Low-Level Detection of Viral Pathogens by a Surface-Enhanced Raman Scattering Based Immunoassay

Abstract

The need for rapid, highly sensitive, and versatile diagnostic tests for viral pathogens spans from human and veterinary medicine to bioterrorism prevention. As an approach to meet these demands, a diagnostic test employing monoclonal antibodies (mAbs) for the selective extraction of viral pathogens from a sample in a chip-scale, sandwich immunoassay format has been developed using surface-enhanced Raman scattering (SERS) as a readout method. The strengths of SERS-based detection include its inherent high sensitivity and facility for multiplexing. The capability of this approach is demonstrated by the capture of feline calicivirus (FCV) from cell culture media that is exposed to a gold substrate modified with a covalently immobilized layer of anti-FCV mAbs. The surface-bound FCVs are subsequently coupled with an extrinsic Raman label (ERL) for identification and quantification. The ERLs consist of 60-nm gold nanoparticles coated first with a layer of Raman reporter molecules and then a layer of mAbs. The Raman reporter molecule is strategically designed to chemisorb as a thiolate adlayer on the gold nanoparticle, to provide a strong and unique spectral signature, and to covalently link a layer of mAbs to the gold nanoparticle. The last feature provides a means to selectively tag substrate-bound FCV. This paper describes the development of the assay, which uses cell culture media as a sample matrix and has a linear dynamic range of 1 x 10(6)-2.5 x 10(8) viruses/mL and a limit of detection of 1 x 10(6) viruses/mL. These results reflect the findings from a detailed series of investigations on the effects of several experimental parameters (e.g., salt concentration, ERL binding buffer, and sample agitation), all of which were aimed at minimizing nonspecific binding and maximizing FCV binding efficiency. The performance of the assay is correlated with the number of captured FCV, determined by atomic force microscopy, as a means of method validation.

Low level epifluorescent detection of nanoparticles and DNA on dielectrophoretic microarrays

“…The ability to detect and identify low levels of analytes is of growing importance in medicine and nanotechnology [1][2][3][4][5][6][7]. Diagnostic monitoring of drug delivery nanoparticle levels [8][9][10], identification of cell-free circulating (cfc) DNA/RNA and other nanoparticulate biomarkers [11][12][13][14][15], and detection of pathogens in clinical and environmental samples [16] are examples where low level detection of analytes are of extreme importance.…”

Bioaffinity detection of pathogens on surfaces

“…One approach is to use reporter molecules with distinct spectra to label pathogens of interest. Porter et al designed a sandwich immunoassay chip format for the detection of feline calcivirus [48]. Antibodies specific to the virus were immobilized on gold nanoparticles along with an extrinsic Raman label 5,5-dithiobis(succinimidyl-2-nitrobenzoate) and were able to detect less than 100 virus surface binding events.…”

Design, Characterization, and Integration of Nanometric Objects with Chip-scale Platforms for Disease Diagnosis

“…An essential aspect of our assay platform relies on the design of our assay label, or extrinsic Raman labels (ERLs). By using an adlayer of an intrinsically strong Raman scatterer (i.e., reporter molecules), full advantage of the enhanced scattering from gold nanoparticles can be realized (28,(31)(32)(33)(34)(35)(41)(42)(43). Additionally, target specificity can be achieved by incorporating an antibody or other molecular recognition element in the design of the ERL.…”

“…We often use flocculation in each process step to evaluate the impact of changes in preparative conditions and to test the effectiveness of antibody coupling (33). The extinction spectra in Figure 3 show an example of results from our work in the development of ERLs for feline calicivirus (FCV), a simulant for the human calicivirus known as norovirus.…”

“…The extinction spectra in Figure 3 show an example of results from our work in the development of ERLs for feline calicivirus (FCV), a simulant for the human calicivirus known as norovirus. By systematically varying the amount of anti-FCV monoclonal antibody (mAb) added to borate-buffered suspensions of 60-nm gold, particle aggregation upon the addition of NaCl to physiological concentration (150 mM) was monitored (33). The as-received suspension has an extinction maximum at 535 nm, consistent with the location of the plasmon resonance of isolated gold particles with an average diameter of 60 nm (50).…”