Advancing COVID-19 Diagnosis: Enhancement in SERS-PCR with 30-nm Au Nanoparticle-Internalized Nanodimpled Substrates.

Real-time polymerase chain reaction (RT-PCR) with fluorescence detection is the gold standard for diagnosing coronavirus disease 2019 (COVID-19) However, the fluorescence detection in RT-PCR requires multiple amplification steps when the initial deoxyribonucleic acid (DNA) concentration is low. Therefore, this study has developed a highly sensitive surface-enhanced Raman scattering-based PCR (SERS-PCR) assay platform using the gold nanoparticle (AuNP)-internalized gold nanodimpled substrate (AuNDS) plasmonic platform. By comparing different sizes of AuNPs, it is observed that using 30 nm AuNPs improves the detection limit by approximately ten times compared to 70 nm AuNPs. Finite-difference time-domain (FDTD) simulations show that multiple hotspots are formed between AuNPs and the cavity surface and between AuNPs when 30 nm AuNPs are internalized in the cavity, generating a strong electric field. With this 30 nm AuNPs-AuNDS SERS platform, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA)-dependent RNA polymerase (RdRp) can be detected in only six amplification cycles, significantly improving over the 25 cycles required for RT-PCR. These findings pave the way for an amplification-free molecular diagnostic system based on SERS.

SERS-based microdevices for use as in vitro diagnostic biosensors.

Advances in surface-enhanced Raman scattering (SERS) detection have helped to overcome the limitations of traditional in vitro diagnostic methods, such as fluorescence and chemiluminescence, owing to its high sensitivity and multiplex detection capability. However, for the implementation of SERS detection technology in disease diagnosis, a SERS-based assay platform capable of analyzing clinical samples is essential. Moreover, infectious diseases like COVID-19 require the development of point-of-care (POC) diagnostic technologies that can rapidly and accurately determine infection status. As an effective assay platform, SERS-based bioassays utilize SERS nanotags labeled with protein or DNA receptors on Au or Ag nanoparticles, serving as highly sensitive optical probes. Additionally, a microdevice is necessary as an interface between the target biomolecules and SERS nanotags. This review aims to introduce various microdevices developed for SERS detection, available for POC diagnostics, including LFA strips, microfluidic chips, and microarray chips. Furthermore, the article presents research findings reported in the last 20 years for the SERS-based bioassay of various diseases, such as cancer, cardiovascular diseases, and infectious diseases. Finally, the prospects of SERS bioassays are discussed concerning the integration of SERS-based microdevices and portable Raman readers into POC systems, along with the utilization of artificial intelligence technology.

SERS-PCR assays of SARS-CoV-2 target genes using Au nanoparticles-internalized Au nanodimple substrates.

The reverse transcription-polymerase chain reaction (RT-PCR) method has been adopted worldwide to diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although this method has good sensitivity and specificity, there is a need to develop a more rapid diagnostic technology, given the virus's rapid spread. However, the RT-PCR method takes a long time to diagnose SARS-CoV-2 because of the required thermocycling steps. Therefore, we developed a surface-enhanced Raman scattering (SERS)-PCR detection method using an AuNP-internalized Au nanodimple substrate (AuNDS) to shorten the diagnosis time by reducing the number of thermocycling steps needed to amplify the DNA. For the representative target markers, namely, the envelope protein (E) and RNA-dependent RNA polymerase (RdRp) genes of SARS-CoV-2, 25 RT-PCR thermocycles are required to reach a detectable threshold value, while 15 cycles are needed for magnetic bead-based SERS-PCR when the initial DNA concentration was 1.00× 105 copies/µL. However, only 8 cycles are needed for the AuNDS-based SERS-PCR. The corresponding detectable target DNA concentrations were 3.36 × 1012, 3.28 × 109, and 2.56 × 107 copies/µL, respectively. Therefore, AuNDS-based SERS-PCR is seen as being a new molecular diagnostic platform that can shorten the time required for the thermocycling steps relative to the conventional RT-PCR.

Recent advances in sensitive surface-enhanced Raman scattering-based lateral flow assay platforms for point-of-care diagnostics of infectious diseases.

This review reports the recent advances in surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) platforms for the diagnosis of infectious diseases. As observed through the recent infection outbreaks of COVID-19 worldwide, a timely diagnosis of the disease is critical for preventing the spread of a disease and to ensure epidemic preparedness. In this regard, an innovative point-of-care diagnostic method is essential. Recently, SERS-based assay platforms have received increasing attention in medical communities owing to their high sensitivity and multiplex detection capability. In contrast, LFAs provide a user-friendly and easily accessible sensing platform. Thus, the combination of LFAs with a SERS detection system provides a new diagnostic modality for accurate and rapid diagnoses of infectious diseases. In this context, we briefly discuss the recent application of LFA platforms for the POC diagnosis of SARS-CoV-2. Thereafter, we focus on the recent advances in SERS-based LFA platforms for the early diagnosis of infectious diseases and their applicability for the rapid diagnosis of SARS-CoV-2. Finally, the key issues that need to be addressed to accelerate the clinical translation of SERS-based LFA platforms from the research laboratory to the bedside are discussed.

SERS imaging-based aptasensor for ultrasensitive and reproducible detection of influenza virus A.

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.

SERS biosensors for ultrasensitive detection of multiple biomarkers expressed in cancer cells.

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.

Performance Evaluation of Surface-Enhanced Raman Scattering-Polymerase Chain Reaction Sensors for Future Use in Sensitive Genetic Assays.

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-time quantitative PCR (RT-qPCR) has been recently considered a gold standard for the quantitative evaluation of a target gene, but it still suffers from the problem of a long thermocycling time. To address this issue, we developed a conceptually new SERS-PCR platform and evaluated its performance by sequentially measuring the Raman signals of B. anthracis DNA after the completion of different thermocycling numbers. According to our experimental data, SERS-PCR has lower limits of detection (LODs) than RT-qPCR under the small cycle number of 20. Particularly, it was impossible to detect a target DNA amplicon using RT-qPCR before the number of cycles reached 15, but SERS-PCR enabled DNA detection after only five cycles with an LOD value of 960 pM. In addition, the dynamic range for SERS-PCR (0.1-1000 pM) is wider than that for RT-qPCR (150-1000 pM) under the same condition. We believe that this SERS-PCR technique has a strong potential to be a powerful tool for the rapid and sensitive diagnosis of infectious diseases in the near future.