SERS/photothermal-based dual-modal lateral flow immunoassays for sensitive and simultaneous antigen detection of respiratory viral infections

Lateral flow immunoassay (LFIA) is one of the most common analytical platforms for point-of-care testing (POCT), which is capable of large-scale primary screening and home self-testing of infectious diseases. However, the sensitivity of conventional AuNPs-based LFIA is relatively low and more prone to false negatives. Herein, we report a novel LFIA based on gold-core-silver-shell bimetallic nanoparticles (Au4-ATP@Ag NPs) emitting Surface-enhanced Raman scatting (SERS) and Photothermal (PT) effect, named SERS/PT-based dual-modal LFIA (SERS/PT-dmLFIA), for the antigen detection of infectious diseases pathogens, which displayed an excellent performance. For influenza A virus (IAV), influenza B virus (IBV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) N protein detection, the limit of detections (LoD) with Raman as signal were 31.25, 93.75, and 31.25 pg mL-1 respectively, and the LoDs with temperature difference (∆T) as signal were as low as 15.63, 187.5, and 15.63 pg mL-1 respectively, which were over 4-fold more sensitive than visual-based LFIA. The proposed SERS/PT-dmLFIA was used for detecting virus antigen in pharyngeal swabs and showed ideal coincidence rate of over 95% compared to the commercialized assays. In addition, we explored the development of multiplex SERS/PT-dmLFIA that can detect IAV, IBV, and SARS-CoV-2 antigens simultaneously without cross reactivity. Overall, the SERS/PT-dmLFIA for antigen detection not only exhibits high sensitivity, accuracy and specificity, but also have characteristics of rapidity and simplicity, which holds high potential for rapid diagnosis of infectious diseases in laboratory testing, mass screening, and home self-testing. © 2023 Elsevier B.V.

INTEGRATION OF MINIVALVES WITH RNA AMPLIFICATION DEVICE FOR SIMULTANEOUS DETECTION OF SARS-COV-2 AND INFLUENZA VIRUSES

We report a point-of-care (POC) device for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A viruses. The device carries out sample preparation using ball-based valves for sequential delivery of reagents. A microfluidic paper-based analytical device (µPAD) in the detection unit enables RNA isolation and enrichment, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and colorimetric detection. The device integrates all the necessary steps for the sample preparation, including virus lysis, RNA enrichment and purification of two virus samples. The device enabled simultaneous detection of SARS-CoV-2 and Influenza A N1H1 viruses in 50 min., with limit of detection of 2 and 6 genome equivalents (GEs), respectively. The device was also capable of detecting environmental sample of the two viruses. Copyright © 2022 by ASME.

Introduction of graphene oxide-supported multilayer-quantum dots nanofilm into multiplex lateral flow immunoassay: A rapid and ultrasensitive point-of-care testing technique for multiple respiratory viruses.

A lateral flow immunoassay (LFA) biosensor that allows the sensitive and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses remains highly desired in the face of the coronavirus disease 2019 pandemic. Here, we propose a multiplex LFA method for the on-site, rapid, and highly sensitive screening of multiple respiratory viruses, using a multilayered film-like fluorescent tag as the performance enhancement and signal amplification tool. This film-like three-dimensional (3D) tag was prepared through the layer-by-layer assembly of highly photostable CdSe@ZnS-COOH quantum dots (QDs) onto the surfaces of monolayer graphene oxide nanosheets, which can provide larger reaction interfaces and specific active surface areas, higher QD loads, and better luminescence and dispersibility than traditional spherical fluorescent microspheres for LFA applications. The constructed fluorescent LFA biosensor can simultaneously and sensitively quantify SARS-CoV-2, influenza A virus, and human adenovirus with low detection limits (8 pg/mL, 488 copies/mL, and 471 copies/mL), short assay time (15 min), good reproducibility, and high accuracy. Moreover, our proposed assay has great potential for the early diagnosis of respiratory virus infections given its robustness when validated in real saliva samples. Electronic Supplementary Material: Supplementary material (Section S1 Experimental section, Section S2 Calculation of the maximum number of QDs on the GO@TQD nanofilm, Section S3 Optimization of the LFA method, and Figs. S1-S17 mentioned in the main text) is available in the online version of this article at 10.1007/s12274-022-5043-6.

Electrochemical detection of Zika and Dengue infections using a single chip.

Zika and Dengue are infectious diseases caused by flaviviruses and transmitted by Aedes mosquitoes. Although symptoms are usually mild, complications such as dengue hemorrhagic fever and microcephaly in newborns -after the pregnant woman becomes infected with the Zika virus-have emerged as a global public health concern. The co-circulation of Zika and Dengue viruses and the overlapping of their symptoms represent a challenge for the accurate diagnosis. A single test for the point-of-care detection of both diseases is crucial. Here we report a single chip that distinguishes between Zika and Dengue infections using the non-structural protein 1 (NS1) as biomarkers. A novel multiplex electrochemical device containing four independent working electrodes was developed. Zika and Dengue biosensors were fabricated separately on different working electrodes. Selectivity tests showed that the two biosensors can distinguish not only the NS1 proteins from Zika and Dengue but also the spike proteins present in the SARS-CoV-2. This is especially relevant as patients with COVID-19 may have symptoms similar to Zika and Dengue. The gold surface was modified with cysteamine and antibodies against the NS1 proteins. Both biosensors detected their respective biomarkers at clinically relevant concentrations and presented a good linear relationship between the percentage change in impedance and the logarithm of the NS1 concentration (R2 = 0.990 for Dengue and R2 = 0.995 for Zika). Upon combining a simple sample preparation with a portable detection method, our disposable multiplex device offers a point-of-care diagnostic test for Zika and Dengue using a single chip. Additionally, two other biosensors can be added to the chip, providing a platform for viral detection.

Simultaneously ultrasensitive and quantitative detection of influenza A virus, SARS-CoV-2, and respiratory syncytial virus via multichannel magnetic SERS-based lateral flow immunoassay.

Respiratory viruses usually induced similar clinical symptoms at early infection. Herein, we presented a multichannel surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-based LFA) using high-performance magnetic SERS tags for the simultaneous ultrasensitive detection of respiratory viruses, namely influenza A virus (H1N1), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) in biological samples. As-prepared magnetic SERS tags can directly enrich and capture target viruses without pretreatment of samples, avoiding the interference of impurities in the samples as well as improving the sensitivity. With the capture-detection method, the detection limits of the proposed assay reached 85 copies mL-1, 8 pg mL-1, and 8 pg mL-1 for H1N1, SARS-CoV-2 and RSV, respectively. Moreover, the detection properties of the proposed method for target viruses in throat swab samples were verified, suggesting its remarkable potential for the early and rapid differential diagnosis of respiratory viruses.

Two Multiplex PCR Methods for Detecting Several Pathogens Associated with Feline Respiratory and Intestinal Tracts.

Respiratory tract and intestinal diseases are common threats to feline health. Coinfection with multiple pathogens is not rare among clinical infectious cases. Rapid diagnosis of these coinfections is of great significance for timely and effective clinical treatment. In this study, two novel multiplex polymerase chain reactions (mPCRs) were established for simultaneous detection of four pathogens associated with the feline intestinal tract (feline coronavirus (FCoV), feline astrovirus (FeAstV), feline panleukopenia virus (FPV) and feline kobuvirus (FeKoV)) and five pathogens associated with the respiratory tract (feline calicivirus (FCV), feline herpesvirus 1 (FHV-1), feline leukemia virus (FeLV), Chlamydia felis (C. felis) and influenza A virus (IAV)). The results of sensitivity analysis revealed that the detection limits for FeKoV, FPV, FeAstV, FCoV, IAV, C. felis, FeLV, FHV-1 and FCV were 103, 104, 103, 103, 103, 104, 104, 105 and 105 copies/µL, respectively. Moreover, the specificity of the two mPCRs was high. When the two mPCRs were applied to clinical samples, the assay worked well. In conclusion, we established two mPCR methods that provide an excellent tool for the diagnosis and monitoring of pathogens associated with the feline respiratory and intestinal tracts.

Simultaneous detection and quantification of spike mRNA and protein in SARS-CoV-2 infected airway epithelium.

Visualizing and quantifying mRNA and its corresponding protein provides a unique perspective of gene expression at a single-molecule level. Here, we describe a method for differentiating primary cells for making airway epithelium and detecting SARS-CoV-2 Spike (S) mRNA and S protein in the paraformaldehyde-fixed paraffin-embedded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected airway epithelium. For simultaneous detection of mRNA and protein in the same cell, we combined two protocols: 1. RNA fluorescence-based in situ hybridization (RNA-FISH) based mRNA detection and 2. fluorescence-based immunohistochemistry (IHC) based protein detection. The detection of mRNA and proteins in the same cell also allows for quantifying them using the open-source software QuPath, which provides an accurate and more straightforward fluorescent-based quantification of mRNA and protein in the microscopic images of the infected cells. Additionally, we can achieve the subcellular distribution of both S mRNA and S protein. This method identifies SARS-CoV-2 S gene products' (mRNA and protein) degree of expression and their subcellular localization in the infected airway epithelium. Advantages of this method include: •Simultaneous detection and quantification of mRNA and protein in the same cell.•Universal use due to the ability to use mRNA-specific primer-probe and protein-specific antibodies.•An open-source software QuPath provides a straightforward fluorescent-based quantification.

VALVE-ENABLED SEQUENTIAL REAGENT DELIVERY AND PAPER-BASED ENRICHMENT FOR SIMULTANEOUS DETECTION OF SARS-COV-2 AND INFLUENZA VIRUSES

We report a point-of-care (POC) testing platform for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus. The POC device integrates sample preparation using ball-based valves for sequential delivery of reagents, viral RNA isolation and enrichment by paper-based filtration, with reverse transcription loop-mediated isothermal amplification (RT-LAMP) and colorimetric detection. The device is capable of detecting both viruses, showing high sensitivity and specificity. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Development of a nano-bioplatform for SARS-CoV-2 specific antigens detection

Lateral Flow Immunoassay (LFIA) has been employed for the development of rapid, low-cost, and relatively simple devices for Covid-19 diagnosis. The proposed approach reports the use of the antibody labeled by Gold nanoparticles for the simultaneous detection of Nucleocapsid protein and Spike protein. A comparison with the results obtained by conventional analytical methods (PCR) has been considered in a significant number of nasopharyngeal swabs;48 and 26 samples from positive and negative individuals respectively. This research provides the basis for the development of a more efficient antigenic test for SARS-Co V-2 detection as a low-cost and quick pre-screening tool. © 2022 IEEE.

Ultrasensitive and Simultaneous Detection of Two Specific SARS-CoV-2 Antigens in Human Specimens Using Direct/Enrichment Dual-Mode Fluorescence Lateral Flow Immunoassay.

Sensitive point-of-care methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens are urgently needed to achieve rapid screening of viral infection. We developed a magnetic quantum dot-based dual-mode lateral flow immunoassay (LFIA) biosensor for the high-sensitivity simultaneous detection of SARS-CoV-2 spike (S) and nucleocapsid protein (NP) antigens, which is beneficial for improving the detection accuracy and efficiency of SARS-CoV-2 infection in the point-of-care testing area. A high-performance magnetic quantum dot with a triple-QD shell (MagTQD) nanotag was first fabricated and integrated into the LFIA system to provide superior fluorescence signals, enrichment ability, and detectability for S/NP antigen testing. Two detection modes were provided by the proposed MagTQD-LFIA. The direct mode was used for rapid screening or urgent detection of suspected samples within 10 min, and the enrichment mode was used for the highly sensitive and quantitative analysis of SARS-CoV-2 antigens in biological samples without the interference of the "hook effect." The simultaneous detection of SARS-CoV-2 S/NP antigens was conducted in one LFIA strip, and the detection limits for two antigens under direct and enrichment modes were 1 and 0.5 pg/mL, respectively. The MagTQD-LFIA showed high accuracy, specificity, and stability in saliva and nasal swab samples and is an efficient tool with flexibility to meet the testing requirements for SARS-CoV-2 antigens in various situations.

Simultaneous detection of SARS-CoV-2 and pandemic (H1N1) 2009 virus with real-time isothermal platform.

The recent ongoing outbreak of novel coronavirus SARS-CoV-2 (known as COVID-19) is a severe threat to human health worldwide. By press time, more than 3.3 million people have died from COVID-19, with many countries experiencing peaks in infections and hospitalizations. The main symptoms of infection with SARS-CoV-2 include fever, chills, coughing, shortness of breath or difficulty breathing, fatigue, muscle or body aches and pains. While the symptoms of the pandemic (H1N1) 2009 virus have many similarities to the signs and transmission routes of the novel coronavirus, e.g., fever, cough, sore throat, body aches, headache, chills and fatigue. And a few cases of serious illness, rapid progress, can appear viral pneumonia, combined with respiratory failure, multiple organ function damage, serious people can die. Therefore, there is an urgent need to develop a rapid and accurate field diagnostic method to effectively identify the two viruses and treat these early infections on time, thus helping to control the spread of the disease. Among molecular detection methods, RT-LAMP (real-time reverse transcription-loop-mediated isothermal amplification) has some advantages in pathogen detection due to its rapid, accurate and effective detection characteristics. Here, we combined the primers of the two viruses with the fluorescent probes on the RT-LAMP detection platform to detect the two viruses simultaneously. Firstly, RT-LAMP method was used respectively to detect the two viruses at different concentrations to determine the effectiveness and sensitivity of probe primers to the RNA samples. And then, the two virus samples were detected simultaneously in the same reaction tube to validate if testing for the two viruses together had an impact on the results compared to detecting alone. We verified the detection efficiency of three highly active BST variants during RT-LAMP assay. We expect that this assay can effectively and accurately distinguish COVID-19 from the pandemic (H1N1) 2009, so that these two diseases with similar symptoms can be appropriately differentiated and treated.

Development of an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobead for simultaneous detection of SARS-CoV-2 antigen and influenza A virus.

Rapid and accurate diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (FluA) antigens in the early stages of virus infection is the key to control the epidemic spread. Here, we developed a two-channel fluorescent immunochromatographic assay (ICA) for ultrasensitive and simultaneous qualification of the two viruses in biological samples. A high-performance quantum dot nanobead (QB) was fabricated by adsorption of multilayers of dense quantum dots (QDs) onto the SiO2 surface and used as the highly luminescent label of the ICA system to ensure the high-sensitivity and stability of the assay. The combination of monodispersed SiO2 core (∼180 nm) and numerous carboxylated QDs formed a hierarchical shell, which ensured that the QBs possessed excellent stability, superior fluorescence signal, and convenient surface functionalization. The developed ICA biosensor achieved simultaneous detection of SARS-CoV-2 and FluA in one test within 15 min, with detection limits reaching 5 pg/mL for SARS-CoV-2 antigen and 50 pfu/mL for FluA H1N1. Moreover, our method showed high accuracy and specificity in throat swab samples with two orders of magnitude improvement in sensitivity compared with traditional AuNP-based ICA method. Hence, the proposed method is a promising and convenient tool for detection of respiratory viruses.

Assay System for Simultaneous Detection of SARS-CoV-2 and Other Respiratory Viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers disease with nonspecific symptoms that overlap those of infections caused by other seasonal respiratory viruses (RVs), such as the influenza virus (Flu) or respiratory syncytial virus (RSV). A molecular assay for accurate and rapid detection of RV and SARS-CoV-2 is crucial to manage these infections. Here, we compared the analytical performance and clinical reliability of Allplex™ SARS-CoV-2/FluA/FluB/RSV (SC2FabR; Seegene Inc., Seoul, South Korea) kit with those of four commercially available RV detection kits. Upon testing five target viral strains (SARS-CoV-2, FluA, FluB, RSV A, and RSV B), the analytical performance of SC2FabR was similar to that of the other kits, with no significant difference (p ≥ 0.78) in z-scores. The efficiency of SC2FabR (E-value, 81-104%) enabled reliable SARS-CoV-2 and seasonal RV detection in 888 nasopharyngeal swab specimens processed using a fully automated nucleic acid extraction platform. Bland-Altman analyses revealed an agreement value of 95.4% (SD ± 1.96) for the kits, indicating statistically similar results for all five. In conclusion, SC2FabR is a rapid and accurate diagnostic tool for both SARS-CoV-2 and seasonal RV detection, allowing for high-throughput RV analysis with efficiency comparable to that of commercially available kits. This can be used to help manage respiratory infections in patients during and after the coronavirus disease 2019 pandemic.

The next-generation coronavirus diagnostic techniques with particular emphasis on the SARS-CoV-2.

The potential zoonotic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2) are of global health concerns. Early diagnosis is the milestone in their mitigation, control, and eradication. Many diagnostic techniques are showing great success and have many advantages, such as the rapid turnover of the results, high accuracy, and high specificity and sensitivity. However, some of these techniques have several pitfalls if samples were not collected, processed, and transported in the standard ways and if these techniques were not practiced with extreme caution and precision. This may lead to false-negative/positive results. This may affect the downstream management of the affected cases. These techniques require regular fine-tuning, upgrading, and optimization. The continuous evolution of new strains and viruses belong to the coronaviruses is hampering the success of many classical techniques. There are urgent needs for next generations of coronaviruses diagnostic assays that overcome these pitfalls. This new generation of diagnostic tests should be able to do simultaneous, multiplex, and high-throughput detection of various coronavirus in one reaction. Furthermore, the development of novel assays and techniques that enable the in situ detection of the virus on the environmental samples, especially air, water, and surfaces, should be given considerable attention in the future. These approaches will have a substantial positive impact on the mitigation and eradication of coronaviruses, including the current SARS-CoV-2 pandemic.

Novel dual multiplex real-time RT-PCR assays for the rapid detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus using the BD MAX open system.

SARS-CoV-2 has spread rapidly, causing deaths worldwide. In this study, we evaluated the performance of the BD MAX Open System module for identifying viral pathogens, including SARS-CoV-2, in nasopharyngeal specimens from individuals with symptoms of upper respiratory tract infection. We developed and validated a rapid total nucleic acid extraction method based on real-time reverse transcription-polymerase chain reaction (RT-PCR) for the reliable, high-throughput simultaneous detection of common cold viral pathogens using the BD MAX Platform. The system was evaluated using 205 nasopharyngeal swab clinical samples. For assessment of the limit of detection (LoD), we used SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) RNA standards. The BD MAX dual multiplex real-time RT-PCR panel demonstrated a sensitivity comparable to that of the World Health Organization-recommended SARS-CoV-2 assay with an LoD of 50 copies/PCR. The LoD of influenza A/B and RSV was 100-200 copies/PCR. The overall percent agreement between the BD MAX panel and laboratory-developed RT-PCR test on 55 SARS-CoV-2-positive clinical samples was 100%. Among the 55 positive cases of COVID-19 analysed, no coinfection was detected. The BD MAX rapid multiplex PCR provides a highly sensitive, robust, and accurate assay for the rapid detection of SARS-CoV-2, influenza A/B, and RSV.

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.