Fluorescence-enhanced dual signal lateral flow immunoassay for flexible and ultrasensitive detection of monkeypox virus.

The outbreak of the monkeypox virus (MPXV) worldwide in 2022 highlights the need for a rapid and low-cost MPXV detection tool for effectively monitoring and controlling monkeypox disease. In this study, we developed a flexible lateral flow immunoassay (LFIA) with strong colorimetric and enhanced fluorescence dual-signal output for the rapid, on-site, and highly sensitive detection of the MPXV antigen in different scenarios. A multilayered SiO2-Au core dual-quantum dot (QD) shell nanocomposite (named SiO2-Au/DQD), which consists of a large SiO2 core (~ 200 nm), one layer of density-controlled gold nanoparticles (AuNPs, 20 nm), and thousands of small QDs, was fabricated instead of a traditional colorimetric nanotag (i.e., AuNPs) and a fluorescent nanotag (QD nanobead) to simultaneously provide good stability, strong colorimetric ability and superior fluorescence intensity. With the dual-signal output LFIA, we achieved the specific screening of the MPXV antigen (A29L) in 15 min, with detection limits of 0.5 and 0.0021 ng/mL for the colorimetric and fluorometric modes, respectively. Moreover, the colorimetric mode of SiO2-Au/DQD-LFIA exhibits the same sensitivity as the traditional AuNP- LFIA, whereas the overall sensitivity of this method on the basis of the fluorescent signal can achieve 238- and 3.3-fold improvements in sensitivity for MPXV compared with the AuNP-based LFIA and ELISA methods, respectively, indicating the powerful performance and good versatility of the dual-signal method in the point-of-care testing of the MPXV.

Development of a Magnetically-Assisted SERS Biosensor for Rapid Bacterial Detection.

Introduction: Ultrasensitive bacterial detection methods are crucial to ensuring accurate diagnosis and effective clinical monitoring, given the significant threat bacterial infections pose to human health. The aim of this study is to develop a biosensor with capabilities for broad-spectrum bacterial detection, rapid processing, and cost-effectiveness. Methods: A magnetically-assisted SERS biosensor was designed, employing wheat germ agglutinin (WGA) for broad-spectrum recognition and antibodies for specific capture. Gold nanostars (AuNSs) were sequentially modified with the Raman reporter molecules and WGA, creating a versatile SERS tag with high affinity for a diverse range of bacteria. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) antibody-modified Fe3O4 magnetic gold nanoparticles (MGNPs) served as the capture probes. Target bacteria were captured by MGNPs and combined with SERS tags, forming a "sandwich" composite structure for bacterial detection. Results: AuNSs, with a core size of 65 nm, exhibited excellent storage stability (RSD=5.6%) and demonstrated superior SERS enhancement compared to colloidal gold nanoparticles. Efficient binding of S. aureus and P. aeruginosa to MGNPs resulted in capture efficiencies of 89.13% and 85.31%, respectively. Under optimized conditions, the developed assay achieved a limit of detection (LOD) of 7 CFU/mL for S. aureus and 5 CFU/mL for P. aeruginosa. The bacterial concentration (10-106 CFU/mL) showed a strong linear correlation with the SERS intensity at 1331 cm-1. Additionally, high recoveries (84.8% - 118.0%) and low RSD (6.21% - 11.42%) were observed in spiked human urine samples. Conclusion: This study introduces a simple and innovative magnetically-assisted SERS biosensor for the sensitive and quantitative detection of S. aureus or P. aeruginosa, utilizing WGA and antibodies. The developed biosensor enhances the capabilities of the "sandwich" type SERS biosensor, offering a novel and effective platform for accurate and timely clinical diagnosis of bacterial infections.

Graphene oxide-based colorimetric/fluorescence dual-mode immunochromatography assay for simultaneous ultrasensitive detection of respiratory virus and bacteria in complex samples.

A point-of-care testing biosensor that supports direct, sensitive, and simultaneous identification of bacteria and virus is still lacking. In this study, an ultrasensitive immunochromatography assay (ICA) with colorimetric/fluorescence dual-signal output was proposed for flexible and accurate detection of respiratory virus and bacteria in complex samples. Colorimetric AuNPs of 16 nm and two layers of quantum dots (QDs) were coated onto the surface of monolayer graphene oxide (GO) layer by layer to form a multilayered dual-signal nanofilm. This material not only can generate strong colorimetric and fluorescence signals for ICA analysis but also can provide larger surface area, better stability, and superior dispersibility than conventional spherical nanomaterials. Two test lines were built onto the ICA strip to simultaneously detect common respiratory virus influenza A and respiratory bacteria Streptococcus pneumoniae. The dual-signal mode of assay greatly broadened the applied range of ICA method, in which the colorimetric mode allows for quick determination of virus/bacteria and the fluorescence mode ensures the highly sensitive and quantitative detection of target pathogens with detection limits down to 891 copies/mL and 17 cells/mL, respectively. The proposed dual-mode ICA can also be applied directly for real biological and environment samples, which suggests its great potential for field application.

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.

Universal and ultrasensitive detection of foodborne bacteria on a lateral flow assay strip by using wheat germ agglutinin-modified magnetic SERS nanotags.

Rapid, direct and sensitive detection of foodborne bacteria in complex samples is still challenging. Here, we reported a universal surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) for highly sensitive detection of foodborne bacteria in food and environmental samples using wheat germ agglutinin (WGA)-modified Fe3O4@Au (Au@MNP-WGA) nanotags. The Au@MNP-WGA tag with numerous intraparticle hotspots was integrated into the LFA system for the first time, which can not only greatly improve the detection sensitivity through the dual amplification effect of magnetic enrichment and SERS enhancement but also achieve the broad-spectrum capture of multiple bacteria. In addition, monoclonal antibodies were separately immobilized onto the test line of different LFA strips to ensure the specific detection of different target pathogens. With this strategy, the proposed assay can achieve the universal and highly sensitive determination of three common foodborne bacteria, namely, Listeria monocytogenes, Campylobacter jejuni, and Staphylococcus aureus, with low detection limit (10 cells mL-1), short testing time (<35 min), and high reproducibility (RSD < 8.14%). Given its good stability and accuracy in complex samples, the Au@MNP-WGA-based SERS-LFA has great potential to be a powerful tool for the universal and on-site detection of different foodborne pathogens.

Ultrasensitive and multiplex detection of four pathogenic bacteria on a bi-channel lateral flow immunoassay strip with three-dimensional membrane-like SERS nanostickers.

A lateral flow immunoassay (LFA) technique for sensitive and multiplexed on-site detection of bacteria remains a challenge. Here, we develop a bi-channel surface-enhanced Raman scattering (SERS)-based LFA by using three-dimensional membrane-like SERS tags as nanostickers (named GO@Au/Ag) for direct and ultrasensitive analysis of multiple pathogens in a single test. The grafting of numerous Ag satellites onto nanosticker significantly increased the relative surface area for bacteria binding and generated efficient SERS hotspots over large area to improve the sensing sensitivity. Antibody-labeled GO@Au/Ag nanostickers can rapidly stick onto the target bacteria and generate superior SERS signals and fluidity on the paper strip, thus conquering the adverse effect of bacteria size and improving the multiplex analysis ability of LFA. The integration of two different Raman reporter molecules into nanostickers allows simultaneous detection of four pathogens on two test lines, which significantly simplifies the reading process of SERS signals. The proposed biosensor can quantitatively detect four different bacteria in real clinical samples with low detection limit (9 cells mL-1 level), short assay time (20 min), high accuracy and excellent stability, indicating its great application potential for on-site detection of pathogens.

Recombinase Polymerase Amplification Combined with Fluorescence Immunochromatography Assay for On-Site and Ultrasensitive Detection of SARS-CoV-2.

This study established a portable and ultrasensitive detection method based on recombinase polymerase amplification (RPA) combined with high-sensitivity multilayer quantum dot (MQD)-based immunochromatographic assay (ICA) to detect the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The RPA-MQD-based ICA method is reported for the first time and has the following advantages: (i) RPA is free from the constraints of instruments and can be promoted in point-of-care testing (POCT) scenarios, (ii) fluorescence ICA enhances the portability of detection operation so that the entire operation time is controlled within 1 h, and (iii) compared with common colorimetric-based RPA-ICA, the proposed assay used MQD to provide strong and quantifiable fluorescence signal, thus enhancing the detection sensitivity. With this strategy, the proposed RPA-MQD-based ICA can amplify and detect the SARS-CoV-2 nucleic acid on-site with a sensitivity of 2 copies/reaction, which is comparable to the sensitivity of commercial reverse transcription quantitative polymerase chain reaction (RT-qPCR) kits. Moreover, the designed primers did not cross-react with other common respiratory viruses, including adenovirus, influenza virus A, and influenza virus B, suggesting high specificity. Thus, the established portable method can sensitively detect SARS-CoV-2 nucleic acid without relying on equipment, having good application prospects in SARS-CoV-2 detection scenarios under non-lab conditions.

Ultrasensitive Fluorescence Lateral Flow Assay for Simultaneous Detection of Pseudomonas aeruginosa and Salmonella typhimurium via Wheat Germ Agglutinin-Functionalized Magnetic Quantum Dot Nanoprobe.

Point-of-care testing methods for the rapid and sensitive screening of pathogenic bacteria are urgently needed because of the high number of outbreaks of microbial infections and foodborne diseases. In this study, we developed a highly sensitive and multiplex lateral flow assay (LFA) for the simultaneous detection of Pseudomonas aeruginosa and Salmonella typhimurium in complex samples by using wheat germ agglutinin (WGA)-modified magnetic quantum dots (Mag@QDs) as a universal detection nanoprobe. The Mag@QDs-WGA tag with a 200 nm Fe3O4 core and multiple QD-formed shell was introduced into the LFA biosensor for the universal capture of the two target bacteria and provided the dual amplification effect of fluorescence enhancement and magnetic enrichment for ultra-sensitivity detection. Meanwhile, two antibacterial antibodies were separately sprayed onto the two test lines of the LFA strip to ensure the specific identification of P. aeruginosa and S. typhimurium through one test. The proposed LFA exhibited excellent analytical performance, including high capture rate (>80%) to the target pathogens, low detection limit (<30 cells/mL), short testing time (<35 min), and good reproducibility (relative standard deviation < 10.4%). Given these merits, the Mag@QDs-WGA-based LFA has a great potential for the on-site and real-time diagnosis of bacterial samples.

An efficient SERS platform for the ultrasensitive detection of Staphylococcus aureus and Listeria monocytogenes via wheat germ agglutinin-modified magnetic SERS substrate and streptavidin/aptamer co-functionalized SERS tags.

A novel surface-enhanced Raman scattering (SERS)-based analytical technique was proposed to simultaneously detect two highly pathogenic bacteria, namely, Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. mono) by using a dual-recognition pattern with wheat germ agglutinin (WGA) and nucleic acid aptamers. WGA was modified onto Fe3O4@Au magnetic nanoparticles (MNPs) for the efficient capture of S. aureus and L. mono in complex samples (orange juice, extracts of lettuce, and human urine) within 15 min. The streptavidin (SA)/aptamers co-functionalized SERS tags were fabricated by covalent attaching two different Raman reporters and SA molecules onto 45 nm Au NPs and then conjugated with two biotin-aptamers that specifically bind to their target bacteria with high affinity and stability. The combined use of high-sensitive SERS tags, WGA-mediated magnetic enrichment, and SA-mediated aptamer conjugation remarkably improved the assay sensitivity. Under optimized conditions, the developed SERS biosensor can simultaneously detect the two target bacteria with high detection sensitivity (<6 cells/mL), favorable linear relation (10-107 cells/mL), and high accuracy (recovery rate <7.03%). Therefore, the proposed SERS platform is rapid, sensitive, easy to use, and thus show potential as a tool for the timely identification of pathogenic bacteria in real samples.