Application of SERS-based Sensing Platforms for Multiplex Bioassays

Multiplex bioassays are in great demand for chemical and biological detection and identification due to their unique advantages. In recent years, surface-enhanced Raman scattering (SERS) has become a promising technique for the detection of multiple analytes. SERS was discovered in the mid-1970s. It is a spectroscopic technique which exploits the interaction of light, molecules and metal nanostructures to boost strong Raman signals, in some cases up to 14 orders of magnitude. In addition, SERS can avoid the rapid photobleaching, and provide rich fingerprint spectra with energy resolution readily up to 1 cm???1 and large dynamic range (5???4000 cm???1). Due to the ultrahigh sensitivity and multiplex detection capability, SERS-based multiplex bioassays will provide an unprecedented opportunity for disease diagnosis, food safety supervision and environmental surveillance, etc. In recent years, SERS-based bioassay technique using functional metal nanoparticles (SERS nanotags) has attracted much interest. When SERS nanotags are used as the detection probe instead of fluorescence dyes or other molecules, their Raman signals are strongly enhanced at SERS-active junctions (named ???hot spots???) due to their electromagnetic and chemical enhancement effects. To date, various ever-increasing demands have promoted SERS-based bioassays from the classical type to those integrated with fascinating automatic platforms, including lateral flow assay (LFA) strips, gold-patterned substrates and microfluidic chips, etc. In this dissertation, we investigated the application of SERS-based LFA and three-dimensional (3D) nanopillar plasmonic substrate platforms for the multiplex bioassays. In chapter 2, we first fabricated a new class of SERS-based LFA biosensor for the simultaneous detection of dual DNA markers. The LFA strip in this sensor was composed of two test lines and one control line. SERS nanotags labeled with detection DNA probes were used for quantitative evaluation of dual DNA markers with high sensitivity. Target DNA, associated with Kaposi???s sarcoma-associated herpesvirus (KSHV) and bacillary angiomatosis (BA), were tested to validate the detection capability of this SERS-based LFA strip. Characteristic peak intensities of SERS nanotags on two test lines were used for quantitative evaluations of KSHV and BA. The limits of detection for KSHV and BA, determined from our SERS-based LFA sensing platform, were estimated to be 0.043 pM and 0.074 pM, respectively. These values indicated approximately 10,000 times higher sensitivity than previously reported values using the aggregation-based colorimetric method. We believe that this is the first report of simultaneous detection of two different DNA mixtures using a SERS-based LFA platform. This novel detection technique is also a promising multiplex DNA sensing platform for early disease diagnosis. In chapter 3, We reported a SERS-based mapping technique for the highly sensitive and reproducible analysis of multiple mycotoxins. Raman images of three mycotoxins, ochratoxin A (OTA), fumonisin B (FUMB), and aflatoxin B1 (AFB1) have been obtained by rapidly scanning the SERS nanotags-anchoring mycotoxins captured on a three-dimensional (3D) nanopillar plasmonic substrate. In this system, the decreased gap distance between nanopillars by their leaning effects as well as the multiple hot spots between SERS nanotags and highly packed nanopillars greatly enhanced the coupling of local plasmonic fields. This strong enhancement effect made it possible to perform a highly sensitive detection of multiple mycotoxins. In addition, the high uniformity of the densely packed nanopillar substrate, fabricated by thermal evaporation method, minimized the spot-to-spot fluctuations of the Raman peak intensity in the scanned area when Raman mapping was performed. Consequently, this made it possible to gain a highly reproducible quantitative analysis of mycotoxins. A patterned 3D plasmonic substrate composed of 49 (7??7 wells) circle wells were fabricated and successfully utilized for the immunoassay of multiple mycotoxins. The LODs were determined to be 5.09 pg/mL, 5.11 pg/mL and 6.07 pg/mL for OTA, FUMB and AFB1, and these values are approximately two orders of magnitude more sensitive than those determined by the ELISA assays. Selectivity tests were also performed for three cocktail samples with different mycotoxin ratios, and our SERS mapping-based assay technique demonstrated a good selectivity for OTA, FUMB and AFB1. Poor SERS signal uniformity has impeded reliable quantitative analysis of target molecules because of the heterogeneous and random distribution of hot spots over the plasmonic substrates. It is difficult to achieve a highly homogeneous SERS mapping image even though the surface morphology and detection conditions are carefully controlled. To resolve this problem, a Raman mapping technique was developed in chapter 3. In most cases of SERS quantitative reports, about 5 to 10 points were averaged to get a calibration curve for quantitative evaluations. Compared with the Raman mapping technique, it only takes several seconds for one sample, and greatly saves the assay time. In chapter 4, a minimum number of Raman mapping points with statistical reliability was investigated to reduce the assay time. Deoxynivalenol (DONs), a type B trichothecene was tested to validate the detection reliability of the Raman mapping method. The experiment result indicated that the standard calibration curve fits very well even when the number of mapping points was reduced to 484 pixels (100 ??m interval). For this number of mapping points, it took 60 s for each concentration to obtain a full calibration curve.Docto