Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-02-16
2003-01-07
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C422S068100, C422S082050, C422S082060, C422S082080, C422S082090, C250S458100, C065S409000, C536S023100
Reexamination Certificate
active
06503711
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed generally to biosensors that are useful in the identification and analysis of biologically significant 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 diagnostics.
BACKGROUND OF THE INVENTION
The detection and identification of microorganisms is a problem common to many areas of human and veterinary health. For example, the detection of pathogenic species such as
Salmonella typhimurium, Listeria monocytogenes
, and
Escherichia coli
, which are causative agents of major food borne epidemics, is a great concern within the food industry with respect to the quality and safety of the food supply. In other areas of human and veterinary health care, detection and identification of infectious diseases caused by pathogenic microorganisms and viruses is a first step in diagnosis and treatment. For example, it is estimated that 10-15 million office visits per year are for the detection and treatment of three major pathogens—Chlamydia ssp.,
Trichomonas vaginalis
and
Gradenerella vaginitis
. Infections of these organisms annually effect 3.75 million, 0.75 million and 1.5 million patients, respectively.
Classical techniques routinely used for the detection and identification of microorganisms are often labor intensive, involving plating procedures which require lengthy analysis times. To illustrate, the method currently employed for the detection of
Listeria monocytogenes
in food and feed commodities involves a three stage analysis. The analysis begins with enrichment of the sample to be analyzed in a nutrient broth for 2 to 4 days. After the enrichment period, plating of the sample onto selective agar media is done and the sample is allowed to incubate for 2 days in order to obtain colonies for biotyping and serotyping, which may take as long as 20 days to complete (McLauchlin et al., 1988, Microbiology Review, 55: 578).
Detection processes based on culturing require analysis times which are too lengthy for effective monitoring and timely intervention to prevent the spread of biohazardous materials or treat disease. In addition, although these methods have been improved over the last decade, the chance of obtaining false negative results is still considerable, and many microorganisms are difficult to culture. Thus, plating/culture methods are limited with respect to their sensitivity, specificity, and lengthy analysis times that are required.
In order to shorten the time required to detect and identify pathogenic bacteria, viruses and genetic diseases, rapid tests such as enzyme immunoassays (EIA) have been developed (Olapedo et al., 1992). Although immunoassay techniques can be very sensitive and effective, there are practical drawbacks which have restricted the use of these methods. Such drawbacks include the need for highly skilled personnel, lengthy analysis and preparation times, and the large quantities of costly reagents that are required to do such analysis.
With the advent of nucleic acid amplification techniques (the polymerase chain reaction), the in-vitro amplification of specific sequences from a portion of DNA or RNA is now possible. Detection of very low numbers of microorganisms has been demonstrated (Rossen et al., 1991; Golsteyn et al., 1991; Wernars, K., et al., 1991). The polymerase chain reaction technique is sensitive and specific but involves complex manipulations in carrying out the tests and is not particularly well-suited for large numbers of samples. Due to the sensitivity of Polymerase Chain Reaction (PCR) technology, special rooms or areas for sample preparation and analysis are required to prevent contamination. In many tests PCR results must be confirmed by additional hybridization analysis. RNAs are difficult to assay by PCR but are very important for human viral detection. In general, PCR needs to be automated for acceptance as a practical diagnostic tool. Hybridization methods require as much as three or four days to complete results. Although the actual hybridization step can be as short as 18 hours, the entire detection process of a DNA/DNA hybrid can take as long as three days with a radioisotope marker.
Thus, there is a great need for simpler, faster and more cost-effective means for detecting specific biologically important RNA and DNA sequences in the fields of human and veterinary in-vitro diagnostics, food microbiology, and forensic applications.
Biosensors developed to date begin to overcome drawbacks associated with the current state of the art in detecting and identifying microorganisms. A biosensor is a device which consists of a biologically active material connected to a transducer that converts a selective biochemical 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 the ease of use (by non-expert personnel), low cost, ease of fabrication, small size, ruggedness, facile 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, other biologically significant materials, biochemical reactions and immunological reactions by using detection strategies that involve immobilization of enzymes, antibodies or other selective proteins onto solid substrates such as quartz and fused silica (for piezoelectric and optical sensors) or metal (for electrochemical sensors) (Andrade et al., 1990, Biosensor Technology: Fundamentals and Applications, R. P. Buck, W. E. Hatfield, M. Umana, E. F. Bowden, Eds., Marcel Dekker Inc., NY, pp. 219; Wise, 1990, Bioinstrumentation: Research, Developments and Applications, Butterworth Publishers, Stoneham, Mass.). However, such sensors are not widely available from commercial sources due to problems associated with the long-term stability of the selective recognition elements when immobilized onto 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 the stability of DNA. With the recent advent of DNA probe technology, a number of selective oligomers which interact with the DNA of important biological species, for instance salmonella, have been identified (Symons, 1989, Nucleic Acid Probes, CRC Press, Boca Raton, Fla.; Bock et al., 1992, Nature, 355: 564; Tay et al., 1992, Oral Microbiology and Immunology, : 344; Sherman et al., 1993, Bioorganic & Medicinal Chemistry Letters, 3: 469). These have been used to provide a new type of biorecognition element which is highly selective, stable, and can be easily synthesized in the laboratory (Letsinger et al., 1976, Journal of the American Chemical Society, 98: 3655; Beaucage et al., 1981, Tetrahedron Letters, 22: 1859; Alvarado-Urbina et al., 1981, Science, 214: 270).
Until recently, the only other research group in existence which has published work done on the fluorimetric detection of nucleic acid hybridization immobilized onto optical substrates is that of Squirrell et al. (C. R. Graham, D. Leslie, and D. J. Squirrell, Biosensors and Bioelectronics 7 (1992) 487-493.) In this work, single-stranded nucleic acid sequences ranging in length from 16-mer oligonucleotides to 204-base oligomers functionalized with an aminohexyl linker at the 5′ terminus were covalently attached to optical fiber sections functionalized with 3-aminopropyl triethoxysilane via a gluteraldehyde linkage. All investigations of nucleic acid hybridization were done by monitoring fluorescence intensity in an intrinsic mode configuration using complementary strands which had been previously labeled with a fluorescein moiety. This yi
Damha Masad
Hudson Robert H. E.
Krull Ulrich J.
Piunno Paul A.
Uddin Andre H.
Fredman Jeffrey
Greenlee Winner and Sullivan P.C.
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