of the immobilized nucleic acid. The present invention provides biosensors for direct analysis of nucleic acid hybridization by the use of an optical substrate such as an optical wafer or an optical fiber, and nucleic acids or nucleic acid analogs that have been immobilized on the optical substrate. The generation of a fluorescence signal by hybridizing to complementary nucleic acids and nucleic acid analogs in a sample will be achieved in a number of different ways. The biosensors of this invention are sufficiently sensitive for the direct detection of smaller amounts of objective nucleic acids in a sample without the need to employ nucleic acid amplification methods such as PCR techniques. The biosensors of this invention may have detection limits for target nucleic acids below 106 molecules. The optical biosensor comprising nucleic acid strands, or nucleic acid analogues of a specific selected sequence immobilized on activated optical supports. The selected immobilized sequences are capable of binding to the target sequences including sequence characteristics, and selective for viruses, bacteria or other microorganisms as well as genetic disorders or other conditions. Biosensors having such characteristic or selective immobilized sequences are useful for the rapid screening of genetic disorders, viruses, pathogenic bacteria and in biotechnological applications, such as cell culture monitoring and gene expression.An important path that has been largely ignored by the nucleic acid biosensor community is the investigation of the formation of multiple strand nucleic acids (>; 3) . For example, it has been reported that triple helical oligonucleotides offer potential use as: sequence-specific artificial nucleases ( { A.}. Moser, HE; Dervan, PB Science 1987, 238, 645.. Strobel, SA, Doucettestamm, LA, Riba, L., Housman, DE, Dervan, PB Science 1991, 254, 1639.), DNA-binding pro-ethin modulators / gene expression regulators ( { A.}. , M., Czernuszewicz, Postel, EH, Flint, SJ, Hogan, ME Science, 1988, 241, 456 { B.}. Durland, RH, Kessler, DJ, Gunnel, S., Duvic, M .; Pettit, BM; Hogan, ME; Biochem., 1991, 30, 9246 {c.}. Maher, LJ; Dervan, PB; Wold, B .; Biochemistry, 1992, 31, 70. { D} Maher, LJ BioEssays, 1992, 14, 807 { E.}. Maher, LJ Biochemistry, 1992, 31, 7587. { F.}. Duvalvalentin, G., Thoung, NT; Héléne, C Proc. Na t. Acad. Sci. USA, 1992, 89, 504. { G.}. Lu, G., Ferl, RJ Int. J. Biochem., 1993, 25, 1529.) materials for map genomic eo (. { to} Ito, T., Smith, C. L .; Cantor, C. R. Proc. Na ti. Acad. Sci. U. S TO . , 1992, 89, 495. { b} Ito, T., Smith, C.L., Cantor, C.R. Nucleic Acids Res. 1992, 20, 3524), and high selective screening reagents for detecting mutations within the DNA doublet (Wang, S.H., Friedman, A.E., Kool, E.T., (1995) Biochemistry 34, 9774-9784). The present invention can also be used to detect the formation of multiple strand nucleic acid hybrids (e.g., triple helical nucleic acid formation) and therefore, could, for example, operate to monitor effectiveness, dose dependence. and intracellular concentration of nucleic acid pharmaceuticals used in gene therapy applications as well as an analysis to identify a multiple strand formation associated with any of the potential applications mentioned above associated with a triple helical oligonucleotide. The invention is a biosensor system for detecting a target nucleic acid, which consists of at least three layers, two of which are a waveguide, wherein a layer includes a nucleic acid or a nucleic acid analog capable of hybridizing to the target nucleic acid, and wherein a fluorophore is attached to the nucleic acid or nucleic acid analogue and wherein the biosensor functions in accordance with direct excitation. The invention also relates to a biosensor for detecting a target nucleic acid, which comprises an inner layer, an intermediate layer and an outer layer, wherein • the inner layer has a refractive index ni, and • the middle layer includes an acid nucleic acid analogue or nucleic acid capable of hybridizing the target nucleic acid and has a refractive index n2, which is greater than, or equal to, the refractive index __ ?, and • the outer layer has a refractive index n ^, which is smaller than the refractive index n2. and wherein a fluorophore is attached to the nucleic acid or nucleic acid analogue of the intermediate layer and wherein the biosensor functions in accordance with direct excitation. In a preferred embodiment, the iris layer is an optical fiber or optical wafer and the outer layer is in an environment. The outer layer is a water-based solution. The biosensor is used to detect the formation of the triplet or the formation of multiple strand nucleic acid. The triplet formation especially involves a branched counterense nucleic acid which inhibits the expression of a target nucleic acid sequence by triplet formation with the sequence. The biosensor is useful for the detection of nucleic acids from bacteria, viruses, fungi, unicellular or multicellular organisms or for the screening of nucleic acids from cells, cell homogenates, tissues or organs. Preferably, a fluorophore is attached to a suitable nucleic acid or nucleic acid analogue which is one of the layers of a biosensor having at least three layers and the biosensor functions in accordance with direct excitation. The invention also includes the use of a fluorophore to detect the target nucleic acid. The invention also relates to a method for detecting an objective nucleic acid comprising: pre-treating a sample so that the characteristic nucleic acids selective of the sample are available for hybridization; • contacting the sample with the intermediate layer of the biosensor of claim 2, so that the target nucleic acids can be hybridized to nucleic acids or nucleic acid analogues from the middle layer; • allowing the fluorophore to bind to the nucleic acids of the intermediate layer to bind by hybridization of the target nucleic acids with the nucleic acids or nucleic analogs of the second layer; • illuminate fluorescent molecules with light so that fluorescence is stimulated; and • detect the emitted fluorescence. so the presence of objective nucleic acids is detected. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (a). Synthetic scheme of Arnold et al. used to activate glass or silica surfaces fused with long-chain aliphatic spacer molecules terminated with 5'-0-dimethoxytrityl-2'-deoxythymidine. Figure 1 (b). Synthetic scheme of Brennan et al. used to create alkylamine substrate binding molecules on hydroxylated fused silica surfaces. Figure 1 (c). Synthetic scheme of Maskos and Southern used to functionalize fused silica surfaces hydroxylated with GOPS followed by the extension of HEG. Figure 1 (d). Closed loop structure relationship possible as a sequence of the synthesis scheme used in Figure 1 (c). Figure 1 (e). Synthetic scheme used to extend the GOPS functionalized substrates of DMT-HEG via a base catalyzed mechanism. Figure 1 (f). Synthetic scheme used to covalently bind DMT-HEG on hydroxylated fused silica surfaces via activation with methanesulfonyl chloride. Figure 2. The phenoxyacetyl protecting group used for the protection of exocyclic amine (R) in nucleoside phosphoramidite synthases. Figure 3 (a). The synthetic scheme used to create bound analog of Ethidium Bromide hydrocarbon. Figure 3 (b) (i). The synthetic scheme is used to create a phosphoramidite analog bound to the Edithide Bromide polyether. Figure 3 (b) (ii). Synthetic scheme used to create an analog linked to polyether of the bis-intercalative fluorescent probe YOYO-1. Removal of the protecting group from DMT followed by treatment with ß-cyanoethyl-N, N-diisopropylphosphityl chloride will produce the attached YOYO-1 phosphoramidite synthon. Figure 4 (a). Schematic diagram of a modality of an apparatus used to measure fluorescence intensity of optical fibers coated with immobilized DNA.

Figure 4 (b). Schematic diagram of an example of a dedicated instrument for analysis of nucleic acid samples for fiber optic nucleic acid biosensor of the present invention. Figure 4 (c). Schematic representation of a biosensor system whose light from the appropriate source is directed through a dichroic mirror separator and focused on a fiber or waveguide of the coupler and then on an optical fiber having a single-stranded nucleic acid. to the surface thereof and in which any resulting fluorescent light travels back through the coupler, and passes through the beam splitter and is directed to a photo-multiplier detector. Figure 5. Illustration of the operating principles of the fiber optic nucleic acid biosensor. Hybridization of single-stranded oligonucleotide complementary to solution with nucleic acid probe generating on the biosensor followed by intercalation of the bound fluorescent ligand that provides transduction from the selective binding process into a measurable analytical signal. Figure 6. Fluorescent intensity as a function of temperature for the functionalized fibers of icosanucleotide of mixed base sequence. Upper curve: response of optical sensor to 20 pmoles of linear complement icosanucleotide in the presence of 2.5 x 10"8M ethidium bromide Lower curve: response of the optical sensor to 2.5 x L0 ~ 8M of ethidium bromide. Figure 7. (a) A triplet formation model (T * AT) using dT? 0 and an optic biosensor functionalized with immobilized dAio. The doublet dT? 0: dA? 0 is first formed by cooling the system below the doublet Tm followed by the formation of the triple-stranded complex with additional cooling below the Tra for the formation of the triplet. (b) The day of the optical sensor capturing the branched "V" compound 1 (see Figure 15). Note now that the fluorescent probe was excluded from the triplet as the temperature cools. Figure 8. Amount of trityl cation released during each detritylation step of dT20 automatic phosphoramidite synthesis in fused silica optical fibers functionalized by the protocols of the examples 1 and 5. Figure 9 (a). Response characteristics of an optical biosensor to complement DNA and without complement. Figure 9 (b). Response characteristics of an optical biosensor to 570 ng-l-1 of complement RNA. Figure 10. Response time of the optical sensor constructed by the protocols in examples 1 and 5 and incubation time effect of ethidium bromide. Figure 11. Response of an optical DNA biosensor (a) after storage for a month used without cleaning and (b) after storage for eleven months and cleaned with sonification treatment in ethanol for 10 minutes. Note: A 1-month sensor that was cleaned by sonification (data not shown) provided a response similar to (b). Figure 12. Thermal denaturation profiles of dA20 + dT20 aqueous and dT2o immobilized with aqueous dA20. Figure 13. Response of optical sensor with immobilized nucleic acid probe for Candida albicans to complement DNA. Figure 14. Biosensor response without reagent as described in Example 14. The graph measures fluorescence of the dye attached at the termination of the immobilized nucleic acid as a function of time after exposure to a sample of 720 ng of cDNA. Figure 15. The structures of dT? 0 and compound 1, a branched oligonucleotide with identical oligo (thymidine) chains linked to the 2 'and 3' portions of a ribose branch point nucleoside, i.e. 2 '? -5' -dtl 0 junctions of rA 3 '? -5' -dT10 to dAio to give a complex of triple strand containing only triplets based on T * AT (Hoogsteen-Watson / Crick inverse). Figure 16 (a). Response (•) of the optical sensor with a recognition sequence terminated at the 5 'end at 40 pmol of linear dT? 0 in the presence of 2-5 x 10 ~ 8M of ethidium bromide. Response (X) of the optical sensor of 2.5 x 10-8M of ethidium bromide and not of dT10. Fusion profile of the same nucleic acid system in bulky solution by measuring the absorbance (260 nm) in 10 mM TRIS and 50 mM MgCl2 at pH 7.3. Figure 16 (b). Response (•) of the optical sensor with a recognition sequence terminated at the 3 'end at 40 pmol dT? Or linear in the presence of 2.5 x 10 ~ 8M ethidium bromide. Response (X) of the optical sensor to 2.5 x 10 ~ 8M of ethidium bromide and no dT? 0. The fusion profile of the same nucleic acid system in bulky solution by measurement of absorbance (260 nm) in 10 mM TRIS and 50 mM MgCl2 at pH 7.3. Figure 16 (c). Response (•) of the optical sensor with a Recognition Sequence completed at the 3 'end 40 pmol of 1 (see Figure 15) in the presence of 2.5 x 10 ~ 8M of ethidium bromide. Response (X) of the optical sensor at 2.5 x 10 ~ 8M with ethidium bromide with the number 1. The fusion profile of the same nucleic acid system in voluminous solution by measuring the absorbance (260 nm) in 10 mM TRIS and 50 mM MgCl2 at pH 7.3. Figure 17 (a) Photograph of a native shaded polyacrylamide gel with UV containing helical complexes of single strand, double and triple of branched and linear controls. The DNA samples were loaded in 50 mM MgCl2, and 30% sucrose. Lines 4-10 are dT? 0, dT? 0: dA? _ (1: 1)., DT? 0: dA10 (2.5: 1), dTi0: dA10 (4: 1), dA10, 1 + dA ? 0, and 1, respectively. As you can see the triplet dT10: dA? 0 (line 7) showed a greater delay in mobility in relation to the corresponding doublet (lines 5 and 6). The slowest mobility was observed in line 9 for l: dA? 0: Note: See Figure 15 for the structure of 1. - Figure 17 (b). Photograph of native polyacrylamide gel obtained with ethidium bromide (same gel as in Figure 17 { A.}.) Containing single-stranded, double or triple helical complexes of linear and branched controls. The DNA samples were loaded in 50 mM MgCl2 and 30% sucrose. Lines 4-10 are dTio, dT? O: dA? O (1: 1), dT? 0: dA? 0 (2.5: 1), dT? O: dA? O (4: 1), dA? O, 1 + dAio, and 1, respectively.

As you can, observing the triplet d? O: dA? O (line 7) showed a slight delay in mobility in relation to the corresponding doublet lines 5 and 6). The slowest mobility was observed in line 9 for l: dA? O- Note that only doublets and triplets showed fluorescence of ethidium bromide. Note: see Figure 15 of the structure of 1. Figure 18. Schematic diagram illustrating the experimental concept for light-sweeping investigations of a two-layer system with a fused nSy¿ice > ^ Film - Figure 19. Schematic diagram illustrating the experimental concept for light-sweeping investigations of a three-layer system with fused nsiiice > Opeiicu? A > n environment • Figure 20. The schematic diagram illustrating the experimental concept for light-sweeping investigations of a three-layer system with fused nSiiice < - ^ Movie - > Environment - Figure 21. The schematic diagram of the instrument used for investigations of light sweep that depends on the angle. Figure 22. Control experiments for the Luminous Sweeping Technique that Depends on Angulation using Known Refractive Index substances. Figure 23. Results of light scanning experiments performed with substrates coated with thin organic films. Figure 24. Results of light scanning experiments performed with substrates coated with covalently immobilized oligonucleotides. Detailed Description of the Invention The invention relates to a biosensor that operates in accordance with an intrinsic mode of operation. Using the chemistry as described in this patent application) to join the interlaced support molecules in optical waveguides (preferably optical fibers) and an automatic DNA synthesizer, an orientation control and a wide density scale are provided. oligonucleotide in the waveguide. In this form, immobilized oligonucleotide films of desired refractive index can be constructed of waveguide carriers so that the oligonucleotide film is made to be an extension of the waveguide. In this way intrinsic operation provides a highly efficient means of signal generation and the collection of excitation of the fluorescence and emission occurs within the waveguide itself providing an expected improvement in sensitivity and decrease in detection limits by six orders of magnitude. The second major improvement provided by our technology is the use of fluorescent dyes attached to or otherwise associated with oligonucleotides. 'Thompson and Krull ( { A.}. M. Thompson and U. J. Krull, Trends in Analytical Chemistry, 3 (1984) 173-178. . { b} M. Thompson and U. J. Krull, Analytical Chemistry, 63 (1991) 393A-405A. ) teaches that biosensors can be defined as devices that consist of a biorecognition element and a transduction element. The biorecognition element can be a biological material capable of participating in highly selective binding to a target, usually a significantly important molecule. The transduction element converts the selective binding reaction into a measurable analytical signal. The Gerdt transduction strategy is not selective for the technology that is classified as a biosensor while the devices of Fodor, Squirrell, Abel et al. , Sutherland et al. , and Hirschfeld do not contain an element of transduction at al. In addition to the requirement for external reactive treatment, in the cases of Fodor, Sutherland et al., And Hirschfeld, there is also the additional drawback that all intercalating colors are known or suspected to be mutagenic. Therefore, after each analysis there are the problematic aspects of collection and disposal of hazardous chemical waste. By associating the transduction element with the biorecognition element, the device can operate without needing the external reactive treatment and make the need to collect and dispose of the hazardous waste. Such technology easily leads to automatic on-line analysis by itself and excludes the need for expert technicians to participate in the residue analysis or disposal procedure (as long as the sample itself is not biohazardous). The other advantages provided for the incorporated colorant is the internal calibration. More specifically, three advantages can be realized: 1) the associated dye provides a means to determine the amount of the fluorophore and the nucleic acid immobilizing in the waveguide; 2) the fluorophore in the presence of single-stranded nucleic acid provides a baseline signal to which all signals can refer, providing here significant analytical data; and 3) the lifetime of the device can be determined from alterations in the background fluorescence signal of the incorporated fluorophore over time. Therefore, including the associated fluorescence transduction unit, an internal reference marker and diagnostic tool for device status is included as an integral part of the optical biosensor. The nucleic acid oligomers are covalently immobilized on optical fibers by first activating the surface of the optical fiber with a long-chain terminator extension terminated with a chemically protected terminator, typically a dimethoxytrityl (DMT) portion followed by solid-phase DNA synthesis automatic The detection of nucleic acids or nucleic acid analogues on the fiber surface after hybridization between the immobilized nucleic acid and its complementary nucleic acid is achieved by measuring the enhanced fluorescence emission of the fluorophore.

The optical fiber can be activated with certain numbers of different compounds. The method of Arnold and co-workers (Arnold et al., 1989, Collect, Czech, Chem. Commun., 5: 523) can be used for the activation of wafers, fused silicas, optical waveguides, and optical fibers. whereby the 25 atom long terminator molecules terminated by a dimethoxytrityl protected nucleoside were immobilized on the clean optical fiber substrate as illustrated in Figure 1 (a). In this method, the length of the separator between the substrate and the first nucleoside is sufficiently long so that the environment of the terminal nucleoside is sufficiently fluid to allow efficient coupling with successive nucleotide monomers during the synthesis of automatic foromadithia of the probe. immobilized nucleic acid. This is according to the report by Beaucage et al. (1992, Tetrahedron, 48_: 2223-2311) where it was established that substrate interleavers of lengths of at least 25 atoms are required to achieve high coupling yields (> 99.5%). The synthetic scheme of Arnold et al. It requires inexpensive chemicals, to be easy to execute and to be carried out as a one-pot process where the insulation is a product and the purification is obvious. Because the interleaver is terminated by a protected nucleoside, any site reactive in the support that could lead to the production of unwanted side products during automatic synthesis can be eliminated by treating the derivatized supports with acetic anhydride prior to synthesis. Finally, the linker coverage on the support is easily determined by determining the amount of trifile cation released during the first trichloroacetic deprotection step (TCA) of the automated synthesis. The methodology however limits the types of protective chemistries of nucleobases that can be used as a strong base treatment separated from the succinate binding between the substrate interleaver and the oligonucleotide probe. An amine-terminated solid support suitable for automatic oligonucleotide synthesis can be prepared according to the method of Brennan et al. (1993, Sensors and Actuators, B, 1_1: 109). A bifunctional amphiphilic subject derivatizing agent can be condensed α-aminopropyltriethoxysilane (APTES) with 12-nitrododecanoic acid. The resulting long chain detached molecule is covalently immobilized on the surface of the optical fibers by a reaction of Sn2 between the hydroxyl groups present on the surface of the fiber and the silane portion of the amphiphilic. With the termination of the substrate interleaver in the non-reactive nitro form, the support can be crowned using standard methods employed during the automatic synthesis (acetic anhydride or with chlorotrimethylsilane (RT Pon Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs, S. Agrawa, De. 1993, Humana Press, Inc. Totowa NJ.), In which other reaction sites that can produce unwanted side products during the synthesis of oligonucleotides are masked.The reduction of the terminal nitro functionalities is then achieved by the treatment of s-oporte derivatized with an acid zinc solution The resulting main amine groups can then be used directly for an automatic synthesis where a resistant base ammonolysis / phosphoramidate binding is made between the active support and the first nucleotide A major part of a synthetic procedure has been used for immobilization hoisting covalently alkylamine monolayers on fused silica substrates as described in Figure 1 (b). The Maskos and Southern hydrolysis resistant ligature can also be used to provide functionalized waveguides with substrate interleavers. The analogue to the natural internucleotide binding, a phosphodiester ligation between the substrate interleaver and the first nucleotide is completely resistant to ammonolysis under the conditions that remove the normal protecting base groups. This ligation is produced by derivatization of optical fibers with a bifunctional silylation reagent of 3-glycidoxypropyltrimethoxy silane via ligation formation of silyl ether with the hydroxylated waveguide surface. This produces a substrate derivatized with spacer molecules with terminal epoxide moieties. The length of the separation extension then extends by nucleophilic attack within a polyether, such as hexaethylene glycol (HEG), in a catalyzed epoxide ring opening reaction, giving a stable ether bond (U. Maskos and EM Southern, 1992, Nucí, Acids Res., 20 (7), 1673), as shown in Figure 1 (c). The polyether chains provide hydration, flexibility for molecular movement and improved biocompatibility in terms of minimization of non-selective binding to biological compounds. Extending the molecule assembly, separator to a compound of at least 25 atoms, achieves optimal phosphoramidite tuning coupling efficiencies (Beaucage et al., 1992 Tetrahedron, 1992, 4_8, 2223). This support, terminated with a hydroxyl functionality, is then used directly for the synthesis of automatic oligonucleotide, obviating the need for functionality of the tedious nucleotide of the support. Since polyethylene glycols are bifunctional, here there is the possibility of creating non-reactive closed-loop structures that can significantly decrease the amount of oligonucleotide loading on the surface of the optical fiber, as shown in Figure 1 (d). To eliminate any problems and improve the prior art, a polyether termination is protected with a suitable blocking group, for example, with a DMT functionality, prior to the extension of the glycidoxypropyltrimethylsilane. In the case where the chromophoric protecting group (such as DMT) is used, an additional advantage is provided where easy determination of the amount of crosslinked supports can be determined by monitoring the absorbance of the deprotection solution (e.g., 504 nm for DMT +). The polyethylene glycols protected with monodimethoxytrityl can be introduced into the surface of fused silica waveguides by a number of methods. The waveguides first functionalized with GOPS, as in the Maskos and Southern method can be treated with a solution of polyethylene monoditethoxytritylated glycol on sodium hydride for polyether bonding to the terminal epoxide portion of the GOPS immobilized via a reaction of epoxy ring opening catalyzed with base as shown in Figure 1 (e). Polyethylene monoditethoxytritylated glycols (such as DMT-HEG) can also be bound to the surface of fused silica waveguides by activation of polyether terminal hydroxyl portion with sulfonylmethane chloride or β-cyanoethyl chloride N, N-diisopropylphosphityl as it is shown in Figures 1 (e) and l (f), respectively. In the latter case, the polyether substrate interleaver binds to a phosphoramidite tuning which can be performed as part of the automatic oligonucleotide synthesis process; thus forming the entire fully automatic biosensor manufacturing protocol after cleaning the waveguide parts that are introduced into the synthesis column of the automatic synthesizer. The biorecognition element that has been bound at the termination of the substrate linker in the described biosensor configuration can include immobilized nucleic acids (DNA and RNA), modified nucleic acids and nucleic acid analogues prepared by well known methods or by -extension or frontal straight modification of these methods. The terminal nucleic acid includes polynucleotides, oligomers, relatively short polynucleotides (up to about 50 bases), long polynucleotides that vary to several hundred bases, and double-stranded polynucleotides. There is no specific size limit on nucleic acids used for immobilization in this invention. However, problems due to self-hybridization and reduced selectivity can occur with modified nucleic acids. As used herein, the term "nucleic acid analogs" includes modified nucleic acids. As used herein, the term "nucleotide analogue" includes nucleic acids wherein the internucleotide phosphodiester binding of DNA or RNA is modified to improve the biostability of the oligomer and "tunes" the selectivity / specificity for target molecules (Ulhmann, et al., 1990, Angew. Chem. Int. Ed. Eng., 90: 543; Goodchild, 1990, J. Bioconjugate Chem., I; 165; Englisch et al, 1991, Angew, Chem. Int. Ed. Eng. ., 3_0: 613). Such modifications may include and are not limited to phosphorothioates, phosphorodithioates, phosphotriesters, fostoroamidates or methylphosphonates. In the present invention, the nucleic acid sequences are covalently bound to the surface of the optical fiber. In a preferred embodiment, an automatic DNA synthesizer is used to develop nucleotide oligomers on the surface of activated optical fibers via the well-established β-cyanoethylphosphoramidite method. Any commercially available automatic DNA synthesizer can be used. The use of an automated synthesizer to develop nucleic acids or nucleic acid analogs on fiber optic substrates provides many advantages over conventional DNA immobilization techniques. Conventionally, nucleic acid strands are absorbed on a suitable support (usually nitrocellulose) with little orientation about the strands. The use of an automated oligonucleotide synthesizer provides complete control of the oligomer sequence, strand orientation, and package density in association with the activation of fiber optic substrates. Control over these parameters is critical in the development of a nucleic acid detection method based on hybridization as the alignment of the immobilized strands with respect to the availability of target nucleotides for hybridization and intermolecular interactions (electrostatic and steric) between oligomers will have direct ramifications on the kinetics and thermodynamics of hybrid formation and dissociation. The use of a gene machine in addition to the chemistry used to activate the surface of the optical fibers allows the creation of membranes of desired density and structural order to allow rapid and reversible hybridization and to control the refractive index. The use of the phosphoramidite method of oligonucleotide synthesis has been extensively reviewed and has become a synthetic method of choice that pertains to high coupling efficiencies and strengthens reagents in addition to the fact that the need for numerous isolation and purification steps is avoided. products (which is required for liquid phase methods). There are two readily available types of phosphoramidites that can be used to synthetically develop oligonucleotides, namely, methylphosphoramidites and β-cyanoethylphosphoramidides . The method using β-cyanoethyl phosphoramidides is preferred since full deprotection of the oligonucleotides can be performed using aqueous ammonia (as opposed to thiophenol) for the case where oligonucleotides are developed in controlled pore glass (CPG). Triethylamine is used to deprotect the "ß-cyanoethyl protected oligonucleotides developed on fused silica wafers or optical fibers without releasing the oligonucleotides from their support." A review of the synthesis of β-cyanoethylphosphoramidide is as follows: The first stage of each cycle The synthesis of solid phase automatic phosphoramidite involves the removal of the protective group of dimethoxytrityl in the immobilized nucleotide.Detritylation is carried out by introducing a solution of 3% trichloroacetic acid (TCA)., in 1,2-dichloroethane (DCE) in the synthesis column in order to give a 5'-hydroxyl functionality on which the monomer of the following nucleotide can be coupled. TCA is the reagent of choice for detritylation due to its rapid reaction regime, so that the oligonucleotide is only exposed to the acid for a short time thus preventing the catalyzed removal of acid from the adenine and guanine portions of the groups of nucleotide sugars through the process of depurination. Once the reaction is complete, the acid is removed by rinsing the column with acetonitrile. The eluent containing the released trifile cation is sent to a fraction collector so that the coupling efficiency of the synthesis can be monitored by absorption spectroscopy. The coupling of the next stage of the synthesis cycle. The content of the synthesis column is dried by washing alternately with acetonitrile and rinsing with dry argon. This ensures that the support is anhydrous and free of nucleophiles. The desired phosphoramidite and tetrazole are then sent to the synthesis column. Tetrazole is a weak acid (pKa = 4.8) that is used to activate phosphoramidite. The nucleophilic attack by the 5'-hydroxyl group on the activated phosphoramidite moiety forms an internucleotide ligation. A ten-fold molar excess of phosphoramidite in excess tetrazole is added to the synthesis column to ensure that coupling yields are achieved. The next step of synthesis is the crowning step. This is done by eliminating the additional growth of sequences in which the coupling occurs. Failing sequences become non-reactive by introducing acetic anhydride in the presence of dimethylaminopyridine, in order to acetylate any remaining unprotected 5'-hydroxyl portion. Because the trivalent internucleotide phosphite moieties are labile for acidic and basic conditions, an aqueous iodide solution is added after rinsing the column crowning assets. This is done in order to oxidize the trivalent internucleotide phosphite moieties to the more stable pentavalent phosphate moieties found in naturally occurring nucleic acids. This procedure is called the oxidation step. After the oxidation step, a nucleotide addition cycle is completed. The process can be repeated many times until the oligonucleotides of desired length and base sequence have been constructed. After the addition of the last nucleotide, a final detritylation step is usually carried out in order to produce a 5'-hydroxyl group in the complete sequence. • Triethylamine is used for the removal of protected groups of β-cyanoethyl in the internucleotide phosphotriester portions of oligonucleotides developed in optical substrates. This method is known to cause quantitative loss of the phosphate protecting groups via a β-elimination mechanism while not separating the nucleic acids in a single strand of the optical fibers. The ammonia treatment of the immobilized oligonucleotides is avoided by choosing a thymine base sequence. Thymine does not contain a primary amine functionality that could require protection during oligonucleotide synthesis. This approach is not limited to the use of phosphoramidite synthons, but is compatible with all commercially available solid phase syntheses such as the H-phosphonate chemistry (Froehler, B. C, 1986, Tetrahedron, Letters, 27: 5575; Stein et al, 1990, Analytical Biochemistry, 188: 11). Contrary to conventional preparation of oligonucleotides by solid phase synthesis, removal after synthesis of the support product is not desired. In order to avoid the separation of supporting oligonucleotide (optical fiber) while the protective groups of the nucleobases are removed, two modifications can be made to the usual synthetic protocol. The approach involves the combination of a hydrolysis resistant ligature between the Oligomer and the support together with the use of labile base protecting groups. Therefore, an oligomer of any sequence can be prepared and deprotected although it remains attached to the support available for partial hybridization. - The phenoxyacetyl family (PAC) of protecting groups represents a convenient method to block the exocyclic amino functions of the guanine, adenine and cytosine residues (thymine or uracil do not require nucleobase protection). The mean time of deprotection with concentrated ammonium hydroxide at 20 ° C is 8 minutes, 7 minutes and 2 minutes, respectively. (Wu et al, 1989). Under these conditions, the cyanoethylphosphate protecting groups are removed within seconds (Letsinger and Ogilvie, 1969), while the ligation that binds the oligomer to the surface of the fused silica fiber (eg, a phosphodiester or phosphoramidate) is completely stable under these conditions. Alternative labile protecting groups are derivatized phenoxyacetyl groups which include alkyl-substituted PAC groups, more specifically t-butylphenoxyacetyl groups. The t-butylphenoxyacetyl group can be rapidly removed compared to the hydrolysis of the ligation to the separator thus reducing the possibility of separation in the immobilized sequence of the surface. N-phenoxyacetyl deoxynucleoside 3'-cyanoethylphosphoramidites • and analogous t-butylphenoxyacetyl phosphoramidites are commercially available. It has been reported by Polushin and Cohen (NN Polushin and JS Cohen, Nucleic Acids Research, 1994, 2_2, 5492-5496) that the t-butylphenoxyacetyl nucleobase protecting groups can be removed quantitatively by treatment with ethanolamine for 10 minutes at room temperature. treatment with a mixture of hydrazine / ethanolamine / MeOH (1: 1: 5 v / v / v) for 3 minutes. Beaucage and co-workers (JH Boal, A. W-ilk, N. Harindranath, EE Max, T. Kempe and SL Beaucage, Nucleic Acids Research, 1996, 2_4, 3115-3117.) Also report the rapid quantitative removal of protecting groups of t-butylphenoxyacetyl by treatment with protected nucleic acid bound to the support with gaseous amines ( { a.}. anhydrous ammonia gas, 10 bar, 25 ° C, 35 minutes, or { b.}. methylamino, 2.5 bar, 25 ° C, 2 minutes). Other possible labile protection groups could include the "FOD" (rapid oligonucleotide deprotection available from Applied Biosystems Inc.) based on N, N-dialkylformamidines (Vinayak et al, 1992, Nucleic Acids Research, 20_: 1265-1269) . Kuijpers et al (Tetrahedron Lett., 1990, 31_6729-6732 and Nucleic Acids Res., 1993, 21, 3493-3500) have described a method of protecting the nucleobase using portions of 2- (acetoxy-methyl) benzoyl (AMB) which can be removed by treatment with anhydrous potassium carbonate in methanol for 90 minutes at room temperature. The use of protecting groups that can be selectively removed under conditions that will not separate the support oligomer, such as the levulinyl group (removed by hydrazine treatment) (Letsinger et al, 1968, Tetrahedron Letters, 22: 2621-2624; Hassner et al, 1975, J. Amer. Chem. Soc., 97: 1614-1615) is also contemplated by the present invention. Even synthesis without the protecting groups and nucleobases is possible for nucleic acid oligomers of up to 20 nuleotides in length using the phosphoramidite approach (Gryaznov et al, 1991, J. Amer. Chem. Soc., 113: 5876) or chemistry of H-phosphonate (Kung et al, 1992, Tetrahedron Letters, 3_3: 5869). Any of these approaches encompasses difficulties in the removal of nucleobase protecting groups while leaving the oligomer attached to the support. Short free strands of nucleic acids can also be covalently bound to the optical fiber directly or via the interlacing molecules. This approach allows the use of DNA or RNA isolated from natural sources, amplified nucleic acids or their analogs, or synthetic samples provided in the fully unprotected form. The protocols provide oligomers bound at the end of a well-defined orientation. The chemically stable bonds between the support and the oligonucleotide can be used to improve the biosensor's greatness. Quartz optical fibers (or interchangeably fused silica) derivatized with linker molecules terminated with hydroxyl or amino groups can serve as carbodiimide-mediated coupling substrates with terminally phosphorylated single-stranded nucleic acids. Coupling to the hydroxyl fiber produces a phosphodiester ligation while coupling to an amine fiber produces a phosphoramidate linkage. Oligonucleotides can be phosphorylated in solution, either chemically via a modification or Ouchi method (Sowa et al Bull. 'Chem. Soc., Japan 1975, 48_ 2084) or enzymatically. The covalent attachment of short free strands of nucleic acid with a single strand to the optical fibers can be achieved by a slight modification of the Ghosh and Musso method (Ghosh and Musso, 1987, Nucí Acids Res. 15: 5353). Coupling of a derivatized oligomer of 5'-aminohexyl with activated carboxyl fibers producing oligomers bound at the end. This method is known to minimize the reduction in the amino groups of the DNA bases (which could potentially compromise the hybridization event) and give the surfaces with excellent nucleic acid coverage. The synthesis of the oligomers modified at the 5 'or 3' terminus can be easily achieved by normal methods (Ghosh and Musso, 1987, Beaucage and Iyer, 1993). The RNA can be assembled on the support or prepared separately and ligated to the support after synthesis. RNA monomers are commercially available as some synthons modified in 2'-0. RNA analogs of 2'-0-methylallyl and 2'-deoxy-2'-fluoro when incorporated into an oligomer show the increased biostability and stabilization of the RNA / DNA doublet (Lesnik et al., 1993, Biochemistry, 32: 7832). As used herein, the term "nucleic acid analogues" also includes alpha anomers (a-DNA), L-DNA (mirror-image DNA), 2'-5'-linked RNA, branched DNA / RNA or chimeras of natural DNA or RNA and the nucleic acids modified above. Nucleic acid analogs replaced in the base structure are also adapted for use in the biosensor of the present invention. For the purpose of the present invention, peptide nucleic acids (PNA) (Nielsen et al, 1993, Anti-Cancer Drug Design, 8:53, Engels, et al., 1992, Angew, Chem. Int. Ed. Eng. , _31: 1008) and morpholino-like nucleotide analogs bound with carbamate (Burger, DR, 1993, J. Clinical Immunoassay, 1_6: 224; Uhlmann, et al., 1993, Methods in Molecular Biology, 20., "Protocols for Oligonucleotides and Analogs, "by Sudhir Agarwal, Humana Press, NJ, USA, pp. 335-389) are also encompassed by the term" nucleic acid analogue ". Both exhibit specific DNA sequence binding with the resulting doublets being more thermally stable than the DNA / DNA doublet. Other nucleic acids replaced in the structure of the base are well known to those skilled in the art and can also be used in the present invention (See for example, Uhlmann et al 1993, Methods in Molecular Biology, 2_0, "Protocols for Oligonucleotides and Analogs," ed. Sudhir Agrawal, Human Press, NJ, U.S.A., pp. 335). Optical substrates such as flat wafers and optical fibers can be used in the present invention. A preferred embodiment uses optical fibers. Optical fibers are particularly advantageous as membrane supports due to their small size, high light transmission capacity, and ability to allow total internal reflection of the (TIR). The optical fibers also provide a rugged compact sensing device and offer the capability for remote spectroscopic measurements (Love et al, 1991, Biosensors with Fiberoptics, DL Wise and LB Wingard (Eds.), Humana, NJ, pp. 139-180). . There are two fundamental configurations in which alterations in fluorescence parameters of the fluorescently contaminated membranes in the optical fibers can be monitored, i.e., extrinsically and intrinsically. The extrinsic mode configurations are those in which the waveguide is simply used as a pipe or light conduit. Investigations in an extrinsic manner at the end are usually done using optical fibers. In a biosensor that uses extrinsic mode configurations at the end, fluorescent dyes and selective chemistry are located near the distal end of the fiber. The fiber is used as a pipe or light conduit, where the excitation or emission radiation is simply guided from the sampling region to the detector. The fluorescence is stimulated by copying the excitation radiation at the near end of a fiber, the emission can be monitored by placing the light sensing equipment directly opposite the distal end of the fiber. Alternatively, the detector is placed at the near end of the fiber as some of the fluorescence can be coupled to the fiber and reflected back completely internally to the trailing end. The extrinsic lateral mode approach is normally used for investigations carried out on flat supports, but can also be used for fibers. The immobilized single stranded nucleic acid and fluorophore are placed along the length of the waveguide / fiber optic wafer. The fiber is illuminated by a localized light source normal in the length of the fiber and the emission of fluorescence is also monitored by the normal placed in the fiber equipment. The extrinsic configurations provide the advantage of simple and inexpensive equipment including conventional light sources and detectors, which are used (Krull et al, 1991, Fiber Optic Chemical Sensors and Biosensors, Vol. II, OS Wolfbeis, Ed., CRC Press , Boca Ratón, pp. 315). However, the extrinsic sampling configuration provides poorer sensitivity that belongs to the shortest path length and sensitivity for interferences present in the surrounding medium. In a preferred embodiment, an intrinsically modeled arrangement, based on careful control of the refractive index, is used to monitor the emission of fluorescence from the surface of optical fibers. Fluorophores present on any surface or below the surface of the fiber can be excited through the formation of an electrical wave electric field that propagates normal to the surface of the fiber, of total internal reflection of radiation in the fiber. The TIR process occurs when the angle of incidence?, To the interference between a fiber of high refractive index, n¿ and the lower average refractive index, n2, is the critical angle? C, defined as: The electric field amplitude of the reflecting radiation decreases exponentially in a vertical wave in a medium having the lower refractive index. This decaying radiation is termed the evanescent wave and can be used to excite fluorophores located near the limit for IRR. The propagation intensity, I, of the evanescent wave depends on the angle of reflection,? , the wavelength of the transmitted radiation,?, and the Fresnel transmission factor, T: where x represents the normal distance to the TIR limit, and dp is the depth of penetration that is given by (Krull et al, 1990, Talanta, 37: 801-807)? d = (3) Ap ^ n] sin2 (?) - n¡ The depth of penetration is defined as the distance at which the intensity of the evanescent field has decayed at 1 / e of the intensity at the reflection limit. Normally, the evanescent wave is prepared in a thin area beyond the surface of the fiber with a penetration surface ranging from about 200 nm to 400 nm for visible light. Fluorophores within the evanescent wave propagation wave are excited by the evanescent wave to emit fluorescence. Additional fluorophores of the fiber optic interface will experience low light intensity at the excitation frequency and a concomitant decrease in resulting fluorescence intensity emitted. A major limitation of the evanescent wave excitation is that less than 0.01% of all the excitation radiation in a fiber currently leaks past the fiber than an evanescent wave and less than 2% of the fluorescence caused by the evanescent wave is currently recovered in the fiber for transmission to the detector by total internal reflection (Love et al, 1991, Biosensors with Fiberoptics, DL Wise and LB Wingard (Eds.), Human, NJ, pp. 139-180). As such, the evanescent background mode of fluorescent signal excitation and recovery is very inefficient and is not the preferred mode of operation for optical sensor devices. For the case where the refractive index of the immobilized layer is effectively the same or greater than the refractive index of the substrate for immobilization (for example, the silica surface of the optical element) the limit for IRR effectively reaches the interface between the immobilized layer and the solution. The fluorophores bound to the nucleic acid in the immobilized layer are exposed directly to the junction of electromagnetic radiation within the waveguide thus providing a vastly improved excitation efficiency and, as a consequence, emitting fluorescence of increased intensity. For example, the refractive index of a monolayer of organic media n monolayer 1. 46 to 1.-5; Ducharme et al, 1990, J. Phys. Chem. 94: 1925) is very similar to fused silica or fused silica (ncuarzo = 1.46, O'Hanian, H.C. 1985, Physics, W. W Norton &Co. N Y. p.835). The fluorophores in the immobilized layer emit the fluorescence radiation within the waveguide by itself to provide the rather improved probability for the transmission of the fluorescence signal by total internal reflection to the detector giving increased sensitivity and nucleic acid detection limits lower objective. Fluorescence is an analytical method chosen for the transduction of hybridization events in a measurable analytical signal, since fluorescence techniques have been known to provide high sensitivity (comparable to radioisotope methods) and detailed information about structure at the molecular level ( Lakowicz, 1983, Principies of Fluorescence Spectroscopy, Plenum Press, NY). Changes in polarity, pH, temperature, microviscosity, or orientation of molecules in the local environment of a fluorophore can result in alteration of the electron structure or collisional probability of the fluorophore. Such environmental changes can be detected by monitoring fluorescent signal parameters such as intensity, wavelength, life time, or polarization. For example, it is not common for fluorescence emission efficiency (quantum yield) and fluorescence lifetime of an intercalating fluorophore is increased by an order of magnitude more when inserted into the stacking region of rigid and hydrophobic base of a double-stranded nucleic acid with respect to the non-bound dye in solution. The present invention utilizes, and is not limited to, the fluorescence intensity response of the bound fluorophore via monitoring in a total internal reflection configuration along the fiber optic substrate to quantitate the presence of hybridized nucleic acids on the surface of the fiber. . The intensity of fluorescence is directly proportional to the amount of target nucleic acid or nucleic acid analog initially present in the solution. It is also possible to use the time dependence of the rate of change of the fluorescence intensity increase of hybridization to determine the concentration of the target nucleic acid. The fluorophore of the present invention may be, for example, ethidium bromide (EB). The ethidium cation (3,8-diamino-6-phenyl-5-ethyl-phenanthrid) is a fluorescent compound which is strongly associated with double-stranded nucleic acids by intercalation in the base stacking region and in medium cases the main groove of the main structure of the double helical structure (Monaco et al., 199'3, Journal of Bimolecular Structure and Dynamics, 10: 675). The ethidium cation is particularly suitable for nucleic acid hybridization investigations for a number of reasons. Firstly, the quantum yield of the dye is known to increase as much as 100 times when it is intercalated in the base stacking region with respect to that unbound dye in aqueous solution (Bauer et al, 1989, Proceedings of the National Academy of Science USA, 5_6: 7937). Second, the binding affinity and the increase in fluorescence of the dye are independent of the base composition (Cuniberti et al, 1990, Biophysical Chemistry, 38_: 11). Third, the intercalation of the ethidium cation is known to increase double stability as the two substituents 3,8-amino hydrogen linked with the internucleotide phosphate groups in each of the DNA strands (while other intercalators are known to significantly decrease double stability) (Cuniberti et al, 1990, Biophysical Chemistry, 38: 11). The maximum absorption of ethidium bromide is 510 nm, which is sufficiently close to the wavelength of 488 nm, of an Ar * laser that is used to excite the fluorophore. The dye has a maximum emission of 595 nm when it binds to the DNA which is a sufficiently large Stoke change to form the emission radiation separation of the excitation radiation directly forward and avoids the effects of internal filter by using a dichroic thickness and other normal optical components (Haugland, 1992, Molecular Probes: Handbook of Fluorescent Probes and Research Chemicals, 5th Ed .:, USA: Molecular Probes Inc.). Due to the reasons mentioned above, the use of EB has been shown to provide a sensitive means to detect the presence of double nucleic acids for this application. -A specific example of a bound fluorophore is illustrated in the synthetic schemes in Figures 3a, b and c. In this case, a modified ethidium dye with binding, here the acid portion of C13, is synthesized as shown (Figure 3a). The acid bound ethidium analog binds to oligonucleotides functionalized with immobilized 5'-hexylamine on the surface of an optical fiber to generate the biosensor with the bound fluorophore zone. For the case where the nucleotides are developed in the support via solid phase phosphoramidite synthesis, the functionalization of 5'-hexyl amine can be easily achieved by the use of commercially available reagent Aminolink 2®. The fluorophore or report group can be linked at the 5 'or 3' end of the oligomer but not only to a hydrocarbon bond but other types of bonds such as polyether, aliphatic / aromatic or peptidic mixture. The binding does not need to be restricted to the 3 'or 5' ends of the oligomer, but can be bound to a terminal or internal ribo-residue via the 2'-hydroxyl (Yamana et al, 1991, Tetrahedron Letters, _32: 6347). Similarly, a binding can be attached to an internal terminal nucleobase using pyrimidines (Pieles et al, 1990, Nucleic Acids Research, 1_8: 4355) or purines (Roduit et al, 1987, Nucleosides and Nucleotides, 6: 349). In addition, the internucleotide ligation can be a site for the attachment of the binding (Agrawal et al, 1990, Nucleic Acids Research, _18_: 5419). Obviously, any combination of these methods could be used to specifically incorporate multiple reporter groups on site. The choice of fluorophores that can be attached to the oligonucleotide include organic intercalating complexes, such as ethidium bromide of commonly used nucleic acid, thiazole orange and analogues thereof as prepared by LG Lee et al (1986, Cytometry 1: 508). and the cyanine YOYO, BOBO and TOTO series based on intercalating fluorophores that are commercially available from Molecular Probes Inc. (Eugene, OR). Inorganic coordination complexes such as the "molecular light switch" Ru (Phen ') 2 dppz PFg developed by Jenkins et al. (1992, J. Amer. Chem. Soc. 11: 8736) can also be used as slit-binding dyes such as Hoechst 33258 and Hoechst 33342, which are commercially available from Aldrich Chemical Co. (Milwaukee, Wl). These fluorophores are chosen so that the fluorescent probe is cooled (non-emissive) when in the presence of single-stranded nucleic acids and provides intense luminescence when in the presence of double-stranded nucleic acids. This change in observed luminescence occurs via changes in the relative regimes of radioactive and non-radioactive probe relaxation processes when environmental changes from the aqueous solution to a highly structured hydrophobic zone in the nucleic acid base stacking region double-stranded Other examples of classes of fluorophores may be used in the present invention include acridine dyes, phenanthrides, phenazines, phenothiazines, quinolines, alphatoxins, polycillic hydrocarbons, oxirane derivatives, actinomycins, anthracyclines, thiaxanthenones, anthramycin, mitomycin, platinum complexes, polyinterlactants, norfilin -A, fluorenones and fluorenones, furocoumarins, benzodipirones and monostral fast blue. Preferred dyes are also those that provide large Stokes changes, can be excited at long wavelengths and have large differences in fluorescence lifetime, quantum efficiency and / or excitation and emission wavelength when in solution compared when they bind to hybridized nucleic acids. The light emitted from the fluorophores (after direct excitation) to the surface of the fiber is preferably applied back to the fiber and can be monitored by a photomultiplier tube (PMT *) or any other suitable detection equipment. The increase in length of the coated fiber results in a longer optical path length and better sensitivity (Krull et al, 1991, Fiber Optic Chemical Sensors and Biosensors, Vol. II, 0. S. Wolfbeis, Ed., CRC Press , Boca Ratón, pp 315). The direct excitation of fluorophores in an immobilized layer extending from the biosensor results in an improved signal-to-noise ratio as background fluorescence interference in the bulky environment is avoided. An instrument used for fluorescence intensity measurements is based on a fluorescence microscope described elsewhere (Brennan et al, 1990, Anal. Chim. Acta 237: 253) and shown in Figure 4 (a). An instrument as shown in Figure 4 (b) can also be used in which the output of a suitable light source, for example, an argon ion laser is directed in an optical fiber via a lens with a numerical aperture that is equal to, or greater than, the numerical aperture of the functionalized nucleic acid waveguide when it is in the regulatory solution of Hybridization used for the detection of analytes. The excitation radiation can be coupled to a supply fiber via a fiber optic waveguide assembly bent so that all modes carried by the first fiber in which the excitation radiation is first coupled can be supplied to the second fiber. to provide the optical excitation of fluorophores associated with the biosensor. The excitation radiation can be internally reflected fully along the length of the supply fiber to a sensor fiber functionalized with immobilized oligonucleotide and fluorophore. The coupling of the radiation between fibers can be achieved by abutting the distal termination of the supply fiber of the proximal end of the sensor fiber in a suitable non-fluorescent fiber coupler. The termination of the coupler is preferably designed as a compression fitting end that provides a hermetic seal to solutions to prevent diffusion of contaminants in the fiber coupler and to cause bypass in the analytical signal. The sensor fiber is placed inside a small volume flow stop hybridization chamber made of a suitable inert material with good thermal conductivity (for example, stainless steel or titanium). The temperature of the hybridization can be controlled by the use of a suitable thermoelectric housing to provide fast thermofixing at the desired temperature and computer control. The temperature of the solutions in the hybridization cell can be determined accurately (± 0.2 ° C) by using a glass encapsulated thermistor incorporated in the hybridization cell. The supply solutions to the hybridization and sensing fiber cell can be realized by the use of a computer controlled pump (for example, peristaltic pump) where all the solutions originate from a computer controlled a.sub.sampling tester. Fluorescence emission from fluorophores associated with fully immobilized nucleic acid complexes is internally reflected within the sensing fiber. The portion of the light coupled on the back in the supply fiber is directed towards an interference filter with the bandpass window appropriate for the emission of fluorophore used in the optical sensor. The fluorescence radiation that passes through the interference filter then enters the photomultiplier tube to provide a quantitative measurement of the fluorescence intensity. In alternative embodiments, the radiation source can be a dual frequency laser, a semiconductor laser, a brightness lamp or LED. The waveguide coupling can be achieved with fiber couplers and the detector can be an avalanche diode instead of a PMT. In one embodiment of the invention, the biosensor operates as follows. The optical fiber with the fluorescently bound single-stranded nucleic acid is flushed through the cell and immersed in the hybridization buffer. When nucleic acids or nucleic acid analogs of a single strand that are complementary to the immobilized strands are introduced to the flow cell, hybridization occurs after intercalation (or other suitable ligand binding motif) and enhanced fluorescence emission of the bound fluorescent probe, as illustrated in Figure 5. The fluorescence intensity is monitored in a total internal reflection configuration where the optical fiber and organic coating form a waveguide to provide excitation to the immobilized nucleic acid and fluorescent probe on the surface as well as collecting the fluorescence emission. Monitoring the fluorescence intensity of 'the fiber, a measurement of the amount of target nucleic acid in the solution can be determined. The regeneration of the biosensor can be achieved by technical methods such as raising the temperature inside the hybridization cell of the flow of flow or by chaotropic methods in which highly polarized salt solutions alter the hydrogen bonding structure of the solution to affect the denaturation of hybridized complex. In any case, the stability of complex in the system is reduced to the point where the hybridization is not energetically favorable and the complement strands dissociate from the covalently immobilized oligomers and can be flushed from the flow cell. The regeneration methods as described herein can be used to recycle the biosensors. The formation of multiple strand nucleic acids (i.e., nucleic acid complexes composed of 3 or more strands), such as triple nucleic acids, can be determined from the temperature dependence of the fluorescent signal. Normally, the fluorescence efficiency of a fluorophore is increased with the decrease in temperature pertaining to reduced collisional deactivation as a consequence of the reduced kinetic energy of the molecules surrounding the fluorophore. Fluorescence efficiencies with negative temperature coefficients are readily observed for fluorophores in solution as well as for fluorophores interspersed with nucleic acids as illustrated in Figure 6. When multiple strand formation occurs (eg, attachment of a third strand in a primary groove of a double-helical nucleic acid), the exclusion of the ligand bound frequently follows the division coefficient for the fluorophore in the multiple strand nucleic acid is often greatly reduced with respect to the same fluorophore in the double-stranded nucleic acid . The ligand exclusion process will also show a temperature dependence when the ligand binding is reduced as the temperature of the system decreases. As such, a positive temperature and fluorescence temperature coefficient can be observed for fluorophores associated with multiple strand nucleic acids as increasing amounts of fluorophores exclude the highly structured environment within the nucleic acid complex in the volume solution. where the probability of collisional fluorescence cooling is much higher. A clearly positive temperature coefficient of fluorescence intensity could be observed for a fluorescent nucleic acid binding ligand in a multiple strand nucleic acid. The temperature at which multiple strand formation occurs could be analyzed from the maximum in a graph of fluorescence intensity versus temperature where the temperature coefficient changes from negative (for the dye attached in the double-stranded nucleic acid) to positive (for the dye that is excluded from the multiple strand nucleic acid complex). This process is illustrated in Figures 7 (a and b) for the formation of the triplet of the sensor surface with linear and branched nucleic acids. The biosensor in the present invention provides a rapid clinical test for viruses (e.g., HIV, lymphotropic T cells, viruses 1 and 2, hepatitis B and C), and pathogenic bacteria (e.g., E. coli, Salmonella, Listeria, Chlamydia ssp. ., Trichomonas vaginalis, Gradenerella vaginitis) as well as other microorganisms. The detection of genetic disorders (e.g., cystic fibrosis and sickle cell anemia) and diseases such as cancer is also made with the method and apparatus of the present invention as well as potential therapies for treating such diseases (e.g., branched antisense nucleic acids). that inhibit the expression of target nucleic acid sequences via the formation of triplets with the particular sequence, effectively protecting the genetic information from being read by transcription enzymes). The biosensor is useful for monitoring the in vivo response of bacteria to an antibiotic treatment to ensure the efficacy of the treatment regimen. The doctor, based on past experiences, chooses the antibiotic and the dose to treat the bacterial infection. Samples of organisms of infection can be sent to the laboratory to be tested for MIC, Minimum Inhibitory Concentration for the lower concentration of this particular antibiotic needed to inhibit the growth of the infecting organism. If the MIC of the in vitro test is lower than the dose given to the patient, the treatment is allowed to continue, somehow an increased dose and / or a change of antibiotic can be ordered. The most important problem with this approach is the time required, where 1 to 3 days are needed to acquire the MIC result. Second, it is a test of an in vitro response to reflect an in vivo situation. The use of larger doses than necessary is not a reasonable treatment, given that most antibiotics are toxic to the patient. In addition, the use of inappropriate antibiotics or inappropriate doses can lead to the development of drug resistance in infectious organisms. The biosensor described above on these problems to determine the concentration of one or more species of bacteria present in the patient. The samples can be taken at intervals and tested for the concentration of the bacteria. The change in bacterial concentration over time could reflect the efficacy of antibiotic treatment against the organism or infectious organisms. This ensures that an adequate amount of an appropriate antibiotic can be provided to the patient without providing excessive amounts of the antibiotic. The method described above can use a variety of situations to monitor the response of organisms such as bacteria or fungi to drugs. EXAMPLES Example 1 Preparation of Fused Optical Fibers of Silica Derivatives with Separated Aliphatic Long Chain Molecules Finished with 5 'Nucleoside r-0-dimethoxytrityl-2'-deoxythymidine. Plastic coated silica optical fibers with a diameter of 400 μm were purchased from 3M Specialty Optical Fiber (North York, ON, Canada). The coating of the fibers was mechanically removed and the fibers were cut to lengths of approximately 1 cm. One face on each fiber was polished by suspending the fiber on (and placing the end face of the fiber in contact with) the turntable of a controlled mixer at the speed of 37600 of the Thermolyne type (Sybron Corporation, Dubuque, 10, USA) in the which the 1200-grade sandpaper was immobilized. All the fused silica optical fibers were cleaned using a Harrick PDG-32G plasma cleaner (Harrick S'cientific Corporation, Ossining, NY, USA) before activation with aminopropyltriethoxy silane ( APTES-). The fibers were then washed with a 1: 1 acetone / methanol mixture and stored in a vacuum desiccator. The optical fibers were cleaned with plasma for 5 minutes at a low power (40 W) and placed in a solution of 1: 200 (v '/ v) aminopropyltriethoxy silane (APTES) in dry toluene. This was done under a nitrogen atmosphere using glass containers that were often dried and pretreated with octadecyltrichlorosilane. The structure of the APTES coatings on the fused silica substrates have been previously investigated by Vandenberg et al. (1991, J. Colloid and Interface Sci., 147: 103). The method of Arnold and co-workers (1989, Collect, Czech, Chem. Commun., 54: 523) was used to synthesize an aliphatic end-terminator terminated with 5'-0-dimethoxytrityl-2'-deoxythymidine. In this method, 1, 10-decanediol was condensed with succinic anhydride to form bisuccinate of 110 decanediol as illustrated in Figure 1 (a). The bisuccinate was reacted with N-hydroxysuccinimide and 5'-0-dimethoxytrityl-2'-deoxythymidine in the presence of N, N '-dicyclohexyl-carbodi-ida (DCC) and 4-dimethylaminopyridine (DMAP) to produce a functionalized separating molecule of nucleoside The separator is then attached to the surface of the optical fiber treated with APTES via the amide formation. EXAMPLE 2 Preparation of Fused Silica Optical Fibers Derived with Extended Glycidoxypropyltrimethoxysilane with Interlaced Molecules of Rotated Mono-Dimetoxy Hexamethylene Glycol Substrate In order to develop oligonucleotides on the surface of silica substrates (such as fused silica) by the synthesis of Automatic solid phase, the surface was functionalized, with separate molecules of at least 25 A in length having an amine or a hydroxyl functionality at the termination of the spacer molecule. A non-hydrolysable chemical separating molecule is used. The method used was a modification of that reported by U. Maskos and E.M. Southern Southern where the silica surface was treated with glycidoxy-propyltrimethoxysilane (GOPS), followed by extension via treatment with hexaethylene glycol (HEG) under acidic conditions. For purposes of creating biosensors with higher sensitivity and lower detection limits, this method is advantageous over the use of hydrocarbon bonds. The water-soluble HEG interleaver provides a more fluid environment (which should not be self-assembled) so as to improve the ability of the immobilized DNA strands to hybridize with a complementary material in solution (in terms of energy and kinetics). The hydrophilicity of the interlayer will also facilitate the removal of absorbent contaminants (eg, proteins, organics) that can clog the surface and contribute to the derivation of the fluorescence intensity. However, since HEG is bifunctional, there is the possibility of creating non-reactive closed-loop structures that can significantly decrease the loading of oligonucleotides on the surface of the fibers. In order to eliminate this problem, a HEG termination is protected with a dimethoxytrityl functionality before extension with GOPS. This strategy allows easy determination of the amount of support interleavers attached to the silica surface. The removal of the trifyl protecting groups by the treatment with highly colored trihalion cation acid, which can be measured quantitatively by monitoring A (5o .m) of the deprotection solution. If a triphenyl group released by the interlacing molecule attached to the surface is known, the HEG charge can be easily determined. The immobilization of a protected interlacing molecule provides the additional advantage that the hydroxyl groups produced after the HEG binding to the epoxide portion and all the surface silanols can be crowned to prevent the growth of unwanted oligonucleotides at these sites. The presence of the oligonucleotides of side products, which are prematurely terminated due to the lack of a suitable support molecule, can decrease the sensitivity and selectivity of the sensor. The additional charge imparted to the structure of the anionic base of a strand of side product can inhibit the hybridization between the analyte strands and the neighboring probe sequences. See: R. T. Pon Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs, S. Agrawa, Ed. 1993, Human Press, Inc., Totowa NJ. In conjunction with the use of non-hydrolyzable partitioning molecules, phosphoramidite synthenes protected with t-butylphenoxyacetyl were used. This labile protective group can be removed quickly (15 min @ 55 ° C or 120 minutes at room temperature compared to 12-16 hours @ 55 ° C using 27% aqueous ammonia) thus reducing the possibility of separating the immobilized sequences by hydrolysis. the silyl ether ligatures that eventually expand the strands of the fiber surface, i) Cleaning the Silica Substrates Prior to Functioning with GOPS: The pH regulator coating was mechanically separated from the previously short fiber optic pieces (400 μm diameter x 44 mm) and the coating was dissolved by treatment with acetone. Fused silica substrates, i.e., optical fibers or wafers were added to the 1: 1: 5 (v / v) solution of 30% ammonium hydroxide / 30% hydrogen peroxide / water and the mixture was stirred at 80 ° C for five minutes. The substrates were then removed and treated with a concentrated 1: 1: 5 (v / v) solution of HCl / 30% hydrogen peroxide / water and the mixture was stirred at 80 ° C for five minutes. The substrates were then sequentially washed with a methanol, chloroform, and diethyl ether, respectively, and dried under vacuum, ii) Synthesis and purification of hexamethylene monodimethoxytritylated glycol (DMT-HEG): Dimethoxytrityl chloride (7.1 g) was dissolved in 10 ml of dry pyridine and added dropwise to a stirred solution of hexaethylene glycol (5.65 ml) in 5 ml of dry pyridine under an argon atmosphere. Stirring was continued for 16 hours after the time the reaction mixture was combined with 50 ml of dichloromethane. The dichloromethane solution mixture was stirred twice with 900 ml portions of 5% aqueous sodium carbonate and then with three 900 ml portions of water in order to remove unreacted HEG, pyridine, and pyridinium salts. The product was purified by chromatography using silica gel and a solvent system of 0.1% triethylamine in 1: 1 dichloromethane / diethylether. The identity of the product was confirmed by NMR proton spectroscopy (200 MHz). iü) Functionalization of Silica Substrates Fused with 3-Glycidoxypropyltrimethoxysilane (GOPS): The clean fused silica substrates were suspended in stirred solution composed of 40 ml of xylene, 12 ml of GOPS, and a Hünig base trace at 80 ° C during the night. The fibers were then sequentially washed with methanol, chloroform, ether and then dried under vacuum. iv) Bonding of Functionalized Silica Substrates from DMT-HEG to GOPS The functionalized GOPS fibers were suspended in a stirred solution of 1: 4: 8 (v / v) DMT-HEG / diethyl ether / toluene containing a catalytic amount of hydride. sodium under an argon atmosphere. The reaction mixture was stirred for 14 days after which time the fibers were removed and washed sequentially with methanol, chloroform, ether, and then vacuum dried, v) Silanol Not Reaction Coating and Hydroxyl Functionalities with Chlorotrimethylsilane : Fused silica fibers functionalized with DMT-HEG were suspended in a solution of 1:10 (v / v) chlorotrimethylsilane / pyridine for 16 hours under an argon atmosphere at room temperature. Example 3 Preparation of Fused Optical Fibers of Silica Derivatives with Interlaced Molecules of Mono-Dimethoxytritylated Hexamethylene Glycol Substrate via Mesylate Activation. The details of the preparation of the fused silica substrates and the synthesis of DMT-HEG are provided in example 2 (i and ii). DMT-HEG (0.5 g) was suspended in 50 ml of anhydrous pyridine. The solution was maintained under an anhydrous argon atmosphere and stirred. While 1.2 equivalents of methanesulfonyl chloride were added dropwise. The reaction was allowed to proceed for 60 minutes at room temperature with stirring. The clean fused silica and silicon substrates were introduced into the solution containing the mesylated DMT-HEG and the substrate functionalization reaction was allowed to proceed for 4 days with stirring at 40 ° C under an argon atmosphere. After the incubation period of 4 days, the solution was decanted from the functionalized substrates which were washed with copious amounts of dichloromethane. The washings continued until there was no discernable absorbance at 504 nm when the solution was observed. washing made acid by treatment with trichloroacetic acid. The functionalized substrates were crowned by both the methods of Example 2 (v) and stored under vacuum and over P205 until needed. EXAMPLE 4 Preparation of Fused Optical Fibers of Silica Derivatives with Interlacing Molecules of the Mono-Dimethoxytritylated Hexethylene Glycol Phosphoramidite Substrate via the Coupling of β-Cyanoethylphosphoramidite in an Automatic Synthesizer. The details for the preparation of the fused silica and DMT-HEG substrates are provided in example 2 (i) and 2 (ii), respvely. DMT-HEG (0.5 g) were suspended in a solution consisting of 12 ml of anhydrous THF and 4 ml of anhydrous N, N-diisopropylethylamine. The solution was kept under an atmosphere of anhydrous argon, and stirred at all times. 1.1 equivalents of 2-cyanoethyl-N, N-diisoproylamino-phosphochloride was added dropwise to the DMT-HEG solution and the reaction was allowed to proceed for 90 minutes at room temperature. TLC analysis (1: 1 CH2Cl2 / diethyl ether) indicated the quantitative formation of phosphoramidite synthase of DMT-HEG (Rf = 0.7). The reaction product was extracted three times in ethyl acetate from a 5% sodium carbonate solution. The organic phase was separated from the aqueous layer, dried over NaSO, filtered and the solvent was removed under reduced pressure. The product was then stored dry and at -20 ° C under an argon anhydride atmosphere until required. The functionalization of the fused silica substrates was then performed as part of a normal coupling cycle using the automatic solid phase DNA synthesizer and a 0.1 M solution of the DMT-HEG phosphoramidite in THF anhydride. The methods for automatic oligonucleotide synthesis are detailed in Example 5. The counting procedure detailed in Example 2 (v) was then performed in order to block any undesired reactive sites in the substrates. Example 5 Synthesis of Oligonucleotides in the Functionalized Substrate Interleaver with Fused Silica Waveguides All DNA syntheses were performed by the well established β-cyanoethylphosphoramidite method with an Applied Biosystems 381A or 391EP DNA Synthesizer using a functionalized substrate interleaver with controlled pore glass beads, fused silica optical fibers, fused silica wafers, flat silicon wafers. The synthesis of automatic solid phase DNA is well known and described in detail (Beaucage et al., 1992, Tetrahedron Letters, 4j3: 2223-2311; Oligonucleotides and Analogues: A Practical Approach, F. Eckstein, Ed. Oxford University Press, NY, 1991). The functionalized optical fibers of the substrate interleaver were placed on an Oligonucleotide Purification Cartridge column from Applied Biosystems (ABI) (OPC column) or 10 μmol scale synthesis column with dead volume taken by inert polyethylene pieces. The final filter papers were replaced (ABI) and the column ends were placed closed using aluminum seals. The synthesis of oligomers in the optical fibers was carried out on a scale of 0.2 μmol, with a pulsed supply cycle in the "detritylation" mode. The β-cyanoethylphosphoramidite cycle was used as supplied by ABI with the exception of the extended nucleoside coupling times (2-10 minutes) and the solution delivery times were increased to accommodate the large synthesis columns. With the exception of thymidine syntones, t-butylphenoxyacetyl protected phosphoramidite synthons were used together with a t-butylphenoxyacetic anhydride coping solution supplied by Millipore Inc. In the case where polyimidyl acid oligonucleotides were developed in the optical fibers, the deprotection of the phosphate blocking groups of the immobilized oligomer was achieved by placing the optical fibers in a 2: 3 solution of triethylamine / acetonitrile at room temperature for 1.5 hours . This procedure p-rovocated the loss of the phosphate blocking group via β-elimination while there was no separation of the single-stranded DNA (ssDNA) from the optical fibers. In the case where the oligonucleotides containing bases other than thymine were developed, the protocol for phosphate and nucleobase deprotection was followed. A solution of 30% ammonium hydroxide and the synthesis column containing the functionalized optical fibers with immobilized oligonucleotides were extracted using a syringe and a female-to-male adapter. The fibers immersed in ammonia were allowed to stand for two hours at room temperature for two hours after the time the ammonia solution was expelled from the column and the contents of the column were washed five times with 5 ml portions of sterile water. The deprotection solutions and the washings were collected and concentrated to a total volume of 1 ml. A260nm of the concentrated deprotection solution was measured in order to determine the amount of DNA released from the fused silica substrate. Based on the results of the trifly cation and A250nm assay of the deprotection solution, it was found that approximately ~ 20% of the oligomers remained bound to the surface after the ammonia deprotection process. In the case where the oligonucleotides of the mixed base sequence were developed in the optical fibers, the oligonucleotide sequence was' (5 '-TAG GTG AGA CAT ATC ACA GA-3'). which is a nucleic acid probe for the next E03 sequence of the Candida albicans genome. The fibers coated in ssDA are either stored dry or maintained in a solution of 1: 1 e ± anol / water. All fibers were cleaned before being used by sound treatment in a 1: 1 ethanol / water solution for 5 minutes in order to remove any fluorescent contaminants adsorbed to the surface of the fibers. Example 6 Characterization of Biosensor by Tri ilo Cation Analysis. All oligonucleotide syntheses were evaluated by electroscopic quantification of triplyl cation released during the trichloroacetic acid treatment steps of the automated synthesis. The fractions collected from the trityl cation are diluted with 2.0 ml of 5% TCA in 1,2-dichloroethane immediately before making the absorbance measurements. Absorption at 504 nm was measured in order to determine the concentration and total number of trityl cation moieties released during each TCA deprotection step of the synthesis. Thus, the total number of oligomers successfully developed in the solid supports is determined. Since there is no discernible decrease in the amount of trityl cation released during the successive deprotection steps, it can safely be assumed that the efficient coupling efficiency of 99.5% or better suggested by the automatic synthesizer manufacturer (ABI) was achieved. The results of a trityl cation assay for a dT20 synthesis in optical fibers of the methods given in Examples 1 and 5 are shown in Figure 8. Example 7 Generation of Complementary and Non-Complementary Nucleic Acids. The synthesis of dA2o and rA2o was carried out using a conventional LCAA-CPG support with the β-cyanoethylphosphoramidite cycle by ABI. A nonadecamer of random base composition (dRig) was also prepared by simultaneously introducing all four phosphoramidite reagents to the column at each coupling step. Normal deprotection with aqueous ammonia (29%, 1.5 ml, 24 hours) was used to release the oligomers from the solid support and remove the base protecting groups. In the case of rA2o, the deprotection of the phosphate-blocking groups, base protecting groups and separators of the CPG support was carried out by treating the oligomers with 1.5 ml of a solution consisting of 4 parts of aqueous ammonia and 1 part of ethanol during 48 hours at room temperature. The aqueous solution containing the oligonucleotides was then collected, evaporated to dryness, and the residue treated with 300 μl of a 1 M anhydrous solution of tetra-N-butyl-ammonium fluoride in THF overnight at room temperature. After the incubation time, the reaction was cooled by adding 1 ml of water to the reaction mixture. The unpurified oligomer was purified by polyacrylamide gel electrophoresis and reverse phase liquid chromatography or significant exclusion chromatography. Example 8. Detection and Quantification of Complement DNA (cDNA), Complement RNA (RNAc) and Nucleic Acid without Complement by the Optical Sensor Manufactured through the Procedures of Examples 1 and 5. An optical fiber functionalized with icosanucleotide of Polymymidilic acid was randomly selected from the fiber bath (approximately 25) created in Example 1 and positioned under the microscope objective, as illustrated in Figure 4 (a). In this orientation, incident laser radiation that enters the proximal terminal and is completely internal is reflected through the fiber. The majority of the fiber is immersed in a hybridization pH buffer solution consisting of 1.0 M NaCl and 50 mM sodium phosphate (pH 7.4) in sterile water contained within a 4 ml plastic cuvette. The hybridization regulator is passed through an acrodisc filter © immediately before introduction into the cuvette. The fluorescent radiation emitted, from the stimulated fluorescent molecules associated with the double-stranded nucleic acids, is directed towards the microscope by the total internal reflection. The emission of fluorescent molecules was separated from the excitation radiation through a dichroic mirror and directed to a photomultiplier tube. The photomultiplier tube provides measurements of the fluorescence emission intensity. The fluorescence intensity values are reported with the system at 25 ° C to avoid uninformed conditions caused by the temperature dependence in the quantum efficiency of fluorescence and as relative quantities, thus obviating the need to control the experimental parameters such as intensity of laser, optical alignment and photomultiplier tube (PMT) gain which is assured beyond the daily control. In order to affect the hybridization with the nucleic acid strands, dA20 ssDNA were added to the plastic cuvette containing the suspended fiber in a fresh hybridization regulator at a temperature of 85 ° C. This temperature is chosen as sufficiently greater than the melting temperature of the doublet of 60 ° C (Tm), the temperature that is average in all the doublets present dissociates) and so follows the boiling point of the regulator. Incubation at lower temperatures Tm has been shown to cause incomplete hybridization in which only a fraction of the bases in each strand interact to form partially hybridized complexes (Rubin et al, 1989, Nucleic Acid and Monoclonal Antibody Probes, B. Swaminathan and G Prakash, Eds., Marcel Dekker, Inc., NY, pp. 185-219) *. Although the covalent immobilization of ssDNA removes a degree of freedom from the oligomer, hybridization at temperatures initially above the doublet Tra ensures the formation of doublets with the greatest possible extent of overlap. In all cases, no appreciable change in intensity from that of the baseline was observed after a period of 90 minutes of incubation. The solution was allowed to settle and cool to room temperature (25 ° C) between 30 and 90 minutes after which the fiber was aligned with 60 ml of hybridization buffer (25 ° C) to remove the excess strands. The intercalation of the fluorophore in • the dsDNA was achieved by injecting 10 μl of a 1 mg-ml-1 aqueous solution of ethidium bromide (EB) into the cuvette and allowing the solution to settle for 15 minutes followed by washing the solution. the fiber aligning the cuvette with 60 ml of fresh hybridization regulator (25 ° C).

The response of the fiber optic DNA biosensor to EB and cDNA was shown in Figure 9. As a control experiment, 10 μL of an aqueous solution of 1 mg-ml1 of EB was added to the cuvette in which the functionalized fiber with ssDNA was suspended. After 15 minutes, 60 ml of fresh hybridization buffer (25 ° C) was aligned through the cuvette in order to remove any nonspecific ethidium cation link and no discernible increase in fluorescence intensity was observed. the fiber. An increase of 104 ± 15% in the fluorescence intensity of the fiber was observed which was exposed to 189 ng-ml-1 of cDNA and was obtained with EB relative to the baseline value of the clean sensor with ssDNA only on the surface Waveguide The fiber was regenerated by rinsing the cubet and the optical sensor 30 ml of hot buffer solution (85 ° C) for a period of about 30 seconds and the system allowed to place for five minutes. After five minutes of waiting, an additional 30 ml of hot buffer was lined up through the cuvette to wash the cDNA strands in dissociated form. This procedure is known to melt DNA doublets as the regulatory temperature as well as the previous Tm of the dsDNA. The fluorescence intensity returned, within the experimental uncertainty, to the intensity observed at the beginning of the experiment. The same hybridization experiment was repeated when the optical sensor was exposed to 757 ng-ml_1 of cDNA during the hybridization procedure. This produced an increase of 720 ± 20% in the fluorescence intensity observed from the sensor with respect to the baseline value. In contrast, a similar concentration of non-complementary sequences (a random base sequence nonadecamer) gave essentially no response, as shown in Figure 9 (a). A solution of 3.8 ng-μl-1 of rA2o '(450 μl) was introduced into a cuvette containing hot hybridization buffer and the sensor provided a solution of 570 ng ml-1 of cRNA. The same hybridization and the obtaining procedure were used for cDNA that follows. The response profile for this hybridization procedure is shown in Figure 9 (b). A comparison of the response of the biosensor with dT2o immobilized to the cDNA and cRNA according to the experimental error. Example 9. Time of Dyeing and Concentration of Editio Bromide (EB) Effective The time of obtaining the sensor with EB was loaded after each hybridization with cDNA. For each determination, injections of 30 μl of 56.8 μg-ml-1 solution of aqueous dA20 were made and the hot hybridization buffer in the cuvette, containing the cDNA strands, was allowed to cool to room temperature for 30 minutes. A solution of 1 -mg-ml-1 of EB in water (10 μl) was added to the cuvette after each hybridization to provide an EB concentration of 8.4 x 10 ~ 3 M. A dyeing time of 20 minutes, with 8.4 x 10 ~ 3 M EB was required to generate > 99% of the filled signal, as shown in Figure 10 (a). To study the effect of EB concentration during dsDNA staining, all the hybridization parameters were the same as those used to study the dyeing time and the 20 minute dyeing time was used. Dyeing with EB solutions of concentrations of 8.5 X 10_3M or higher was required to generate; > 99% of the full dyeing in 20 minutes, as shown in Figure 10 (b). Example 10. Long Term and Thermal Stability of the Nucleic Acid Sensor. The greatness of the optical sensors, and the DNA as a biorecognition element, were made evident by the maintenance of the activity after long term storage and vigorous cleaning conditions. Fibers that were stored for 1 year under vacuum in 1: 1 ethanol / water solutions, absolute ethanol or dried at -20 ° C provide identical response characteristics for prepared fibers Recently. The adsorbed fluorescent contaminants which were accumulated by long term storage were completely removed (as confirmed by fluorescence microscopy) through the sound of the fibers in a 1: 1 ethanol / water solution with full activity maintenance and sensitivity. Figure 11 (a) showed the response of a fiber in 1 month (stored in vacuo) used without cleaning the surface and (b) and fiber • in 11 months (stored dry at -20 ° C) which had been cleaned through sound in ethanol solution. It should be noted that the cleaning sensitivity of the fiber in 11 months is identical to that of the fibers in 1 month cleaned by the same procedure (data not shown). The cleaning of the sensor by previous sound to use has been consistently observed to increase the sensitivity of the device by a factor of approximately 2.5. The sensors have provided femtomolar detection limits and a response which is linear with the cDNA concentration (P.M. = 6199 g mol-1). The regression lines shown in Figure 11 show good aptitude to the data points with r2 values of 0.965 and 0.968 for the fibers of 1 month and 11 months, respectively. From these data, the sensitivity of the optical sensor (11 months, manufactured by the protocols of Examples 1 and 5) was determined to be an increase in fluorescence intensity of 203% per 100 ng-ml "1 of cDNA with a detection measurement limit of 86 ng-ml-1 Calibration maintenance has been observed for all far-away experiments in which 5 regenerations as many have been performed for durations of up to 12 hours The ability to clean and sterilize Such a biosonde device or biosensor that can be usable in an in-line configuration is a significant advantage.As the specific binding properties of nucleic acids are based on a secondary structure, the use of nucleic acids in biosensor manufacturing charges the devices which are not stable for prolonged storage, but also severe washing conditions and sterilization A summary of cleaning effects through sound in absolute ethanol (15 minutes) and autoclave (120 ° C for 20 minutes at 4 atmospheres pressure in sterile water) in response to the sensors (approximately ~ 400 ng-ml-1) is shown in the Table as follows. Ethanol sonification and autoclaving are observed to improve the sensor response, most likely through the removal of contaminants on the sensor surface (stored dry for 11 months, or stored in ethanol). Table 1. Effect of storage, cleaning and sterilization conditions in response of the sensor to cDNA of 400 ng-ml -i Example 11 Studies of Thermal Denaturation of the cDNA: Complex of Immobilized DNA in the Sensors Created from Protocols of Examples 1 and 5 and in Comparison to that of Same Oligonucleotide Complex in Solution. i) Thermal Denaturation Investigations of dT20 Aqueous with Aqueous dA2p The equimolar amounts of each oligomer in the hybridization buffer (1M NaCl, 10 mM P04, pH = 7.0) were mixed in such a way that the final concentration was about 1 μM in each strand Prior to the thermal-fusion studies the oligonucleotide mixture was heated briefly to 80 ° C and slowly cooled to 20 ° C in order to hybridize all the strands. The samples were kept at a low temperature limit for 15 minutes before starting the fusion studies, by allowing thermal equilibration. The temperature was then excessive at 0.5 ° C intervals in a 0.5 ° C / minute ratio, while the absorbance was recorded at 260 nm. ii) Fusion Curve Investigation of Immobilized dc with cDNA. The dT2o sequences were immobilized on flat fused silica wafers (5 mm x 10 mm x 1 mm) according to the protocols in Examples 1 and 5. The immobilized dT2O was hybridized with complementary dA2o sequences by wafer immersion in solution of 56.8 ng-ml "1 of dA20 at 85 ° C and allowing the wafer to cool down to room temperature (25 ° C)." The wafer was then removed from the cDNA solution and washed with a buffer solution. Hybridization at room temperature The wafer was then suspended in a quartz cuvette which was placed in the controlled temperature of the cuvette housing of the UV-visible spectrometer.The wafer placement was adjusted as well as the rest in the path of the light beam The finished volume under the wafer is taken up to an inert packing material.The absorption spectrum was collected at approximately 2 ° C temperature increments in the range of 29 ° C to 76 ° C. The cuvette was fixed by programming an external circulation bath at a specific temperature and the temperature of the regulator solution around the fused silica wafer was measured quantitatively using an encapsulated silanized glass thermistor. Absorption measurements at each temperature were made integrating 100 spectra in the wavelength range between 220 nm and 320 nm. iii) Hypochromicity Thermodynamics and Fusion Curve The transition between an ordered doublet state and the disordered denatured state for complementary nucleotide systems can be monitored and analyzed by UV-visible absorbance spectroscopy to determine the doublet fusion temperature (Tm). ). The extent of hybridization (ie, the number of base pairs formed by doublet) is determined by a comparison of fusion profiles for the immobilized oligonucleotides at similar reported values and dA20 + dT20 in solution. The fraction of a single strand present in the system at any temperature (fss (T)) can be determined through the use of the following equation: A (T) -A? (T) Ja} A "(DA? (G) where A (T), ASS (T), and Ads (T) are the absorbances of the obtained experimental fusion curve, the upper baseline (single-stranded oligomers) and the line lower base (double-stranded oligomers) respectively at temperature T (Nelson, JW, Martin, FH, Tinoco Jr., I. Biopolymers 1981, 20, 2509-2531) By conspiring the temperature again of fss, the doublet fusion temperature can be obtained by determining the temperature which fss = 0.5. i) Fusion Curve Studies of DNA Doublet Support Link and DNA Aqueous Phase. The purpose of thermal denaturation studies to examine both the binding of an oligonucleotide to a solid support through a terminal nucleotide phosphate could result in the loss of degrees of freedom with respect to the availability of each nucleotide to be separated in the formation of the double-stranded structure. The melting profiles of the thermal denaturing of dsDNA immobilized on the surface of the fused silica wafer and dsDNA in solution were obtained, and the results of these investigations are summarized in Figure 12. The double melting temperature of the strands immobilized with complementary strands of aqueous phase was 62.4 ± 0.3 ° C. The Tm value for the aqueous phase of doublet dA20 + dT20 was determined to be 60.5 ° C using the software supplied by Varian. Kibler-Herzon et al., (Kibler-Herzong, L., Zon, G., Whittier, G., Mizan, S., Wilson, WD anti-Cancer Durg Design 1993, 8, 65-79.) Have reported the melting temperature of a doublet of dAig + d ig in 1.02 M NaCl to be 61.1 ° C. This suggests that the immobilized oligomers investigated in this work, to extend the hybridization were completed with base pairs of 20 bases per strand. The small differences in three Tm values can be counted by the fact that these experiments were performed on a different instrument at different times, and the salt concentration used in this work was slightly lower than that used by Kibler-Herzog et al. Since the stability of doublets in low ionic strength regulators is lower than that in high ionic strength regulators, one would expect that the melting temperature of immobilized dT2o + dA20 doublets could be high in the lower stringency regulator (Puglisi). , JD; Tinoco Jr., I; Methods in Enzymology, 1989, 180, 304-325). In addition to this, a large value of Tm for the doublet immobilized on the aqueous phase doublets should not be considered unusually as only one of the strands would experience a significant gain in entropy until the fusion of the immobilized doublet. These factors conclude that, within the experimental uncertainty, immobilized dT2o + dA20 doublets were more stable, but were as stable as the doublet aqueous phase dA20 + dT2o and dAi. + dTiq. This also suggests that hydration of doublet formation is not observed with respect to the availability of the hybridization bases. This is in accordance with the investigations of Wolf et al. (Wolf, SF; Haines, L., Fisch, J .; Kremsky, JN; Dougherty, JP; Jacobs, K. Nucleics Acids Research 1987, 15, 2911-2926), wherein oligonucleotides linked to solid supports via a Long-chain aliphatic binding to the terminus of the strand (3 'end) was not observed to be impaired with respect to hybridization efficiency. Example 12 Detection of cDNA with, and Functionalized Optical Sensor with an Oligonucleotide Probe Sequence for Candida albicans. The optical sensors created by the protocols in Examples 2 and 5 wherein the oligonucleotide sequence (5 '-TAG GTG AGA CAT ATC ACA GA-3'), which is a nucleic acid probe for the sequence E03 forward of the Candida albicans genome, join in the functionalized fibers of the substrate linker. The hybridization and dyeing protocols are continued as reported in example 9. The response of the cDNA sensor (20 nucleotides in length) as shown in Figure 13. The linear calibration (r2 = 0.988), good sensitivity (100%) fluorescence intensity increased by 89 pM increased in concentration in 4 ml of solution surrounding the optical sensor) and low detection limits (6 x 1010 molecules) are observed by the device. Example 13 Manufacture of Optical Sensors with Polymicidyl Acid Icosanucleotides Functionalized at the 5 'Term with 3, 8-Diamino-6-Phenylphenanthridium N5-Tacked Cation. i) Synthesis of methyl- (12-hydroxy) dodecane to. 12-Hydroxydodecanoic acid (5 g) was dissolved in 100 ml of dry methanol to which a solution of p-toluenesulfonic acid (88 mg) in 5 ml of methanol was added dropwise over a period of 15 minutes. The solution is refluxed for 16 hours after which the solvent is removed under reduced pressure. The product is then extracted twice in chloroform from a 5% aqueous solution of sodium bicarbonate. The organic phase is recovered, dried over NaSO, and the solvent is removed under reduced pressure. ii) Tosylation of methyl- (12-hydroxy) dodecane to Methyl- (12-hydroxy) dodecanoate (1.6 g, 7 mmol) is placed in a dryer flask furnace cooled under argon and treated with 3 ml of a sodium chloride solution. p-toluenesulfonyl (1 equivalent, 7 mmol, 1.31 g) in dry pyridine. The solution is stirred at 25 ° C under an inert atmosphere for 16 hours. The solvent is then removed under reduced pressure and the tosylated product is stored dry at 20 ° C until required. iii) N-Alkylation of 3,8-dinitro-6-phenyl-enantridine with methyl- (12-hydroxy) dodecanoate tosylate. 3, 8-Dinitro-6-phenyl-phenanthridine (3.5 mmol, 1.2 g) is combined with the tosylation of methyl- (12-hydroxy) dodecanoate (7 mmol, 2.7 g) in dry nitrobenzene and the solution is stirred for 6 hours at 160 ° C under an argon atmosphere. The alkylated quaternary ammonium salt is precipitated from the mother liquor by the addition of diethyl ether and collected by filtration. The product is further purified by silica gel column chromatography (25% methanol in chloroform) and recovered as a dark purple solid. iv) Chloride Reduction of 3,8-dinitro-5-methyldodecanoa to-6-f nil-f enant ridio 3,8-dinitro-5-methyldodecanoate-6-phenyl-phenanthrid chloride (1.3 mmole, 0.72 g) was dissolved. ) in ml of THF and stirred over NiCl2 »6H20 (10.68 g) and converted to Al powder (0.81 g). Water was then added (0.3 ml) to start the formation of the black Ni / Al catalyst and the reaction allowed to proceed for 15 minutes. The solution containing the reduced product was recovered by filtration, followed by removal of the solvent under reduced pressure. The product was purified by silica gel column chromatography (25% methanol in chloroform) and recovered as a dark purple solid (4%, 0.03 g). v) Tritylation of 3,8-diamino-5-methyldodecanoa to-6-phenyl-phenanthrid chloride. 3,8-Diamino-5-methyldodecanoate-6-phenyl-phenanthridium chloride (0.03 g, 62 pmol) was dissolved in pyridine. dried and treated with dimethoxytrityl chloride (3 equivalents, 68 'g) suspended in dry pyridine (4 ml).

The reaction was allowed to proceed for 16 hours at 25 ° C with stirring under an inert atmosphere. The solvent was then removed under reduced pressure and the product purified (58%, 36 pmol) by reverse phase CLAP (isocratic elution with methanol / water 1: 1). vi) Deprotection of the methyl ester protecting group in 3,8-Bis (dimethoxytrithylamino) -5-methyldodecanoate-6-f-enyl-phenyl ester chloride 3,8-Bis (dimethoxytrithylamino) -5- chloride was suspended. methyldodecanoate-6-phenyl-phenanthridide (36 μmol) in 80 ml of a water / methanol 1: 3 solution. The solution was degassed and treated with KOH (4 equivalents, 160 μmoles) for 16 hours with stirring at 25 ° C. The reaction was cooled and the pH neutralized by treatment with HCl (1 equivalent, 15 μl concentration). vii) Synthesis of Functionalized Optical Sensors of 5'-aminohexyl-dT? Functionalized optical fibers of DMT-HEG-GOPS (prepared according to the method of Example 2) were functionalized with polymyxidic acid icosanucleotide (according to the method of the example 5) terminated in a protected aminohexyl portion of N-trifluoroacetamide to the 5 'end by the use of an Aminolink 2® phosphoramidite synthesizer commercially available from ABI. Deprotection of the phosphate blocking groups of the immobilized oligomers was achieved by obtaining the fibers in a 2: 3 (v / v) triethylamine / acetonitrile solution at room temperature for 1.5 hours. Removal of the trifluoroacetamide protecting group on the aminohexyl functionality located at the 5 'end of the immobilized strands was performed by exposing the fibers in a 10"3M solution of sodium borohydride in absolute ethanol for 1 hour at room temperature. The fibers were then washed once in a 10-3M HCl solution followed by washing with copious amounts of sterile water, viii) Bonding of Protected Trityl Etidium Analog to Functionalized Aminohexyl Fibers, Fused Silica Optical Fibers. completely deprotected with 5'-aminohexyl polyimidylic acid icosanucleotides are immersed in a solution containing 5 mg of the ethidium analogue of unprotected tethered DMT, 40 μl of 1-methylimidazole, and 1.91 g of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 50 ml of water After an incubation period of 7 days at room temperature, the fibers were washed five times each with 50 ml of water. e portions of water, ethanol, and dichloromethane respectively. The proportion of functionalized dry oligonucleotides was determined by measuring the amount of dimethoxytrityl released from each detritylation step during automatic synthesis and the deprotection procedure used to store the primary amine portions in the dye. From these tests it is determined that 63% of immobilized oligonucleotides were functionalized with bound dye. ix) Characterization of the Fluorescent Response of Reagent Sensors with Fluorophore Tied: The response of reactive sensors to 720 ng of complementary DNA is shown in Figure 14. Hybridization was performed at 40 ° C in a regulator consisting of 1 M NaCl and 50 mM phosphate (pH 7.0). It should be noted that this sensor has a significantly improved response time on the sensors without attached dye. The analytical signal filled with 99% was reached in approximately 6 minutes after the injection of the complementary strands for the reagent system as long as 45 minutes were required for the generation of full signal by the sensors without attached dye (see figure 10. { . to} ). Example 14. Detection of Triple Helix TAT DNA Using a Fiber Optic Biosensor i) Background An important path not yet explored by the fiber optic nucleic acid biosensor community is the triple strand oligonucleotide formation research. Usually, a number of spectroscopic techniques (CD, NMR, UV and fluorescence spectroscopy) in addition to the gel mobility assays need to be implemented in order to study the formation of triple helical nucleic acids. However, each of these methods have problems in terms of either the amount of material that is required for the analysis (NMR, CD and gel mobility tests), or that they are limited for investigations of only certain triple systems (for example, example, only TAT triplets can be monitored by UV absorption spectroscopy at 284 nm). Several groups have developed methods for triplet detection ( { I.}. Geselowitx, D. A.; Neumann, R. D. Bioconjugue Chem. , 1995, 6, 502,. { ii} Bates, P.J. Dosanij, H.S., Kumar, S .; Jenkins, T. C.; Laughton, C. A .; Neidle, S. Nucleic Acids Res. , 1995, 23, 3627.). The use of linked nucleic acid ligands to identify DNA structures and morphology is the use of such a method. Many ligands are known to interact in a non-covalent manner with the target oligonucleotide. The linkage modes can be characterized as: (i) intercalation of the ligand, in which normally a flat aromatic portion decreases between the DNA bases - stabilized by pp stacking and dipole interactions, or (ii) lower or higher slot interaction which is stabilized by hydrogen bonding, hydrophobic and / or electrostatic interaction (Long, E.C., Barton, JK Acc. Chem. Res. 1990, 23, 273). The ethidium bromide bonds for both doublets and triplets via an intercalative mode (Waring, M. J. Biochim, Biophys. Acta, 1966, 114, 234), and - this has been studied extensively by fluorescence methods. The fluorescence quantum efficiency of the ethidium cation is increased when it is intercalated in the doublets (LePecq, JB, Paoletti, CJ Mol. Biol., 1967, 27, 87), and triplets (Mergnay, JL; Collier, D .; Rougée , Montenay-Garestier, T., Hélén, C. Nucleic Acids Research, 1991, 19, 1521., Scaria, PV, Shafer, RHJ Biol. Chem., 1991, 266, 5417), however, showed that there is a marked difference in link efficiency and therefore fluorescence intensity between the two types of complexes. LePecq and Paoletti were the first to observe that _the improvement of ethidium fluorescence during the interaction with the doublets (poly rA) - (poly-rU) was significantly greater than the union of the triplet (poly rA) - (poly rU) 2 ( LePecq, JB; Paoletti, CC R. Acad. Sc. Paris 1965, 260, 7033). More recent studies have confirmed that the fluorescence intensity of the intercalated ethidium bromide is greater for the doublets than the ribonucleic acid triplets, and that a temperature dependence exists for the deoxyribonucleic acids (Mergny, JL; Collier, D .; Rougée, M .; Montenay-Garestier, T., Helene, C. Nucleic Acids Res., 1991, 19, 1521., Scaria, PV, Shafer, RHJ Biol. Chem., 1991, 266, 5417., Fang, Y .; Bai, Cl .; Zhang, P. C; Cao, EH; Tang, YQ Science In China (Ser. B), 1994, 37, 1306). The results of molecular model studies suggest that a reduced binding affinity of ethidium for triplets (relative to doublets) exists due to the energy cost of unstacked base triplets compared to successive base pairs (Sun, JS; Lavery, R Chomilier, J.; Z.akrzewska, K .; Montenay-Garestier, T., Helene, CJ Biomol., Struct. Dynam., 1991, 9, 425). This is partially compensated by the quantum efficiency of ethidium bromide in triple DNA which is greater than that for double DNA. The short homopolymer T * AT triplets have been subjected to seminal fluorescence studies. Letsinger et al. (Salinkhe, M., Wu, T. &Letsinger, RL (1992) J. Am. Chem. Soc. 114, 8768-8772.) Have shown that for parallel T * AT triplets, the fluorescence intensity of the Ethidium cation decreases dramatically compared to the fluorescence intensity of the ligand binding to AT doublets. Independent confirmation of the fluorescence intensity decreased by the ethidium bond bound to the parallel T * AT triplets (2xdT? 0: dA10) relative to the doublets (dT? 0: dA? O) has appeared (Fang, Y .; Bai, CL; Zhang, P.C; Cao, EH; Tang, YQ Science In China (Ser. B), 1994, 37, 1306).

TACE parallel and antiparallel triplets are chosen for the investigation as those sequences that have been well documented in the literature (Plum, G. E., Pilch, DS, Singlton, SF, Breslauer, KJ Annu, Rev. Biophys, Biomol. Struct. 1995, 24, 319, and references thereof). The branched nucleic acids as described in Damha et al. (Hudson, RHE; Damha, MJ Nucleic Acids Res. Symp. Ser. 1993, 29, 97., R. Hudson, A. Uddin, and M. Damha J. Am. Chem. Soc., 1995, 117, 12470) were used in this study as the only architecture of bNAs that has been used to stabilize the TAT triplets of Hoogsteen inverse and Hoogsteen. Investigations were also conducted to determine the best orientation of oligonucleotide in the support for the detection of TAT triplets. The motivation behind the effort reached was then to create a rapid, releasable, reproducible assay for the detection of triple helical nucleic acid formation. The development of a triple helix test, is an extension of the work initiated for the detection of nucleic hybridization (Watson-Crick motif) using fluorescence sensors (TIRF) of total internal reflection of optical fiber functionalized with deoxyribonucleic acid probes of a single strand (ssDNA). ii) Synthesis of Oligonucleotides in Optical Fibers.

The optical fibers were activated by protocols given in Example 2 and the polyadenyl decanucleotides are bound in the substrate linker molecules on the fiber surface as per the methods given in Example 5. Two fiber baths were created, the first using N6. -phenoxyacetyl-5 '-O-DMT-2' -deoxy-adenosine-3 '-0- [(β-cyanoethyl) -N, N-diisopropyl] phosphoramidite commercially available from Millipore, Inc., to bind de-nucleotides with terms 5' away of the fiber surface. N6-phenoxyacetyl-3 '-O-DMT-2' -deoxy-adenosine-5 '-O- [(β-cyanoethyl) N, N-diisopro-pyl] phosphoramidite was prepared via standard protocols using the oligonucleotide developed in the functionalized fibers in an inverse orientation (substrate-fiber-linker > 5'-dA10-3 '). iii) Synthesis of Branched Oligonucleotides The branched sequence "V" 1 (Figure 15) was synthesized in an Applied Biosystems 381A instrument using a 1 pmoles scale synthesis cycle and β-cyanoethylphosphoramidite chemistry. Purification, desalination, and analysis of branched oligonucleotide 1 by polyacrylamide gel electrophoresis was achieved by these detailed protocols (Damha, MJ, Ganeshan, K., Hudson, RHE, Zabarylo, SV, (1992) Nucleic Acids Res 20, 6565 -6573; Damha, MJ; Ogilvie, KK in Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs; Agrawal, S., Ed .; Human Press, Inc .: Totowa, NJ, 1993, pp. 81-114 ). Typical yields of this branched oligomer were 5-15 A26o units (15-25%). The dA10 complement for thermal denaturation studies was obtained from Dalton Laboratories (Toronto, Canada). iv) Thermal Denaturation Profiles. The absorbance against the temperature profiles of the nucleic acid complexes was measured at 260 nm using a Varian Cary I UV-VIS spectrophotometer equipped with a variable temperature cell holder controlled by an external variable temperature circulation bath. The data was collected with the fixed spectrophotometer in double beam optical mode to reduce optical movement. The data were collected at 260 nm at 0.5 ° C intervals with an equilibrium time of 60s for each measurement point. The absorption coefficients of the branched molecules are assumed to be similar to their corresponding linear sequences and were calculated from those of the mononucleotides and dinucleotides according to the nearest-neighbor approach (Puglisi, JD; Tinoco, I., Jr. Methods in Enzymology; Dahlberg, JE Abelson, JN Eds .; Academic Press, Inc .: San Diego, 1989; Vol. 180, 304.). The thermal denaturation analysis samples were prepared by mixing the pyrimidine containing the strand with the target (2 mM), lyophilizing the solution to dryness, and dissolving the oligomers in 10 mM Tris, 50 mM MgCl 2, pH 7.3 adjusted with HCl . The mixtures were then transferred to Hellma QS-1,000-104 cells. The oligonucleotide solutions were heated at 80 ° C for 15 minutes and then cooled slowly to room temperature before the fusion experiments. The standardized graphs were constructed according to the method of Kibler-Herzog et al. (Kibler-Herzog, L., Zon, G., Whittierm, G., Shaikh, M., Wilson, W. D. Anti-Cancer Drug Des. 1993, 8, 65) based on. { (At - A0) / (Af - A0)} : where A0 is the initial absorbance, Af is the final absorbance and At is the absorbance at any temperature. All the complexes showed defined fusion transitions. The melting temperature (Tm) was determined from the first derivative of each thermal curve. A precision in Tm values, determined from the variance in repeated experiments, of ñ0.5 ° C or higher was obtained for all the denatured profiles investigated. v) Instrument Set and Fluorescent Measurements Laser radiation excites the immersion lens of the fluorescent microscope (as described in Example 9) was coupled to a fiber supplied from a similar numerical aperture 0.48) aligned below the objective, as illustrated Figure 4 (c). The light was reflected internally throughout the fiber delivered to a functionalized fiber sensitive to the immobilized oligonucleotide.

The coupling of the radiation between the fibers was achieved by confining the distal term of the supplied fiber to the close term of the sensitive fiber. A loss in optical transmission no greater than 2% was observed by the coupled system. The terminal of the Teflon fiber coupler was designed as the suitable compression ends which provide a narrow solution signal that prevents contaminants from diffusing into the fiber coupler and causing movement in the analytical signal. The sensitive fiber was placed in a small volume, stopped flow, stainless steel hybridization chamber (1.5 mm i.d. x 48 mm) which provides a volume of solution of 79 μl exposed to the sensitive fiber. The temperature of the hybridization cell was controlled by placing the cell in a thermosetting housing in which the glycol solutions of the circulating baths of the external variable temperature were made to flow. The temperature of the solutions in the hybridization cell were determined truthfully (± 0.2 ° C) by the use of an encapsulated glass thermistor incorporated in the hybridization cell and in contact with the solution at the exit of the hybridization chamber. The solutions containing hybridization buffer, ethidium bromide, and complementary nucleic acid sequences were supplied to the hybridization cell and sensitive fiber by the use of a peristaltic pump. The fluorescent emission of ethidium bromide that was interspersed in the immobilized nucleic acid complexes was reflected internally completely within the sensitive fiber. The portion of light coupled to the side in the supplied fiber was directed towards the objective microscope where it was collimated and was directed to the dichroic mirror. The fluorescent radiation was of large wavelength (? Max = 595 nm) than the cut, and was transmitted through the mirror and was directed towards a photomultiplier tube, where the fluorescence intensity could be measured quantitatively. The movement caused by the variations in optical coupling efficiency, laser intensity and photomultiplier gain were obviated by the normalization of all the signals either from a normal solution of ethidium bromide at 25 ° C prior to, and to the constitution of each analysis. vi) Mobility Retardation Test PAGE. The oligonucleotide solutions were lyophilized to dryness, incubated in 10 μl of 30% sucrose with 50 mM MgCl 2 at 75 ° C for 15 minutes, and then cooled to room temperature slowly. After 4 days of incubation at 4 ° C, the samples were loaded on the gel. The run buffer contained 90 mM of tris-borate buffer (pH 8.0). The non-denaturing of 15% polyacrylamide gels contained 90 mM tris-borate (pH 8.0), and 50 mM MgCl2. The native gels were run at 12.5mA for 12 hours. Following the electrophoresis, the gels were covered with Saran Wrap © and photographed with a Polaroid MP4 Land camera on a fluorescent TLC plate (Merck, distributed by EM Science, Gibbstown, NJ) illuminated by a UV lamp (Mineralight lamp, model UVG- 54, San Gabriel, CA). The instant film (# 52, medium contrast, ISO 400/21 ° C) was used and exposure (f4.5, 1.5s) made through a Kodak Wratten gelatin filter (# 58). The gels were subsequently stained for 5 minutes in a solution of 5 μg / ml ethidium bromide and destained in distilled water for 30 seconds. The gels were then covered with Saran Wrap®, illuminated by a UV lamp and photographed (f4.5, 2s) through an orange Hoya filter on a target background. vii) Considerations of Triplet TAT parallel and An tiparalelo In the formation of the intermolecular triplet 2 x dTio / d -io, the third strand dTio interacts by means of hydrogen bonds Hoogsteen with the dAio strand in doublet Watson-Crick goal, and is oriented in parallel. In the fused experiments (Mg + 2 buffer), the triplet 2 x dTio / dAio has two clearly resolved transitions, one for dissociation of the third strand from the doublet, that is, dT? o * dA10 / dT? or? -dT? 0 + dA? 0 / dT? 0 (Tm 18 ° C), and one for dissociation of the doublet in its component strands, that is, dAio / dTio - »dAi0 + dT? 0 (Tm 32 ° C). Thus for this complex, the association of the third strand (dTio) with the doublet (dAio / dTio) is thermodynamically weak to the formation of doublet by itself. Branched oligonucleotides are useful probes for the stabilized DNA triplet (R. Hudson, A. Uddin, and M. Damha J. Am. Chem. Soc., 1995, 117, 12470). The branched oligomer 1 (Figure 15) for example, binds to dAio to give a novel triple TAT complex in which both dTio strands are antiparallel to the strand (dA? 0). Although this motif has been observed by TAT bases in complexes dominated by pur »pur / per link (eg, G» GC, A »AT) ( { i.}. Moser, H. E., Dervan, P. B. Science, 1987, 238, 645 { ii.}.

Strobel, S. A .; Doucettestamm, L. A .; Riba, L .; Housman, D.

AND.; Dervan, P. B. Science, 1991, 254, 1639. { iii} Hoogsteen, K. Acta Crystallogr, 1959, 12, 822-823; (b) Felsenfeld, G .; Davies, D. R .; Rich, A. J. Am. Chem. Soc. , 1957, 79, 2023-2024; . { iv} R. Hudson, A. uddin, and M. Damha J. Am. Chem. Soc. , 1995, 117, 12470.), has not previously been observed for the dTn / dAn complexes. The formation of this triplet is induced by bonding two strands dT? Or through its 5 'ends via coupling to riboadenosine to the neighboring 2' and 3 'oxygen atoms (Figure 15). This arrangement causes the initial direction of the two threads dT? Or to be parallel, and forces the formation of a triplet in which the third strand dTio runs antiparallel to the strand dA? O, and is joined to the inverse Hoogsteen interactions. The thermal denaturation profiles of a mixture of 1 and dAio (1: 1) in Mg + 2 regulator, shows a single transition of the bond to the complex without bond, consistent with this formation involving quite a few 'more than two bimolecular reaction stages , that is, 1 + dAio - > triplet 1 / dA? o (Tm 35 ° C). viií) Triple Studies Using Optical Fibers Derivatized with Oligonucleotide Orientation (Fibra-3 '' -dA10-5 '). . The characterization of the triple helix complexes via thermal denaturation studies were performed. The day was developed in the conventional 3 'to 5' direction of the fiber surface. The solutions of ethidium bromide, ethidium bromide with dTio or ethidium bromide with 1, were heated in the hybridization chamber containing the functionalized optical fibers of decaadenylic acid. Until slow cooling, fluorescent measurements were taken at various temperatures. Figure 16 (a) illustrates that since the doublet of Dt? O: dA? O was formed by decreasing the temperature, there is an increase in the fluorescence intensity corresponding to the intercalation of ethidium bromide and improved quantum yield of the ligand in this complex. After the additional decrease in temperature, the indicative exclusion of the ligand as the result of the triple formation (2 x dT? 0: dA? O). This process is illustrated in Figure 7 (a). In order to verify that the triple formation is solely responsible for the exclusion of the ethidium cation and therefore the decrease in fluorescence intensity of the fiber, a control experiment was performed using functionalized optical fibers with twenty nucleotide probe sequence of mixed base composition unable to form triple structures. The hybridization experiment was carried out using the same methodology as the functionalized decaadenylic acid fibers with the exception of the hybridization regulator (1M NaCl, 50mM P0 pH 7.0). Having this nucleic acid sequence and regulatory composition, only double-stranded complexes could be formed between the immobilized probe sequence and the complementary sequence. As can be seen in Figure 6, a fluorescent intensity with a negative temperature coefficient was observed for the double system over the temperature range studied. The denaturation temperature for this nucleic acid system and hybridization regulator was determined to be 73 ° C by UV-visible thermal denaturation studies. Only double-stranded complexes exist over the investigated temperature range, as indicated by the increased fluorescence intensity for the experiment using ethidium bromide and the complementary oligonucleotide. The control experiment with ethidium bromide and non-complementary oligonucleotide shown did not have such dramatic increased intensity. Upon exposure of the optical sensor for the formation of inverse Hoogsteen 1, the non-significant increase and intensity of fluorescence over that of ethidium bromide alone in solution was observed. The geometrical constrictions of compound 1 are such thatIf a complex is formed with the dAio strand immobilized in this particular orientation (fiber-3 '- »5'), the branching point of the riboadenosine should be oriented towards the surface of the fiber presenting a spherical barrier to the triple formation. In order to test the spherical interference tied around the branch point by avoiding the formation of a triple helix, an optical fiber having a dAio strand is synthesized in the opposite orientation of the surface (ie fiber-5 '- >).;3' ) . ix) Triple Studies Using Derived Optical Fibers with Reverse Oligonucleotide Orientation (Fibra -5 '-dAip-3'). From Figure 16 (b), the fluorescent intensity against the temperature profile indicated with dT? 0 was an initial increase in the fluorescence indicator of the double formation. This was followed by a decrease in intensity which was indicative of the triple formation having occurred. Upon treatment of the optical sensor with 1, a decrease in fluorescence intensity for the inverse Hoogsteen complex at temperatures below the Tm (35 ° C) was observed in Figure 16 (c), which is consistent with the triple formation. From the data of Scaria and Shafer (Scaria, PV, Shafer, RHJ Biol. Chem., 1991, 266, 5417), it can be deduced that the lower temperature of 25 ° C is required for the process of exclusion of the ethidium cation for master the fluorescent intensity. Since the amount of ethidium cation that can be accommodated by triplets is lower than that of the doublets (where intercalation occurs once every 2.8 base triplets and once by 2.4 base pairs at 25 ° C) a reduction of 15% in the amount of ethidium intercalated to triple-strand formation results (Scaria, P.V., Shafer, R.H. J. Biol. Chem., 1991, 266, 5417). However, within the triple structure the fluorescent quantum production of the intercalated ethidium cation has been observed to increase to 19% of the Si-> transition. S0, resulting in a global change in fluorescence intensity of + 2.3%. Therefore, the direct correlation between the Tm for the triple formation and the beginning of the decreased fluorescence intensity of the optical sensor will be observed for the nucleic acid systems which have Tm a, or low 25 ° C values. This is consistent with these conclusions. Figures 16 (a and b) where the decrease in the fluorescence intensity of the sensor correlates well with the Tm at 17 ° C for the triple formation. As can be seen in Figure 16 (c), although the transition for triple strand formation between 1 and immobilized dAio occurs at 35 ° C, a decrease in fluorescence intensity was not observed until the system was cooled to below 25 ° C. In this respect, fluorescence studies involving the binding of ethidium bromide to triple helices are in agreement with several previous conclusions. However, this system is then limited in terms of being able to identify only the double or triple transition temperature for nucleic acid systems with Tm values below 25 ° C. x-) Mobility Retardation Test PAGE. The gel exchange experiments provide us with the opportunity to confirm the interaction of ethidium bromide with the complexes observed in these studies. The electrophoretic mobility of the Watson-Crick base pair of doublet dT? 0 / dA? 0, (the pair of Hoogsteen and inverse Hoogsteen of the TAT triplets, and those of its component strands), were studied at 4 ° C in a regulator that contained magnesium. Following the electrophoresis, the gels were visualized by UV overshadowing, and by staining with ethidium bromide, as shown in Figures 17 (a and b), respectively. The triplet of Hoogsteen emigrated more slowly than the doublet due to the presence of the third strand dT? 0. Triple inverse Hoogsteen has the slowest mobility of all, and is characteristic of branched nucleic acid structures [ref Hudson and Damha, JACS 1993; Wallace, J. C .; Edmons, M. PNAS vol. 80, 950-954, 1983). The association of 1 and dAio was quantitative as evidenced by the disappearance of compound 1 and dAi0 when mixed in equimolar quantities. The stoichiometry of interaction between d? 0 and dA0 for the doublet and triplet of Hoogsteen was also confirmed by studies at different concentrations of two oligonucleotides. When the gel shown in Figure 17 (a) was stained with ethidium bromide and illuminated by a UV lamp, fluorescence was observed only in the bands corresponding to the complexes (without strands). This is consistent with the well-known mechanism of ethidium bromide intercalation (Lim, C. S. BioTechniques, 1994, 17, 626). As previously suggested by the biosensor studies, the 1 / dA? Or triple of inverse Hoogsteen gave the lowest fluorescence intensity, which could be related to the limited ability of ethidium to bind to this complex. Example 15. Optical Sensors That Work by the Intrinsic Operation Mode. Background The angularly dependent light scanning experiments were performed to determine the refractive index of covalently immobilized oligonucleotide monolayers on fused silica substrates. With the knowledge of the refractive index of the immobilized oligonucleotide film, the mode of operation of the devices, ie intrinsic or total evanescent internal fluorescence reflection, can be clarified. The concept of the experiments carried out is based on the optical theory with respect to those alterations in the direction of a beam or collimated beam of light that crosses an interface between two dielectric materials that can be predicted based on the difference in the refractive index of two materials, and vice versa. In particular, Snell's law of refractive states that a ray of light travels in a plane normal to that defined by the interface between two materials of refractive index ni and n2, the angular trajectory of the transmitted ray, p plane normal to interfacial will be different from that of the incident ray,? p, by a quantity dependent on the difference in refractive index of two materials. This can be solved mathematically using the following equation (Ohanina, H. C; Physics, W. W. Norton and Company, New York (1985) p. 837): n ^ sßn (? ^ = N2 sin (?) (4) Figure 18 (a) illustrates this concept for the case where the upper environment is fused silica and the lower environment is the environment, as characterized by fused and distinct nSiiice. respectively, where nSiiice Merged > Ambiguity As shown in Figures 18 (a) and 18 (b), as the angle of incidence increases, the angle of the refractive beam will be deflected by increasing quantities towards the interface, where all the times? I < ? t. This trend is continued to the point where the refracted beam is directed in the interfacial plane (ie,? T = 90 °). The angle of incidence for which this occurs is known as the critical angle,? C, and can be calculated from the following relationship:. "Ambient £ = sin - (5) n S, ffic. Íusiorudí 'For the case where i? G? C, the incident ray experiences the total internal reflection (IRR) to the interface.The beam angle reflected with respect to the normal interface is then equal to that of the angle of incidence for all? i> c, as illustrated in Figure 18 (c) .If a detector for optical radiation is placed directly below the point of intersection of the ray of light with the interface and the intensity is recorded as a function of incident angle, a continuously decreased intensity with increased incidence angle will be observed.This observation is the result of the refraction beam that increases the deflection outside the optical axes of the detector. The illustration that represents the trends in detector response is included in the right hand side of each ray diagram in Figures 18-20. Since the closed refraction beam focuses the interface between the two dielectric materials, a local maximum in the response of the detector will be observed as a result of the beam, being swept by the roughness and other imperfections in the interface. This could continue until? I =? C, after which the point of the incident beam would be subjected to TIR and therefore provide negligible amounts of signal to the detector, with the exception that the outer hole via the sweep of the imperfections to the interphase and within the waveguide medium. As such, the critical angle for IRR can be determined directly from this point on the sweep intensity plot against the angle of incidence (Figure 18 (c)). For the case where the refractive index of one of the medium is known, the refractive index of the other can be solved using equation (5). A three-layer model can be considered for the case where a thin film of organic material is placed at the interface, as shown in Figures 19 and 20. Each type of medium is herein characterized by the refractive index of the material , as given by nsiiice Fusionada / nPeiiCuia. and environment. respectively for fused silica, organic film, and environment. The interaction of a ray of light to each interface must be considered independently. A ray incident on the silica medium fused at an angle ą relative to the normal interfacial will be refractive at a different angle after traversing each interface. The angle of propagation of the ray will then be the color and temperature in the organic film and the environment, respectively, relative to the interfacial normal. For the case where nsiliCe merged > nPeiicuia > nnmbiente / the reciprocal tendency will be observed with respect to the propagation direction of the refractive rays, where? i < ? tpeiicuia < ?you too. As shown in Figure 19. As? i is increased, a local maximum in the detector response will be observed as? tPeiicuia passing through the critical angle for TIR to the film-environment interface. This is illustrated in Figure 19 (b and c). A second local maximum will be revealed as? I passing through the critical angle for TIR to the fused silica film interface, as shown in Figure 19 (d). Giving nsiiice merged and nambiente. The refractive index of the organic film can be determined directly from the analysis of sweep intensity traces against? i. The critical angle for the TIR to the fused silica film interface can be directly obtained from the point where the second local maximum intersects the baseline sweep intensity, as previously described for the two-layer model and shown in the Figure 19 (d). When Substituting? Csilice Fused / Film And nsilice Merged into Equation 5, nPeiiCuia can be solved directly. The verification of this result can be acquired by substitution to the calculated value of nPeii < Then, Y ^ Ambient, and in equation 5 to determine the value of? cpeicuy / ñmbiente -Using the value of? i from the point where the first local maximum intersects the intensity of the baseline sweep, nSiiice Fused. nPeiiCU? a and equation 4, a second method to calculate? cPeiiCuia / Environment is provided. The benefits of agreeing between the two values of? Cpeiicuia / Environment could indicate the validity of the calculated value of nPeiiCu? A. For the case where nSiiiCe Merged < nPelicula > nAmient. an estimate of the value for nPeiiCuia can be attached provided to the values of nSiiice Fused / ramiente is known. A determination of nPencu? A can not be achieved in this case as TIR will not occur at the interface between the fused silica and the organic film for the incident light in the fused silica, without producing any mechanism for the determination of? TPeiicuia- The diagrams of The ray and response trend of the detector for this scenario are shown in Figure 20. A slight estimate for the value of nPeiicuia may have to assume that? cPeiiCuia / Environment is equal to the value of? i to the point of completion of the local maximum from the sweep intensity plot against the angle of incidence. This overestimation of? CPeiiCuia / Environment and Environment can be used in equation 5 to provide an estimate of the value of nPelicu? A. This value of nPencula along that for nSiiiCe merged and i i can then be substituted in equation 4 to provide an estimate of? Cpeiicuia / ymierte • This estimate of? CPeiiCuia / Environment and "Ambient can again be used in the equation 5 to provide an overestimation of npeiicuia- An average of these two values should provide a good estimate of the true value of nPeiicuia within the uncertain limits set by the high and low endpoints Materials and Methods The fused silica wafers Planas Suprasil ® (Heraues Amersil, Duluth, GA, USA) with dimensions of 10 x 5 x 1 mm, a refractive index of 1.46008 and a surface smoothness of 10 waves / inches, were functionalized with the bound molecules of the substrate by the methods of examples 2 and 3. Similarly, silicon wafers (Heraeus Amersil, Duluth, GA, USA) with dimensions of 10 x 5 x 1 mm were functionalized with molecules bound from the substrate by the m Etodes of Example 3. Polymycidyl acid icosanucleotides were then bound in the functionalized wafers by automated solid phase oligonucleotide synthesis, as per the methods provided in Example 5. All the water used in the light scavenging experiments were obtained from a 5-stage cartridge purification system Milli-Q (Millipore Corp., Mississauga, ON, Canada) and have a specific resistance not less than 18 MO-cm. The hybridization regulator was the same as that used for hybridization experiments on optical fibers and described in example 8. The refractive index of the hybridization regulator was determined by the use of a Bausch & Refractometer. Lomb Abbe-3L (Fisher Scientific, Nepean, ON, CA) within the reported veracity of 0.0001. Octadecyltrichlorosilane (OTS), ethylene glycol, hexadecane, carbon tetrachloride, chloroform and cyclohexane were of analytical or higher grade from Aldrich Chemical Co. (St. Louis, MO, USA) and used as received in the state at least from other way. OTS functionalization of fused silica wafers. The fused silica wafers were cleaned by treatment with solutions of NHOH / H20 / H202 and HC1 / H20 / H202 respectively, as per the method detailed in Example 2 (i). Before use, carbon tetrachloride and chloroform were dried by refluxing over P20s under an argon atmosphere followed by distillation under the same conditions. The functionalization of the substrates with the OTS monolayers was carried out later as by the methods of von Tschamer and McConnell (von Tschamer, V. and McConnell, H. M. Biophys. J., 36 (1981) 421) and as described in the following. The cleaned substrates were treated with a solution of 80% hexadecane, 12% carbon tetrachloride, 8% chloroform and 0.1% OTS (v / v) for 15 minutes at 25 ° C with stirring under an atmosphere of anhydrous argon. . The reaction mixture was then decanted and the fused silica wafers were then washed three times with distilled chloroform and stored in vacuo and on P2Os until required. Instrumentation used for the Light Scanning Experiments The wafers were placed in a custom-made stopping flow cell, under a Harrick EA 7X89 fused hemispherical prism with a radius of 8 mm (Harrick Scientific Corp., Ossington, NY , USA) as illustrated in Figure 21. The optical contact between the fused silica hemispherical prism and the fused silica wafer functionalized with an oligonucleotide monolayer was done by applying a thin film of free fluorescence refractive index Zeiss Im ersionsoel 518C oil that combines well (n = 1515, Carl Zeiss Canada Ltd., Don Mills, ON, CA) to the interface between the two. The other side of the wafer was exposed to a shared solution with the dimensions of 9 x 2 x 1 mm (1 x w x h). The flow cell was mounted at the apex of a modified goniometer element obtained from a Type of Thin Film Elipsometer type 43702-200E (Rudolph Research Corporation, Flanders, NJ, USA) with an angular accuracy and a precision of 0.005 ° , 543 nm of optical radiation from a Gre-Ne ™ Laser (Melles Griot, Carisbad, CA, USA lmW performance power, 1.5 mm beam diameter, O.Ommrad divergence beam) mounted on a gionometer arm is passed through the hemispherical prism and impacts on the flat fused silica wafer. The hemispheric prism ensures that the alterations in the angle of incidence that is due to refraction to the air-prism interface where it is eliminated as the beam invariably introduced to the prism normal to the prism / air interface. A M062-FC03 Slo-Syn detection engine (Superior Electric Co., Bristol, CT, USA, 200 steps per revolution) was coupled via a set of gears to the fixed screw of the goniometer used when driving the pivot mechanism of the arms of goniometer. The gear ratio used provided the motor with a 7 x mechanical advantage so that it reduces the load on the motor and prevents oversight. The TTL signals from a parallel interface of normal PC were used to operate the advancing mechanism of the augmentation motor either for the accuracy of the control of the angle of the goniometer arms and the incidence of the laser beam. One end of a fiber optic bundle - (Oriel Corp., Stratford, CT, USA model No. 77533) was mounted at the base of the flow cell about 1 mm from the exposed face of the fused silica wafer. The other term of the fiber bundle was directly at 630 n of colloidally colored long pitch of the glass filter (Schott Glass Technologies, Duryea, PA, USA) placed before the window of a photomultiplier tube R-928 (Hamamatsu Corp., Bridgewater, NJ, USA) operated using a DZ-112 Photoelectric Indicator (Rudolph Research, Flanders, NJ, USA). The long-pass filter provided by attenuation of a light intensity transmitted by the fiber bundle by a factor of 105. This was done in order to avoid an overload condition in the PMT that occurs, guaranteeing the linearity of response and preserving the lifetime of the detector. The actuality of the PTM was converted to an analog performance voltage (0-5 VDC) of the electronic processing signal contained within the Photoelectric Indicator and passed to a 12 bit analog to become digital (Metra-Byte, Taunton, MA, USA) for acquisition data on a PC computer using software created at home to acquire intensity graphs against angle of incidence. Results and Discussion . In order to test the validity of the scanning approach for the determination of the refractive index, the known refractive index samples were introduced into the flow cell below the hemispherical prism and analyzed by ramp of the angle of incidence of the laser mounted on the goniometer arm of the low and high incidence angles while the sweep intensity observed is recorded. The results of experiments performed using samples of air (n = 1,0003), water (n = 1.33) and cyclohexane '(n = 1.4266) are shown in Figure 22 (ac), respectively and summarized in Table 2. Knowing the Refractive index value of the prism material (fused silica, n = 1.46) and the analyte, equation 5 was used to determine the critical angle values for IRR in each system. The good correlation with the predicted values was observed in all three cases with no more than 1% error between the experimental and theoretical values for? C. An unmodified fused silica wafer was then coupled to the base of the prism and the same three control samples were again analyzed. The identical results within the resolution of the experimental technique were observed between the theory and the observed values of the critical angle for the analyzes carried out with and without the fused silica wafer. This indicates that there are no additional modifications to the instrument or the correction factors may need to be applied as a result of moving the intersection of the laser beam 1 mm below the base of the prism. The hybridization regulator (n = 1.35) was also analyzed by the light sweep method (Figure 22 (g)) and provides good agreement with the calculated value for? C based on the refractive index of the regulator as determined using a refractometer Abbe normal. Table 2. Summary of the Results of the Angularly Dependent Light Sweeping Experiments and Correlation with the Known Controls of the Refractive Index.

Experiments where they were then performed using films of organic medium of known refractive index in order to prove the validity of the technique as applied to the three-layer model previously described. Thin films (10 - 50 μm) of refractive index of oil combining well and ethylene glycol were applied to the exposed surface of the hemispherical prism and the analysis was then performed when air was used as the ambient in both cases. The results of the light scanning experiments for these samples are shown in Figures 23a and 23b, respectively. As you can see in figure 23a, the experimental value determined by? Cpeicuia. environment = 41.7 ° for the oil-air interface, based on the value of? i of 43.5 °, and that predicted from the theory, will agree after attracting the refraction on account of the beam through the fused silica - interface of oil. However, for this particular example, the two-step approach method for determining nPeiiCuia can not be used in this example. The first assumption that? I =? T used in these treatment charges to a first estimate of npeiiCuia which is lower than that of nSice merged- This could load to the result that the beam transmitted in the oil may be the case for nPeiicuia > nSiiiCe Merged - This causes the next approximation of nPeiiCuia to be an exaggerated estimate of the true value. As such, the films of the refractive index, index slightly higher than that of the fused silica substrate can not be resolved. The results for the light scan experiment using an ethylene glycol film provide very good state between the values of? C at each interface with respect to that calculated from the theory. Of significance in this graph the intensity of sweep against the angle of incidence is the appearance of two different maxims. The observation of two maxims coincides with that proposed for the three-layer model (Figure 19) for the case where nS iice Fused. >; "film "" Ambie te - As such, the information regarding the refractive index doubt of the organic film is greater than, or less than that of the substrate material that can be obtained by quick inspection. An OTS monolayer film was covalently bonded to the surface of a fused silica wafer by a previously known method to provide dense surface packing and a theoretical refractive index in the range of 1.4-1.6. ( { a.}. Ducharme, D. et al., J. Phys. Chem. 94 (1990) 1925 { b.}. von Tschamer, V. and McConnell, H. M: Biophys, J ., 36 (1981) 421). The results of the light scan experiments are shown in Fig23 (c) and 23 (d), respectively, for functionalized OTS fused silica wafers exposed to air and water as the environment. Using the following rearrangement of equation 5: n _ w 'A_sense? (6) The value of the refractive index for the OTS monolayer could be solved for Values of 1.44 and 1.45 for the refractive index of the monolayer were determined from the analysis using air and water as the environment, respectively. Since the refractive index values determined for the OTS monolayer differ by approximately ~ 1% with that of the fused silica substrate, the same limitation as observed by the oil refractive index that combines well with the layers applied in it . As such, it can only be assumed that the refractive index of the OTS overcoat is only slightly greater than that of the fused silica. This is reinforced by the fact that only a local maximum in the graph of the sweep intensity versus the angle of incidence was observed. If the refractive index of the film was actually lower than that of the fused silica substrate then the two local maxims must have been observed, according to the concept of the three-layer model and as clearly demonstrated by the experiment using ethylene glycol for the film ( Figure 23 (b)).

Samples of the functionalized silica wafer fused to molecules bound by the methods of Example 2 and 3 wherein the polymycidyl acid icosanucleotides are assembled by the method of Example 5 were analyzed by the angularly dependent light sweep technique. The results of the analysis are shown in Figure 24 (a) 24 (b) by the samples prepared by the mesylate activation scheme as detailed in example 3. The results of the samples prepared by the GOPS-HEG protocol given in Example 2 are shown in Figure 24 (c). For samples prepared by the activation of mesylate for which water and ethylene glycol 3: 1 in water solution is used as the environment, a value of 1.57 was determined in both cases for the estimated water, based on the assumption that? tPeiicuia =? i. Subsequent to the recalculation of thepeople based on the estimated value of nPeiiCuia. overestimated for nPeiiCuia of 1.67 and 1.68, respectively, were determined from the cases where water and ethylene glycol 3: 1 in water were used as the environment. This provides an average value for the 1.62 ± 0.05 level. Similarly, the functionalized water analysis of silica fused with polymymidotide acid icosanucleotide in GOPS-HEG substrate linkers (example 2) yielded an average nPeiiCUα value of 1.48 ± 0.01. The fact that the estimates of npeiiCuia for both types of nucleic acid - substrate linker overcoating can be solved by strongly reinforcing the fact that these overlays on the fused silica substrates actually possess a large refractive index value that that of the substrates in which they are immobilized. In addition to the light scanning experiments, the ellipsometry was performed in order to provide a second confirmation of the experimentally determined values of the refractive index of the oligonucleotide monolayers. Ellipsometry was performed on silicon functionalized wafer samples with bound molecules of substrate by the methods of Example 3 in which polymycidyl acid icosanucleotide molecules are pooled by automatic solid-phase oligonucleotide synthesis as detailed in Example 5 The silicon wafers were necessarily used for the light scanning experiments that provide a 'small reflection of the laser beam incident on the 70 ° angle in the environment. The surface of the silicon wafers were made similar to those of fused silica via the cleaning procedure used before the substrate functionalization. This known cleaning procedure for providing a layer of oxidized silicon to the surface of the silicon wafers (Kern, W. and Puotinen, D. A.; RCA Review, 31 (1970) 187-206). As such, the silanol portions present in the oxidized silicon-environment interface provide binding sites for the substrate binding molecules. McCracken (F. L. McCracken, NBS Technical Note 479, Washington DC (1969)) has developed software capable of providing values of thickness and refractive index of the ellipsometric measurements of these films using the exact equations of Drude for ellipsometry. The software film 85 provided with the reflection ellipsometer AutoELII nuil (Rudolph Research Corp., Flanders, NJ, USA) is based on that originally developed by McCracken and used for the analysis of ellipsometric data of these experiments described herein. Ellipsometric analysis of the clean substrate reveals the formation of a thin layer of 20 A of oxidized silicon on the surface of the wafers. Three functionalized silicon wafers with substrate linker and oligonucleotide were then analyzed. Ten different locations on the wafer surfaces were chosen randomly and the results of the ellipsometric analysis are summarized below in Table 3. Table 3 - Results of Ellipometric Analysis of Functionalized Substrates of Oxidized Silicon with Linked Substrate Molecules by Methods of Example 3 Polymicidyl Acid Icosanucleotide by the Methods of Example 5.

As can be seen by the inspection of the data shown in Table 3, the determination of the thickness and refractive index coincidentally via the iterative process provides a large degree variation. This is largely based on the fact that the covalently immobilized nucleic acid membrane system is not ideal for ellipso- metric analysis because it violates many of the assumptions of the Drude equations. Of particular significance is the fact that an oligonucleotide film packed densely with nucleic acid strands oriented perpendicular to the boundary of the air film will be anisotropically uniaxial. This could cause alterations in the velocity of the p and s-polarized components of the light beams up to the passage through the oligonucleotide film. This fact has been known to produce relative errors in thickness of up to 10% (R. M. A. Azzam and N. M. Bashara, Ellipsometry and Polarized Light, North Holland Publishing Company, New York (1977)). A higher estimate of the refractive index of nucleic acid films can be achieved by applying the Maxwell-Garnet theory (RMA Azzam and NM Bashara, Ellipsometry and Polarized Light, North Holland Publishing Company, New York (1977), p.359) . The concept of the Maxwell-Garnet theory, as applied herein, is based on the notion that the monolayer film formed partially of the cover T, is optically equivalent to the formed monolayer film filled with a refractive index ( npeii_uia) and relative thickness (Tf) such that the observed film thickness (T) is related to that of the formed film filled by: T = TTf (7) Likewise, the same adjustment factor, T can be applied to the refractive index value given from the ellipometric analysis of those values more representative of that of the current immobilized layers, can be obtained. The results after applying this correction are given in Table 3 and provide an average value of 1.6 ± 0.1 for nPeiiCuia- The good correlation between the result of the light scanning experiments and the ellipsometer provides unequivocal evidence that oligonucleotide monolayers they can be joined in the functionalized substrate linker of the fused silica substrates of the high refractive index to that of the substrate material. The oligonucleotides bound in the fused silica wafers functionalized with the bound molecules of the substrate via the mesylate activation scheme, as well as in contour in Example 3 were observed to provide the immobilized nucleic acid monolayers with a refractive index value of 1.62. ± 0.05 obtained by ellipometric investigations. This correlates well with the refractive index value of 1.6 ± 0.01 obtained by the ellipsometric investigations. The scanning investigation of the oligonucleotides bound to the fused silica wafers functionalized with substrate linker molecules via the methods of Example 2, revealed a refractive index of nucleic acid film of 1.48 ± 0.01, which is also higher than that of the Substrates of fused silica where there is a covalently binding. As such, the optical sensors created by the methods reported herein will then function by, and provide the signal 'through the advantages associated with the intrinsic TIRF motif described previously. The present invention has been described in terms of particular modalities found or proposed by the present inventor for the preferred modes comprised for the practice of the invention. It will be appreciated by those skilled in the art that, in light of the present disclosure, numerous modifications and changes may be made in the particular embodiments exemplified without departing from the intended scope of the invention. Such modifications are intended to be included within the scope of the appended claims. All publications, patents and patent applications are hereby incorporated by reference in their entirety to the same extent, as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference in their entirety. This previously claimed application of the North American application no. 60 / 050,970, and the Canadian application no. 2,208,165, both of which are incorporated by reference.

Claims (11)

1. CLAIMS 1. A biosensor system to detect target nucleic acid,. which is characterized in that it comprises at least three layers, two of which are a waveguide, wherein a layer in the waveguide includes a nucleic acid or nucleic acid analog capable of hybridizing to the target nucleic acid, so that a fluorophore can associate and interact with the nucleic acid or nucleic acid analogue and the target nucleic acid, and in which the biosensor functions according to direct excitation.
2. 2. The biosensor according to claim 1, characterized in that it comprises an inner layer, an intermediate layer and an outer layer, wherein the waveguide includes the inner layer and the intermediate layer and • the inner layer has a refractive index n . • the intermediate layer includes a nucleic acid or nucleic acid analogue capable of hybridizing to the target nucleic acid and has a refractive index of n2 which is greater than, or equal to, the refractive index nor, following hybridization to the target nucleic acid and • the The outer layer has a refractive index n3, which is smaller than the refractive index n2. and wherein a fluorophore is attached to the nucleic acid or nucleic acid analogue of the intermediate layer and wherein the biosensor functions according to direct excitation.
3. 3. The biosensor according to claim 1 or claim 2, characterized in that the fluorophore is bound to the nucleic acid or nucleic acid analogue. 4. The biosensor according to claim 2, characterized in that the inner layer is an optical fiber or optical wafer and the outer layer is an environment. 5. The biosensor according to claim 3, characterized in that the outer layer is a water-based solution. 6. The use of the biosensor according to claim 1 or claim 2, for the detection of triple formation or nucleic acid formation of multiple strands. The use of the biosensor according to claim 5, characterized in that the triple formation or nucleic acid formation of multiple strands includes a linear nucleic acid or a branched nucleic acid. 8. The biosensor according to claim 5, characterized in that the triple formation implies a branched antisense nucleic acid which inhibits the expression of a target nucleic acid sequence by triple formation with the sequence. The use of the biosensor according to claim 1 or claim 2, for the detection of nucleic acid from bacteria, viruses, fungi, unicellular or multicellular organisms or for the projection of nucleic acids from cells, cell homogenates, tissues or organisms . 10. A method for detecting an objective nucleic acid, characterized in that it comprises: • pre-treating a sample so that objective nucleic acids characterized from, or selected from the sample are available from hybridization; • contacting the sample with the intermediate layer of the biosensor of claim 2, so that the target nucleic acids can hybridize to the nucleic acids or nucleic acid analogs of the intermediate layer; • allowing the biosensor fluorophore of claim 2 to interact with the target nucleic acid hybridized to the nucleic acid or nucleic acid analogue of the intermediate layer so that the fluorophore is capable of being stimulated by electromagnetic radiation and emitting fluorescence; • illuminate the fluororph with electromagnetic radiation so that the fluorescence is stimulated by direct excitation; and • detect the emitted fluorescence, so that the presence of the target nucleic acid is detected. The method according to claim 10, characterized in that the target nucleic acid comprises nucleic acid from an organism selected from the group consisting of bacteria, viruses, fungi, unicellular and multicellular organism.
4. 4 SUMMARY A biosensor is described for direct analysis of nucleic acid hybridization through the use of an optical fiber functionalized with nucleic acid molecules and fluorescence transduction. Nucleic acid probes are immobilized on the surface of optical fibers and undergo hybridization with complementary nucleic acids introduced into the local environment of the detector. Hybridization events are detected by the use of compounds 10 fluorescents, which are linked in the nucleic acid hybrids. The invention finds use in the detection and screening of genetic disorders, viruses and pathogenic microorganisms. Applications in biotechnology include monitoring of gene cultures and expression of genes and the 15 efficacy (eg, dose-response) of pharmacists for gene therapy. The invention includes biosensing systems in which fluorescent molecules are connected to the immobilized nucleic acid molecules. The preferred method for nucleic acid immobilization is by 20 synthesis of solid phase nucleic acid in situ. The control of the refractive index of the nucleic acid immobilized by the support derivative chemistry and the nucleic acid synthesis is achieved. The preferred fiber optic derivation produces a DNA coating of higher refractive index than the fiber core on the surface of the fiber.