TY - JOUR
AU - El Shami,, Saïd
AB - The completion of the Human Genome Project (1) is likely to accelerate the demand for nucleic acid diagnostics, one of the fastest growing segments of in vitro diagnostics. To meet this demand, fully automated, sensitive, and specific technologies for nucleic acid detection are needed. Surface plasmon resonance fluorescence (SPRF) is an optical detection technology that offers kinetic analysis of biomolecular interactions in real time (2). SPRF technology uses a sensor composed of a dielectric material coated with a thin layer of a noble metal. An evanescent field is generated at the metal-dielectric interface through interaction of p-polarized light with surface plasmons (oscillating electrons) under a resonance condition. The evanescent field excites a fluorophore localized near the metal surface, which in turn emits evanescent waves, generating surface plasmons at the fluorescence emission frequency. These propagating waves (plasmon-mediated emission) radiate through the metal film and dielectric material. This emission emerges as a cone of radiation in the dielectric material. Application of SPRF for nucleic acid testing involves immobilization of synthetic oligonucleotide capture probes on the sensor surface (Fig. 1 ). Biotin-tagged nucleic acid amplicons are hybridized to target-specific capture probes and detected with a streptavidin-fluorophore conjugate by monitoring of the plasmon-mediated emission over time. Because the evanescent field penetrates a short distance into the medium above the metal surface, the hybridized amplicons are selectively excited and detected. This feature of SPRF allows sensitive detection without a wash step to separate the free from the hybridized nucleic acids. The fluorescent emission is collected by a patented optical system, providing higher sensitivity than other similar techniques (2). The rate of increase in fluorescence emission is reported as the SPRF signal in mV/min. The hybridization and fluorescence detection steps can be completed in less than 2 min, making this technology one of the most rapid nucleic acid detection methods available. Figure 1. Open in new tabDownload slide SPRF format for detection of nucleic acids. Thiolated oligonucleotide probes, immobilized on the gold surface of SPRF sensor, capture biotin-tagged nucleic acid amplicons. Binding of streptavidin–fluorophore conjugate to hybridized amplicons localizes the fluorophore near the gold surface. Because of the short penetration depth of the evanescent field into the medium above the gold surface, the fluorophores bound to the hybridized amplicons are selectively excited. The excited fluorophores generate surface plasmons, which radiate into the substrate at defined angles. The plasmon-mediated emission emerges from the substrate in the shape of a hollow cone. Figure 1. Open in new tabDownload slide SPRF format for detection of nucleic acids. Thiolated oligonucleotide probes, immobilized on the gold surface of SPRF sensor, capture biotin-tagged nucleic acid amplicons. Binding of streptavidin–fluorophore conjugate to hybridized amplicons localizes the fluorophore near the gold surface. Because of the short penetration depth of the evanescent field into the medium above the gold surface, the fluorophores bound to the hybridized amplicons are selectively excited. The excited fluorophores generate surface plasmons, which radiate into the substrate at defined angles. The plasmon-mediated emission emerges from the substrate in the shape of a hollow cone. To evaluate the potential of SPRF for detecting nucleic acids, model systems using Chlamydia trachomatis, Neisseria gonorrhoeae, and coagulant factor V DNA sequences were developed. Thiol-modified oligonucleotide capture probes were synthesized on an Applied Biosystems EXPEDITE™ Nucleic Acid Synthesis System and immobilized on the gold surface of SPRF sensors. Targets were purified from specimens by binding to silica in the presence of a chaotropic salt (3) with use of the Qiagen QIAamp® DNA Blood Mini Kit or EMD Biosciences MagPrep® silica particles. The sequences were amplified under standard conditions with the Stratagene Brilliant® QPCR Plus Kit. The sensitivity of the SPRF technology for measuring nucleic acids was evaluated with plasmid DNA molecules. Various amounts (106, 10, 5, 1, and 0 copies) of cloned C. trachomatis plasmid DNA were amplified and detected with C. trachomatis-specific capture probes. All reactions containing plasmid DNA produced strong positive SPRF signals, ranging from 800 mV/min for the amplified products of a single copy to 4520 mV/min when starting with 106 copies. In marked contrast, the sample lacking plasmid DNA produced no significantly measurable SPRF signal. These results show that SPRF is a very sensitive technology for nucleic acid detection when combined with DNA amplification. In fact, the combination can detect a single copy of the target sequence. The specificity of the technology was evaluated by use of cloned DNA from C. trachomatis and N. gonorrhoeae. The products were generated by a duplex amplification reaction containing primers specific for each analyte and were detected with SPRF sensors individually coated with capture probes specific for either target. The assay for C. trachomatis was positive (SPRF signal = 1870 mV/min) only when C. trachomatis plasmid was present. Background signal obtained by measurement of these amplicons on the N. gonorrhoeae-specific sensors was very low (30 mV/min) or negative. In addition, a positive signal (2520 mV/min) was obtained for N. gonorrhoeae only when the N. gonorrhoeae target was present. Again, background signal obtained with the C. trachomatis sensor was close to 0 (10 mV/min). When both plasmids were present in a mixed-target sample, positive SPRF signals were obtained with both the C. trachomatis (2100 mV/min) and N. gonorrhoeae (2260 mV/min) sensors. These results demonstrate that the SPRF system is very specific and can readily differentiate between the C. trachomatis and N. gonorrhoeae nucleic acid sequences. The SPRF system was further evaluated for its ability to measure amplicons generated from target sequences in clinical specimens. Cervical swab and urine samples were obtained from SLR Research Laboratory. All had been certified as positive for either C. trachomatis or N. gonorrhoeae infection by SLR by use of a molecular diagnostic test (e.g., GenProbe PACE®, Abbott LCx®, or Roche Amplicor®). After isolation, the nucleic acid targets were amplified with the previously described duplex amplification reaction before SPRF detection. A total of 271 clinical specimens were evaluated, and the SPRF system produced complete agreement with the results reported by the vendor. These results suggest that the SPRF technology could be used for in vitro diagnostic applications. The specificity of the system was further evaluated for its ability to detect nucleic acid sequences that differ only at a single nucleotide position, such as the factor V Leiden mutation. Genomic DNA was extracted from patient blood samples and used to generate both wild-type and mutant amplicons with a pair of primers common for both sequences. The amplicons were then measured with SPRF sensors individually coated with capture probes specific for either sequence. A total of 20 blood samples obtained from ARUP Laboratories were used for the evaluation. Genotypes of the samples were certified by ARUP or determined by direct nucleotide sequencing on an Applied Biosystems 373 XL DNA Sequencer. Initially, three patient samples were tested, representing the wild-type, heterozygous, and Leiden homozygous genotypes, respectively. The results showed that the wild-type sample produced a much stronger SPRF signal with the wild-type sensor (2930 mV/min) than with the Leiden mutation sensor (180 mV/min). In contrast, the homozygous Leiden sample produced a much stronger signal with the Leiden mutation sensor (3420 mV/min) than with the wild-type sensor (80 mV/min), whereas the heterozygous sample produced SPRF signals that were approximately the same for both types of sensors (1480 mV/min for wild-type and 1020 mV/min for Leiden). Testing of the remaining samples confirmed the trend. The wild-type samples produced a signal with the wild-type sensor that was at least 8 times higher than the signal observed with the Leiden mutation sensor, whereas the homozygous Leiden samples produced a signal with the Leiden mutation sensor that was at least 16 times higher than the signal observed with the wild-type sensor. Again, the heterozygous samples produced approximately equal SPRF signals for both sensor types. Such results indicate that the SPRF technology is specific enough to readily distinguish between sequences that differ by only a single nucleotide and that the technology thus has potential for use in the detection of genetic diseases and single-nucleotide polymorphisms. In summary, SPRF is suitable for rapid, sensitive, and specific detection of amplified nucleic acids. The system sensitivity was demonstrated by its ability to detect a single copy of plasmid DNA. The system specificity was demonstrated by its ability to differentiate between various infectious disease agents and to detect the single nucleotide mutation in the factor V Leiden sequence. The SPRF system does not require a wash step, and the assay can be completed in less than 2 min. This technology can easily be integrated into a fully automated nucleic acid testing system for high-throughput clinical screening. We would like to acknowledge Maral Poladian, Alicia Drummond, and Natalie Lopez for excellent technical assistance. References 1 Collins FS, Green ED, Guttmacher AE, Guyer MS. A vision for the future of genomics research. Nature 2003 ; 422 : 835 -847. Crossref Search ADS PubMed 2 Lin J-N, Wilson CJ, inventors. Method and apparatus for immunoassay using fluorescent induced surface plasma emission. US Patent No. 5,776,785; 1998.. 3 Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen PME, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990 ; 28 : 495 -503. Crossref Search ADS PubMed © 2004 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Nucleic Acid Testing Using Surface Plasmon Resonance Fluorescence Detection
JF - Clinical Chemistry
DO - 10.1373/clinchem.2004.036830
DA - 2004-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/nucleic-acid-testing-using-surface-plasmon-resonance-fluorescence-HZlyjpb1j8
SP - 1942
VL - 50
IS - 10
DP - DeepDyve
ER -