Search

We have visualized surface-enhanced Raman scattering (SERS) nano-gap domains with electrostatic force microscopy (EFM). We constructed the SERS substrate using reactive-ion etching of a polyethylene terephthalate (PET) film, with subsequent sputtering of Au onto the etched PET. Because the nano-gap is more strongly charged than the native Au-nanoparticle (NP) surface, we can identify the SERS nano-gap due to the longer dwell time of the tapping EFM tip and the related phase shift of the signal. This produces a differentiating image contrast between the SERS nano-gap and the Au NP surface, with a spatial resolution of ~14 nm. More specifically, differences in the mode-independent Raman-signal enhancement and the peak shift of an adsorbed malachite green isothiocyanate (MGITC) molecule are only observed in the smaller SERS nano-gap (found in the surface with Ar+-sputtered Au nano-pillars). We interpret this in terms of the differing morphologies of the underlying Au substrates and the related mechanical compressive strains, which result in charge transfer (CT)-based single-molecule Raman-reporter detection sensitivity.

In the last two decades, microfluidic technology has emerged as a highly efficient tool for the study of various chemical and biological reactions. Recently, we reported that high-throughput detection of various concentrations of a reagent is possible using a continuous gradient microfluidic channel combined with a surface-enhanced Raman scattering (SERS) detection platform. In this continuous flow regime, however, the deposition of nanoparticle aggregates on channel surfaces induces the "memory effect," affecting both sensitivity and reproducibility. To resolve this problem, a SERS-based gradient droplet system was developed. Herein, the serial dilution of a reagent was achieved in a stepwise manner using microfluidic concentration gradient generators. Then various concentrations of a reagent generated in different channels were simultaneously trapped into the tiny volume of droplets by injecting an oil mixture into the channel. Compared to the single-phase regime, this two-phase liquid/liquid segmented flow regime allows minimization of resident time distributions of reagents through localization of reagents in encapsulated droplets. Consequently, the sample stacking problem could be solved using this system because it greatly reduces the memory effect. We believe that this SERS-based gradient droplet system will be of significant utility in simultaneously monitoring chemical and biological reactions for various concentrations of a reagent.

We developed a new surface-enhanced Raman scattering (SERS)-based aptasensor platform capable of quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lysates with a high sensitivity. In this study, a spike protein deoxyribonucleic acid (DNA) aptamer was used as a receptor, and a self-grown Au nanopopcorn surface was used as a SERS detection substrate for the sensible detection of SARS-CoV-2. A quantitative analysis of the SARS-CoV-2 lysate was performed by monitoring the change in the SERS peak intensity caused by the new binding between the aptamer DNA released from the Au nanopopcorn surface and the spike protein in the SARS-CoV-2 virion. This technique enables detecting SARS-CoV-2 with a limit of detection (LoD) of less than 10 PFU/mL within 15 min. The results of this study demonstrate the possibility of a clinical application that can dramatically improve the detection limit and accuracy of the currently commercialized SARS-CoV-2 immunodiagnostic kit.

This minireview reports the recent advances in surface-enhanced Raman scattering (SERS)-based assay devices for the diagnosis of infectious diseases. SERS-based detection methods have shown promise in overcoming the low sensitivity and multiplex detection problems inherent to fluorescence detection. Therefore, it is interesting to investigate the current status, challenges, and applications associated with SERS-based microdevices for the point-of-care (POC) diagnosis of infectious diseases. The majority of this review highlights three different types of microdevices, namely microfluidic channels, lateral flow assay strips, and three-dimensional nanostructured substrates. Furthermore, the integration of portable Raman spectrophotometry with microdevices provides an ideal platform for the diagnosis of various infectious diseases in the field. Integrated SERS-based assay systems also enable measurements in minimal sample volumes and at low analyte concentrations of viral or bacterial samples. A significant number of studies using the SERS-based assay system have been performed recently to realize POC diagnostics, especially under resource-limited conditions. This portable SERS sensor is expected to be a next-generation POC assay system that could overcome the limitations of current fluorescence-based assay systems. This minireview summarizes recent advances in the development of SERS-based microdevices for the diagnosis of infectious diseases. Lastly, challenges to overcome and future perspectives are discussed.

The design and fabrication of multifunctional surface-enhanced Raman scattering (SERS) nanotags are key issues in their application to biological imaging of cells and tissues. In this study, highly sensitive, reproducible and long-term stable SERS nanotags were developed for the identification of localized distribution of multiple protein biomarkers expressed on breast cancer cells. To enhance the surface electromagnetic fields of Raman reporter molecules, Ag-encapsulated Au (Ag-Au) hollow nanospheres were synthesized. Strong Raman signal enhancement effects could be achieved by positioning Raman reporter molecules in nanogaps between the Au hollow nanospheres and silver shell. In addition, the signal was also enhanced due to the localization of surface electromagnetic fields through the pinholes on the surface of Au hollow nanospheres. To maintain the long-term stability of the Au hollow-Ag core/shell nanospheres, their surface was coated with a polyethylene glycol (PEG) layer. The biocompatibility of PEGylated Ag-Au hollow nanospheres was investigated using the premix water soluble tetrazolium salt (WST-1) cell viability test. These SERS nanotags also enabled a high-resolution multiplexed live cell imaging. Our proposed SERS imaging technique using the new SERS nanotags provides a new platform for fast and accurate classification of different phenotypes of breast cancer cells.

Bacterial pathogens such as Yersinia pestis, Francisella tularensis, and Bacillus anthracis are classified into the highest rank of potential bioterrorism agents. Colorimetric lateral flow assay (LFA) strips are commercially available but these conventional strips have drawbacks in terms of low sensitivity and limit of quantitative analysis. Therefore, there is an urgent need for a new sensing platform to detect these pathogens in the early contamination stage. In this study, a novel surface-enhanced Raman scattering (SERS)-based LFA strip was developed for sensitive detection of bacterial pathogens. Target-specific SERS nanotags (Raman reporter-labeled gold nanoparticles) were used as an alternative to the gold nanoparticles in conventional LFA strips. Using these SERS nanotags the presence of bacteria could be identified through a simple color change in the test line. Additionally, highly sensitive and accurate quantitative analysis could be performed by monitoring the characteristic Raman peak intensity of SERS nanotags that were captured in the test line. This highly sensitive method required a short assay time (15 min) and a tiny volume of pathogen sample (40 μL). We believe that the proposed SERS-based LFA technique has great potential as a valuable tool in the early detection of specific bacterial pathogens in the field due to its excellent analytical sensitivity.

We report the development of amplification-free surface-enhanced Raman scattering (SERS)-based DNA assays for the rapid and highly sensitive detection of prostate cancer antigen 3 (PCA3) mimic DNA. This technique does not require any DNA amplification process using thermo-cycles in conventional polymerase chain reaction (PCR) due to its highly sensitive detection capability. Herein, the PCA3 mimic DNA, composed of 45 nucleotide sequences, was sandwiched between two probe DNA-immobilized particles (ASO 735 -conjugated detection HGNs and ASO 683 -immobilized capture magnetic beads) by hybridization reactions. Its quantitative analysis was performed by monitoring the characteristic Raman peak intensity of sandwich DNA complexes. The limit of detection (LOD) is estimated to be 2.7 fM, which is about four orders of magnitude more sensitive than that of conventional PCR. This SERS-based DNA assay technique is expected to be a potentially useful tool for early disease diagnosis.

Accurate analysis of specific biomarkers in clinical serum is essential for early diagnosis and treatment of cancer. Here, a surface-enhanced Raman scattering (SERS)-based immunoassay, using magnetic beads and SERS nano tags, was developed for the determination of free to total (f/t) prostate specific antigen (PSA) ratio to improve the diagnostic performance of prostate cancer. To assess the clinical applicability of the proposed method, SERS-based assays for the simultaneous detection of dual PSA markers, free PSA (f-PSA) and complexed PSA (c-PSA), were performed for clinical samples in the gray zone between 4.0 and 10.0 ng/mL. Our assay results for f/t PSA ratio showed a good linear correlation with those measured using the electrochemiluminescence (ECL) system installed in the clinical laboratory of the University Hospital. In addition, the simultaneous assay provided better precision than parallel assays for the detection of f-PSA and c-PSA in 13 clinical serum samples. Therefore, our SERS-based assay for simultaneous detection of dual PSA markers in clinical fluids has strong potential for application in the accurate diagnosis of prostate cancer.

Rapid and sensitive detection of botulinum neurotoxins (BoNTs) is important for immediate treatment with proper antitoxins. However, it is difficult to detect BoNTs at the acute phase of infection, owing to its rarity and ambiguous symptoms. To resolve this problem, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique for the rapid and sensitive detection of BoNTs. Magnetic beads and SERS nanotags as capture substrates and detection probes, respectively, and Nile Blue A (NBA) and malachite green isothiocyanate (MGITC) as Raman reporter molecules were used for the detection of two different types of BoNTs (types A and B), respectively. The corresponding limits of detection (LODs) were determined as 5.7 ng/mL (type A) and 1.3 ng/mL (type B). Total assay time, including that for immunoreaction, washing, and detection, was less than 2 h.

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.

Surface-enhanced Raman scattering (SERS)-based aptasensors display high sensitivity for influenza A/H1N1 virus detection but improved signal reproducibility is required. Therefore, in this study, we fabricated a three-dimensional (3D) nano-popcorn plasmonic substrate using the surface energy difference between a perfluorodecanethiol (PFDT) spacer and the Au layer. This energy difference led to Au nanoparticle self-assembly; neighboring nanoparticles then created multiple hotspots on the substrate. The localized surface plasmon effects at the hot spots dramatically enhanced the incident field. Quantitative evaluation of A/H1N1 virus was achieved using the decrease of Raman peak intensity resulting from the release of Cy3-labeled aptamer DNAs from nano-popcorn substrate surfaces via the interaction between the aptamer DNA and A/H1N1 virus. The use of a Raman imaging technique involving the fast mapping of all pixel points enabled the reproducible quantification of A/H1N1 virus on nano-popcorn substrates. Average ensemble effects obtained by averaging all randomly distributed hot spots mapped on the substrate made it possible to reliably quantify target viruses. The SERS-based imaging aptasensor platform proposed in this work overcomes the issues inherent in conventional approaches (the time-consuming and labor-intensiveness of RT-PCR and low sensitivity and quantitative analysis limits of lateral flow assay kits). Our SERS-based assay for detecting A/H1N1 virus had an estimated limit of detection of 97 PFU mL−1 (approximately three orders of magnitude more sensitive than that determined by the enzyme-linked immunosorbent assay) and the approximate assay time was estimated to be 20 min. Thus, this approach provides an ultrasensitive, reliable platform for detecting viral pathogens.

A surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip using silica-encapsulated gold nanoparticles (Si-AuNPs) was developed for the diagnosis of mosquito-borne diseases—dengue fever, Zika virus, or malaria—which are endemic to tropical regions. To investigate the thermal stability of the SERS nanoprobes, Raman peak intensity variations for AuNPs and Si-AuNPs were measured and compared at 45°C. SERS mapping images of target human immunoglobulin (IgG) on the test line of the Si-AuNP-loaded LFA strip were also evaluated at the same temperature to test the reproducibility of detection. In this case, the distribution of Raman mapping points on the test line was relatively uniform compared with the AuNP-loaded LFA strips at 45°C. This indicates that the silica encapsulation on the surface of the AuNPs improves the reproducibility of the SERS nanoprobes at high temperatures. The limit of detection (LOD) value of the AuNP-loaded LFA strips was significantly increased when the temperature was increased from 25°C to 45°C. In the case of the Si-AuNP-loaded LFA strips, however, the LOD value at 45°C was almost the same as that at 25°C. This result also shows that the silica encapsulation of the AuNPs improves the thermal stability of the LFA strips at high temperatures. Thus, Si-AuNPs are reproducible and thermally stable labeling agents for use in LFA strips at high temperatures and are suitable for the preparation of rapid kits, which can be used in tropical areas.

Many efforts have been invested in the development of rapid and sensitive detection methods for the quantification of disease biomarkers in human blood. Although luminescence- and fluorescence-based detection methods, combined with an automatic sampling system, are routinely used for immunoassays of specific biomarkers, a more sensitive detection technique is needed to track disease progression in its early stage. Recently, a surface-enhanced Raman scattering (SERS)-based immunoassay technique has been considered as a strong candidate to resolve the problem of low sensitivity in conventional luminescence- or fluorescence-based clinical immunoassays. Due to its highly sensitive detection capability and the feasibility of automatic assay, the SERS-based assay technique has strong potential to substitute for currently available fluorescence or luminescence assay systems in clinical laboratory settings. To date, a variety of clinical biomarkers such as proteins, DNAs, hormones, viruses, bacteria, and toxins have been measured using the SERS-based assay technique. Several different types of assay platform have been developed for rapid and reproducible SERS-based assays. In this chapter, four different SERS-based assay platforms—gold-patterned microarray-type substrates, magnetic bead-based assay platforms, microfluidic devices, and lateral flow assay platforms—will be introduced. In addition, the diagnostic feasibility of these assay platforms for various biomarkers will be described.