Development of spike protein-based fluorescence lateral flow assay for the simultaneous detection of SARS-CoV-2 specific IgM and IgG.

The pandemic outbreak of the 2019 coronavirus disease (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still spreading rapidly and poses a great threat to human health. As such, developing rapid and accurate immunodiagnostic methods for the identification of infected persons is needed. Here, we proposed a simple but sensitive on-site testing method based on spike protein-conjugated quantum dot (QD) nanotag-integrated lateral flow immunoassay (LFA) to simultaneously detect SARS-CoV-2-specific IgM and IgG in human serum. Advanced silica-core@dual QD-shell nanocomposites (SiO2@DQD) with superior luminescence and stability were prepared to serve as fluorescent nanotags in the LFA strip and guarantee high sensitivity and reliability of the assay. The performance of the SiO2@DQD-strip was fully optimized and confirmed by using 10 positive serum samples from COVID-19 patients and 10 negative samples from patients with other respiratory diseases. The practical clinical value of the assay was further evaluated by testing 316 serum samples (114 positive and 202 negative samples). The overall detection sensitivity and specificity reached 97.37% (111/114) and 95.54% (193/202), respectively, indicating the huge potential of our proposed method for the rapid and accurate detection of SARS-CoV-2-infected persons and asymptomatic carriers.

Development of a SERS-based lateral flow immunoassay for rapid and ultra-sensitive detection of anti-SARS-CoV-2 IgM/IgG in clinical samples.

The accurate and rapid screening of serum antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the key to control the spread of 2019 coronavirus disease (COVID-19). In this study, we reported a surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-LFIA) for the simultaneous detection of anti-SARS-CoV-2 IgM/IgG with high sensitivity. Novel SERS tags labeled with dual layers of Raman dye were fabricated by coating a complete Ag shell on SiO2 core (SiO2@Ag) and exhibited excellent SERS signals, good monodispersity, and high stability. Anti-human IgM and IgG were immobilized onto the two test lines of the strip to capture the formed SiO2@Ag-spike (S) protein-anti-SARS-CoV-2 IgM/IgG immunocomplexes. The SERS signal intensities of the IgM and IgG test zones were easily recorded by a portable Raman instrument and used for the high-sensitivity analysis of target IgM and IgG. The limit of detection of SERS-LFIA was 800 times higher than that of standard Au nanoparticle-based LFIA for target IgM and IgG. The SERS-LFIA biosensor was tested on 19 positive serum samples from COVID-19 patients and 49 negative serum samples from healthy people to demonstrate the clinical feasibility of our proposed assay. The results revealed that the proposed method exhibited high accuracy and specificity for patients with SARS-CoV-2 infection.

Duplex-specific nuclease signal amplification-based fluorescent lateral flow assay for the point-of-care detection of microRNAs.

MiRNAs play important regulatory roles in numerous biological processes and serve as significant biomarkers for the development and prognosis of several diseases. Their unique characteristics, such as short size, high sequence homology among family members, low abundance, and easy degradability, have hindered their specific and highly sensitive detection. Herein, a duplex-specific nuclease (DSN)-assisted target recycling signal amplification-based fluorescent lateral flow assay was demonstrated for the point-of-care detection of cancer-related miRNA-21. In this assay, digoxin/biotin-labeled DNA probes were selectively cleaved by the DSN enzyme in the rounds of hybridization with the miRNA-21 target and cleavage cycle. Subsequently, the resulting mixture, containing the miRNA-21 target and intact and cleaved DNA probes, was loaded onto the lateral flow strip with digoxin antibody-conjugated quantum dot nanobeads and the streptavidin-coated test line. The increase in the proportion of cleaved DNA probes can induce a weakened response signal, which is directly associated with the amount of the miRNA target. Thus, highly sensitive quantification of miRNA-21 was achieved at a low limit of detection of 0.16 pM within 2 h of assay time. Assay specificity toward miRNA-21 was validated by testing several other miRNAs, including let-7b, let-7d, miRNA-141, and miRNA-200a. Moreover, the assay can quantify miRNA-21 spiked in human serum samples with acceptable recovery values, thus indicating its considerable clinical feasibility.

Rapid Enrichment and Ultrasensitive Detection of Influenza A Virus in Human Specimen using Magnetic Quantum Dot Nanobeads Based Test Strips.

Influenza A virus (IAV) possesses a high infectivity and pathogenicity, and can lead to severe respiratory infection with similar symptoms caused by some other common respiratory viruses. Lateral flow assay (LFA) has been widely deployed in remote settings as a rapid and reliable approach for point-of-care detection of infectious pathogens. However, it still remains challenging to detect IAV virions using LFA from clinical samples such as nasopharyngeal or throat swabs, because their various components and high viscosity can decrease flow velocity and lead to the nonspecific adsorption of nanoparticle labels on the sensing membrane. Herein, we demonstrated a magnetic quantum dot nanobeads (MQBs) based LFA for magnetic enrichment and fluorescent detection of IAV virions in clinical specimens. In this study, MQBs were synthesized and then conjugated with IAV-specific antibody to efficiently enrich IAV virions from complex biological matrix, but also serve as highly bright fluorescent probes in lateral flow strips. This assay can achieve quantitative detection of IAV virions with a low limit of detection down to 22 pfu mL-1 within 35 minutes, and show good specificity between influenza B virus and two adenovirus strains. Furthermore, the presented platform was able to directly detect IAV virions spiked in nasopharyngeal swab dilution, indicating its stability and feasibility in clinical applications. Thus, this point-of-care detection platform holds great promise as a broadly applicable approach for the rapid diagnosis of influenza A.

Magnetic quantum dot based lateral flow assay biosensor for multiplex and sensitive detection of protein toxins in food samples.

Protein toxins, such as botulinum neurotoxin type A (BoNT/A) and staphylococcal enterotoxin B (SEB), easily pollute food and water and are ultra-toxic to humans and animals, thus requiring a sensitive on-site detection method. In this study, we reported a novel lateral flow assay (LFA) strip on the basis of magnetic quantum dot nanoparticles (MagQD NPs) for sensitive and multiplex protein toxin detection in food samples. A new type of MagQD NP was prepared by fixing the dense carboxylated QDs on the surface of polyethyleneimine-modified Fe3O4 magnetic NPs (MNPs) and applied in LFA with the following functions: capture and enrich target toxins from sample solutions and serve as advanced fluorescent labels for the quantitative determination of targets on the strip. Through this strategy, the assay realized quantified BoNT/A and SEB detection in 30 min with the limits of detection of 2.52 and 2.86 pg/mL, respectively. The selectivity and the ability of quantitative analysis of the method were validated in real food samples, including milk and juice. This MagQD-LFA biosensor showed considerable potential as a point-of-care testing tool for the sensitive detection of trace toxins.

Dual-color magnetic-quantum dot nanobeads as versatile fluorescent probes in test strip for simultaneous point-of-care detection of free and complexed prostate-specific antigen.

Simultaneous detection of free and complexed prostate-specific antigen (f-PSA and c-PSA) is critical to the prostate cancer (PCa) diagnostic accuracy for clinical samples with PSA values in the diagnostic gray zone between 4 and 10 ng mL-1. Herein, red and green magnetic-quantum dot nanobeads (MQBs) with superior magnetic property and high luminescence were fabricated via polyethyleneimine-mediated electrostatic adsorption of numerous quantum dots onto superparamagnetic Fe3O4 magnetic cores, and were conjugated with f-PSA antibody and c-PSA antibody, respectively, as versatile fluorescent probes in test strip for immune recognition, magnetic enrichment, and simultaneous detection of f-PSA and c-PSA analytes in complex biological matrix with t-PSA antibody on the test line. A low-cost and portable smartphone readout device with an application was also developed for the imaging of dual-color test strips and data processing. This assay can simultaneously detect f-PSA and c-PSA with the limits of detection of 0.009 ng mL-1 and 0.087 ng mL-1, respectively. Clinical serum samples of PCa and benign prostatic hyperplasia patients were evaluated to confirm the clinical feasibility. The results suggest that the proposed dual-color MQBs-based fluorescent lateral flow immunoassay is a promising point-of-care diagnostics technique for the accurate diagnosis of PCa even in resource-limited settings.