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Surface-enhanced resonance Raman scattering (SERRS) is based on nanostructures supporting localized surface plasmon resonances as well as electronic resonances of the molecular adsorbate. Bright NIR-SERRS nanotags require dyes with high Raman scattering cross-sections upon NIR laser excitation in combination with functional groups for chemisorption. Here, we report on the role of isothiocyanate (ITC) and sulfonate (SO) groups in five cyanine dyes on sensitivity and performance of nanotags in SERRS-based LFA. Dye concentration-dependent SERRS and zeta potential measurements on bare gold nanostars (AuNS) revealed strong differences in their sensitivity and stability as a function of the number of ITC and SO groups. A positive correlation with the number of ITC groups due to stronger chemisorption and a negative correlation with the number of SO groups due to stronger desorption because of higher water solubility was observed. The application in SERRS-based biosensing requires additional functionalization steps, for example, co-adsorption of PEG with the NIR dye. The comparison of the Raman intensities before and after bioconjugation showed that the presence of at least one ITC group is necessary for minimizing Raman signal losses, irrespective of the additional presence of SO groups. In a SERRS-based lateral flow assay (LFA) for sensing the pregnancy marker beta-hCG, the two dyes NIR 4f and NIR 5e (both ITC = 2, SO = 0) showed the best performance. In contrast, we do not recommend to use IR-783 (ITC = 0, SO = 2) for SERRS-based biosensing. Overall, this study highlights the importance of surface-seeking moieties such as the common ITC group for optimal performance in SERRS-based biosensing platforms. in press

Surface-enhanced Raman scattering (SERS)-based assays have been recently developed to overcome the low detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SERS-based assays using magnetic beads in microtubes slightly improved the limit of detection (LoD) for SARS-CoV-2. However, the sensitivity and reproducibility of the method are still insufficient for reliable SARS-CoV-2 detection. In this study, we developed a SERS-based microdroplet sensor to dramatically improve the LoD and reproducibility of SARS-CoV-2 detection. Raman signals were measured for SERS nanotags in 140 droplets passing through a laser focal volume fixed at the center of the channel for 15 s. A comparison of the Raman signals of SERS nanotags measured in a microtube with those measured for multiple droplets in the microfluidic channel revealed that the LoD and coefficient of variation significantly improved from 36 to 0.22 PFU/mL and 21.2% to 1.79%, respectively. This improvement resulted from the ensemble average effects because the signals were measured for SERS nanotags in multiple droplets. Moreover, the total assay time decreased from 30 to 10 min. A clinical test was performed on patient samples to evaluate the clinical efficacy of the SERS-based microdroplet sensor. The assay results agreed well with those measured by the reverse transcription-polymerase chain reaction (RT-PCR) method. The proposed SERS-based microdroplet sensor is expected to be used as a new point-of-care diagnostic platform for quick and accurate detection of SARS-CoV-2 in the field.

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.

The surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip has been considered a high-sensitivity sensor that can overcome the low sensitivity and the difficulty of quantitative analysis problems inherent in the colorimetric LFA sensor. In the SERS-based LFA strip reported so far, a liquid sample flows through the nitrocellulose membrane in a single pathway. In some cases, however, this single-flow approach still has a limitation in detection sensitivity. This study developed a conceptually new SERS-based dual-flow LFA sensor to improve the detection sensitivity in a single-flow LFA sensor. First, a 25 nm Raman reporter-labeled gold nanoparticle (AuNP) solution flowed through one way, and a 45 nm AuNP solution continuously flowed through another path. This sequential flow of two different AuNP solutions enables forming additional bright hot spots between 25 and 45 nm AuNPs in the test line, and the SERS signal is strongly enhanced. Using this SERS-based dual-flow LFA sensor, it was possible to detect thyroid-stimulating hormone less than 0.5 μIU/mL that cannot be measured with a SERS-based single-flow LFA sensor.

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.

We report a surface-enhanced Raman scattering (SERS)-based polymerase chain reaction (PCR) assay platform for the sensitive and rapid detection of a DNA marker (pagA) of Bacillus anthracis. Real-ti...

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 a surface-enhanced Raman scattering (SERS)-based immunoassay platform for the rapid diagnosis of scrub typhus and murine typhus, which are the most common acute febrile diseases in South Korea. A microarray device, composed of multiple gold nanopopcorn substrates capable of detecting ultra-sensitive biomarkers, was used as a multiplex SERS-based assay platform. Sequentially diluted titers of Orientia tsutsugamushi and Rickettsia typhi specific human IgG/IgM antibodies, which are biomarkers of two typhus diseases, were analyzed by Raman spectroscopy, and the peak intensity was plotted against the different titer concentration range (0-2048 and 0-1024 for O. tsutsugamushi IgG/IgM and 0-8192 and 0-256 for R. typhi IgG/IgM) to generate calibration curves. The sensitivities and limits of detection (LODs) determined for four different IgG/IgM antibodies were significantly lower than those for the ELISA method. The LODs of titer concentrations for O. tsutsugamushi IgG/IgM and R. typhi IgG/IgM are determined to be 20.4, 7.03, 16.8 and 12.5, respectively. The LOD values were all lower than the cut-off values (256, 16, 128, and 64) used for clinical diagnosis, which means that this assay platform can diagnose two typhus diseases with high sensitivity. When the microarray sensors are combined with portable Raman spectrophotometers, which are commercially available worldwide, it is also possible to directly diagnose a patient in the field without sending their blood sample to a hospital.

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.

We report a rapid and sensitive recognition technique for explosive compounds using Raman spectroscopy and principal component analysis (PCA). Seven hundreds of Raman spectra (50 measurements per sample) for 14 selected explosives were collected, and were pretreated with noise suppression and baseline elimination methods. PCA, a well-known multivariate statistical method, was applied for the proper evaluation, feature extraction, and identification of measured spectra. Here, a broad wavenumber range (200–3500 cm−1) on the collected spectra set was used for the classification of the explosive samples into separate classes. It was found that three principal components achieved 99.3% classification rates in the sample set. The results show that Raman spectroscopy in combination with PCA is well suited for the identification and differentiation of explosives in the field.

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.

The use of silica shells offers many advantages in surface-enhanced Raman scattering (SERS)-based biological sensing applications due to their optical transparency, remarkable stability in environmental media, and improved biocompatibility. Here, we report a novel layer-by-layer method for the preparation of silica-hollow gold nanosphere (HGN) SERS tags. Poly(acrylic acid) was used to stabilize Raman reporter-tagged HGNs prior to the adsorption of a coupling agent, after which a silica shell was deposited onto the particle surface using Stöber's method. Importantly, competitive adsorption of the Raman reporter molecules and coupling agents, which results in unbalanced loading of reporter molecules on individual nanoparticles, was avoided using this method. As a result, the loading density of reporter molecules could be maximized. In addition, HGNs exhibited strong enhancement effects from the individual particles because of their ability to localize the surface electromagnetic fields through pinholes in the hollow particle structures. The proposed layer-by-layer silica-encapsulated HGN tags showed strong SERS signals as well as excellent multiplexing capabilities.

Silver-coated magnetic microspheres (Fe3O4/Ag core/shell) were fabricated for use as a surface-enhanced Raman scattering (SERS) substrate. The surface morphology of the microspheres was characterized via field emission scanning electron microscopy and field emission transmission electron microscopy. Energy-dispersive spectroscopy was also employed to analyze the elemental composition of the structures. Malachite green was used as a model analyte to evaluate the performance of the microspheres as a SERS-active substrate and validate the SERS effect. The prepared microspheres possessed both magnetic and SERS properties. In addition, a microfluidic device with solenoids was designed and fabricated for a facile trace analysis. The solenoid chip allowed us to control the alignment of the magnetic microspheres on the wall of the microfluidic channel. Target samples were introduced into the channel and adsorbed on the surface of trapped silver-coated magnetic microspheres. SERS signals were then measured using a...

We report a rapid and highly sensitive trace analysis of paraquat (PQ) in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aqueous samples of PQ, silver nanoparticles, and NaCl as the aggregation agent were introduced into a microfluidic channel and were encapsulated by a continuous oil phase to form a microdroplet. PQ molecules were adsorbed onto particle surfaces in isolated droplets by passing through the winding part of the channel. Memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, were removed because the aqueous droplets were completely isolated by a continuous oil phase. The limit of detection (LOD) of PQ in water, determined by the SERS-based microdroplet sensor, was estimated to be below 2×10(-9) M, and this low detection limit was enhanced by one to two orders of magnitude compared to conventional analytical methods.

We report the fast and sensitive trace analysis of diaquat dibromide monohydrate (DQ) in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. This sensor is composed of two compartments: the first one is for droplet generation for fresh silver nanoparticle (AgNP) synthesis and the second for droplet merging for SERS detection. Silver ions were nucleated and grown to large size AgNPs in droplets, and then each droplet was synchronously merged with another droplet containing DQ for SERS detection. This two-phase liquid–liquid segmented flow system prevented memory effects caused by the precipitation of nanoparticle aggregates on channel walls because the aqueous droplets were isolated by a continuous oil phase. The limit of detection (LOD) of DQ in water was determined to be below 5 nM, which satisfies the maximum contaminant level defined by the United States EPA. This method was also validated successfully in DQ-spiked tap water. The SERS-based integrated sensing system is expected to be useful as an in-the-field sensing platform for fast and reproducible trace analysis of environmental pollutants in water.

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.

We report the development of a programmable surface-enhanced Raman scattering (SERS)-based micro-network gradient platform to simultaneously detect two different types of DNA oligomer mixtures. The utility of this platform was demonstrated by quantitative analysis of two breast cancer-related (BRCA1) DNA oligomer mixtures. To generate on-demand concentration gradients, the microfluidic circuit was designed using an electric-hydraulic analogy. Then a multi-gradient microfluidic channel was fabricated based on the theoretical design of the concentration control module. These micro-network structures automatically produce a series of different concentration gradients by continuously mixing Cy3-labeled DNA oligomers (BRAC1-Mutation) with TAMRA-labeled DNA oligomer (BRAC1-Wild). The SERS signals for different ratios of duplex DNA oligomer mixtures, adsorbed on the surface of silver nanoparticles, were measured under flowing conditions. Total analysis time from serial mixing to SERS detection takes less than 10 min because all experimental conditions are automatically controlled inside the exquisitely designed microfluidic channel. This novel SERS-based DNA sensing technology in a micro-network gradient channel is expected to be a powerful analytical tool to simultaneously detect multiple DNA oligomer mixtures.