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Biosens Bioelectron ; 197: 113736, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34741957


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.

Técnicas Biossensoriais , COVID-19 , Nanopartículas Metálicas , Ouro , Humanos , Reação em Cadeia da Polimerase , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Sensibilidade e Especificidade
Biosens Bioelectron ; 167: 112496, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32818752


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.

Técnicas Biossensoriais , Vírus da Influenza A Subtipo H1N1 , Influenzavirus A , Nanopartículas Metálicas , Ouro , Reprodutibilidade dos Testes , Análise Espectral Raman
Biosens Bioelectron ; 164: 112326, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32553352


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.

Técnicas Biossensoriais , Nanopartículas Metálicas , Neoplasias , Biomarcadores , Ouro , Prata , Análise Espectral Raman
Anal Chem ; 92(3): 2628-2634, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31939280


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.

DNA/genética , Reação em Cadeia da Polimerase em Tempo Real , DNA/química , Ouro/química , Humanos , Nanopartículas Metálicas/química , Tamanho da Partícula , Análise Espectral Raman , Propriedades de Superfície