Quantitative Serodiagnosis of Scrub Typhus Using Surface-Enhanced Raman Scattering-Based Lateral Flow Assay Platforms.

A surface-enhanced Raman scattering-based lateral flow assay (SERS-LFA) technique has been developed for the rapid and accurate diagnosis of scrub typhus. Lateral flow kits for the detection of O. tsutsugamushi IgG (scrub typhus biomarker) were fabricated, and the calibration curve for various standard clinical sera concentrations were obtained by Raman measurements. The clinical sera titer values were determined by fitting the Raman data to the calibration curve. To assess the clinical feasibility of the proposed method, SERS-LFA assays were performed on 40 clinical samples. The results showed good agreement with those of the standard indirect immunofluorescence assay (IFA) method. SERS-LFA has many advantages over IFA including the less sample volume, simpler assay steps, shorter assay time, more systematic quantitative analysis, and longer assay lifetime. As SERS strips can be easily integrated with a miniaturized Raman spectrophotometer, field serodiagnosis is also more feasible.

Sensitive and Reproducible Immunoassay of Multiple Mycotoxins Using Surface-Enhanced Raman Scattering Mapping on 3D Plasmonic Nanopillar Arrays.

A surface-enhanced Raman scattering-based mapping technique is reported 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) are obtained by rapidly scanning the surface-enhanced Raman scattering (SERS) nanotags-anchoring mycotoxins captured on a 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 nanopillars greatly enhances the coupling of local plasmonic fields. This strong enhancement effect makes it possible to perform a highly sensitive detection of multiple mycotoxins. In addition, the high uniformity of the densely packed nanopillar substrate minimizes the spot-to-spot fluctuations of the Raman peak intensity in the scanned area when Raman mapping is performed. Consequently, this makes it possible to gain a highly reproducible quantitative analysis of mycotoxins. The limit of detections (LODs) are determined to be 5.09, 5.11, and 6.07 pg mL-1 for OTA, FUMB, and AFB1, and these values are approximately two orders of magnitude more sensitive than those determined by the enzyme-linked immunosorbent assays. It is believed that this SERS-based mapping technique provides a facile tool for the sensitive and reproducible quantification of various biotarget molecules.

Culture-Free Detection of Bacterial Pathogens on Plasmonic Nanopillar Arrays Using Rapid Raman Mapping.

We utilized a fast Raman spectral mapping technique for fast detection of bacterial pathogens. Three-dimensional (3D) plasmonic nanopillar arrays were fabricated using the nanolithography-free process consisting of maskless Ar plasma treatment of a polyethylene terephthalate substrate and subsequent metal deposition. Bacterial pathogens were immobilized on the positively charged poly(l-lysine)-coated 3D plasmonic substrate through electrostatic interactions. Then, the bacterial surfaces were selectively labeled with antibody-conjugated surface-enhanced Raman scattering (SERS) nanotags, and Raman mapping images were collected and statistically analyzed for quantitative analysis of bacteria. Salmonella typhimurium was selected as a model pathogen bacterium to confirm the efficacy of our SERS imaging technique. Minimum number of Raman mapping points with statistical reliability was determined to reduce assay time. It was possible to get a statistically reliable standard calibration curve for 529 pixels (laser spot with 60 µm interval), which required a total mapping time of 45 min to get a standard calibration curve for five different concentrations of bacteria in the 0 to 106 CFU/mL range. No amplification step was necessary for quantification because low-abundance target bacteria could be measured using the Raman spectral mapping technique. Therefore, this approach allows accurate quantification of bacterial pathogens without any culturing or enrichment process.

Integrated SERS-Based Microdroplet Platform for the Automated Immunoassay of F1 Antigens in Yersinia pestis.

The development of surface-enhanced Raman scattering (SERS)-based microfluidic platforms has attracted significant recent attention in the biological sciences. SERS is a highly sensitive detection modality, with microfluidic platforms providing many advantages over microscale methods, including high analytical throughput, facile automation, and reduced sample requirements. Accordingly, the integration of SERS with microfluidic platforms offers significant utility in chemical and biological experimentation. Herein, we report a fully integrated SERS-based microdroplet platform for the automatic immunoassay of specific antigen fraction 1 (F1) in Yersinia pestis. Specifically, highly efficient and rapid immunoreactions are achieved through sequential droplet generation, transport, and merging, while wash-free immunodetection is realized through droplet-splitting. Such integration affords a novel multifunctional platform capable of performing complex multistep immunoassays in nL-volume droplets. The limit of detection of the F1 antigen for Yersinia pestis using the integrated SERS-based microdroplet platform is 59.6 pg/mL, a value approximately 2 orders of magnitude more sensitive than conventional enzyme-linked immunosorbent assays. This assay system has additional advantages including reduced sample consumption (less than 100 µL), rapid assay times (less than 10 min), and fully automated fluid control. We anticipate that this integrated SERS-based microdroplet device will provide new insights in the development of facile assay platforms for various hazardous materials.

Simultaneous Detection of Dual Nucleic Acids Using a SERS-Based Lateral Flow Assay Biosensor.

A new class of surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) biosensor has been developed 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 nano tags 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 nano tags 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 and 0.074 pM, respectively. These values indicate 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.

Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor.

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.

Highly sensitive SERS-based immunoassay of aflatoxin B1 using silica-encapsulated hollow gold nanoparticles.

Aflatoxin B1 (AFB1) is a well-known carcinogenic contaminant in foods. It is classified as an extremely hazardous compound because of its potential toxicity to the human nervous system. AFB1 has also been extensively used as a biochemical marker to evaluate the degree of food spoilage. In this study, a novel surface-enhanced Raman scattering (SERS)-based immunoassay platform using silica-encapsulated hollow gold nanoparticles (SEHGNs) and magnetic beads was developed for highly sensitive detection of AFB1. SEHGNs were used as highly stable SERS-encoding nano tags, and magnetic beads were used as supporting substrates for the high-density loading of immunocomplexes. Quantitative analysis of AFB1 was performed by monitoring the intensity change of the characteristic peaks of Raman reporter molecules. The limit of detection (LOD) of AFB1, determined by this SERS-based immunoassay, was determined to be 0.1 ng/mL. This method has some advantages over other analytical methods with respect to rapid analysis (less than 30 min), good selectivity, and reproducibility. The proposed method is expected to be a new analytical tool for the trace analysis of various mycotoxins.

A flow cytometry-based submicron-sized bacterial detection system using a movable virtual wall.

Detection of pathogenic bacteria requires a sensitive, accurate, rapid, and portable device. Given that lethal microbes are of various sizes, bacterial sensors based on DC (direct current) impedance on chips should be equipped with channels with commensurate cross sections. When it comes to counting and interrogation of individual bacteria on a microfluidic chip, very narrow channels are required, which are neither easy nor cost-effective to fabricate. Here, we report a flow cytometry-based submicron-sized bacterial detection system using a movable virtual wall made of a non-conducting fluid. We show that the effective dimension of a microfluidic channel can be adjusted by varying the respective flow rates of a sample solution as well as the liquid wall therein. Using such a virtual wall, we have successfully controlled the channel width and detected submicron-sized Francisella tularensis, a lethal, tularemia-causing bacterium. Since the system is capable of monitoring changes in DC impedance and fluorescence simultaneously, we were also able to discriminate between different types of bacterial mixtures containing F. tularensis and E. coli BL21 that have different gamuts of size distributions. The proposed flow cytometry-based system represents a promising way to detect bacteria including, but not limited to, submicron-sized pathogenic microbes.

Synthesis, characterization, and directional binding of anisotropic biohybrid microparticles for multiplexed biosensing.

Anisotropic microarchitectures with different physicochemical properties have been developed as advanced materials for challenging industrial and biomedical applications including switchable displays, multiplexed biosensors and bioassays, spatially-controlled drug delivery systems, and tissue engineering scaffolds. In this study, anisotropic biohybrid microparticles (MPs) spatio-selectively conjugated with two different antibodies (Abs) are first developed for fluorescence-based, multiplexed sensing of biological molecules. Poly(acrylamide-co-acrylic acid) is chemically modified with maleimide- or acetylene groups to introduce different targeting biological moieties into each compartment of anisotropic MPs. Modified polymer solutions containing two different fluorescent dyes are separately used for electrohydrodynamic co-jetting with side-by-side needle geometry. The anisotropic MPs are chemically stabilized by thermal imidization, followed by bioconjugation of two different sets of polyclonal Abs with two individual compartments via maleimide-thiol coupling reaction and Huisgen 1,3-dipolar cycloaddition. Finally, two compartments of the anisotropic biohybrid MPs are spatio-selectively associated with the respective monoclonal Ab-immobilized substrate in the presence of the antigen by sandwich-type immunocomplex formation, resulting in their ordered orientation due to the spatio-specific molecular interaction, as confirmed by confocal laser scanning microscopy. In conclusion, anisotropic biohybrid MPs capable of directional binding have great potential as a new fluorescence-based multiplexing biosensing system.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment.

Highly sensitive detection of thrombin using SERS-based magnetic aptasensors.

This paper reports a method of highly sensitive detection of thrombin using a surface-enhanced Raman scattering (SERS)-based magnetic aptasensor. Magnetic beads and gold nanoparticles (Au NPs) were used as supporting substrates and sensing probes, respectively. For this purpose, 15-mer thrombin-binding aptamers (TBA15) were immobilized onto the surface of magnetic beads, and then thrombin antigens and 29-mer thrombin-binding aptamer (TBA29)-conjugated Au NPs were sequentially added for the formation of sandwich aptamer complexes. Quantitative analysis was performed by monitoring the intensity variation of a characteristic SERS signal of Raman reporter molecules. Because all of the reactions occur in solution, this SERS-based immunoassay technique can solve the diffusion-limited kinetic problems on a solid substrate. The limit of detection (LOD) of thrombin, determined by the SERS-based aptasensor, was estimated to be 0.27pM. The proposed method is expected to be a good clinical tool for the diagnosis of a thrombotic disease.

Trace analysis of mercury(II) ions using aptamer-modified Au/Ag core-shell nanoparticles and SERS spectroscopy in a microdroplet channel.

We report the rapid and highly sensitive trace analysis of mercury(ii) ions in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aptamer-modified Au/Ag core-shell nanoparticles have been fabricated and utilized as highly functional sensing probes. All detection processes for the reaction between mercury(II) ions and aptamer-modified nanoparticles were performed in a specially designed microdroplet channel. Small water droplets that included sample reagents were separated from each other by an oil phase that continuously flowed along the channel. This two-phase liquid-liquid segmented flow system prevented the adsorption of aggregated colloids to the channel walls due to localized reagents within encapsulated droplets. The result was reduced residence time distributions. The limit of detection (LOD) of mercury(II) ions in water was determined by the SERS-based microdroplet sensor to be below 10 pM, which is three orders below the EPA-defined maximum contaminant level. This combination of a SERS-based microfluidic sensor with aptamer-based functional nanoprobes can be used for in-the-field sensing platforms, due to its size and simplicity.

SERS-based immunoassay of tumor marker VEGF using DNA aptamers and silica-encapsulated hollow gold nanospheres.

A novel SERS-based sandwich immunoassay using DNA aptamers, silica-encapsulated hollow gold nanospheres (SEHGNs) and a gold-patterned microarray was developed for sensitive detection of VEGF (vascular endothelial growth factor) angiogenesis protein markers. Here, a DNA aptamer conjugated to SEHGN was used as a highly reproducible SERS-encoding nanoprobe, and a hybrid microarray including hydrophilic gold wells and other hydrophobic areas was used as a SERS substrate. Target specific DNA aptamers that fold into a G-quadruplex structure were used as a target recognition unit instead of VEGF antibodies. The detection sensitivity was increased by 2 or 3 orders of magnitude over the conventional ELISA method. In particular, the dynamic concentration range was 3 or 4 orders of magnitude greater than that of conventional ELISA. The results demonstrate that this sensing strategy using DNA aptamers is a powerful platform for the design of novel immune-sensors with high performance. In particular, SERS-based detection using SEHGNs provides great promise for highly sensitive biomarker sensing with unprecedented advantages.