Atomic-shell engineering in manganese-doped palladium@iridium nanozymes: d-Band optimization for enhanced peroxidase-like activity in ultrasensitive lateral flow immunoassays

Authors report on a carbon nanotube (CNT)-based lateral flow immunoassay (LFI) for ultrasensitive detection of proteins. Shortened multiwalled CNTs were used as a colored (black) tag. The detection antibody was covalently immobilized on the CNT surface via diimide-activated conjugation between the carboxyl groups on the CNT surface and amino groups of antibodies. The assay involved the capture of target protein in a sandwich-type format between an immobilized capture antibody on the test zone of LFI and a CNT-labelled detection antibody. CNTs were thus captured on the test zone of the LFI and gave a black colored line to enable visual detection of protein. Quantitative results were obtained by reading the test line intensities with a portable strip reader. Rabbit IgG was used as a model target to demonstrate the proof-of-concept. Combining the advantages of lateral flow assay with the unique physical properties of CNT (color, high aspect-to-size ratio and ease of surface modification), the optimized LFI can detect of 1.3pgmL-1 of rabbit IgG (S/N = 3). This is three orders lower than that of gold nanoparticle-based LFI. Rabbit IgG spiked into human plasma samples was successfully detected with this LFI. Conceivably, this method can be extended to various other proteins for which adequate antibodies do exist. Graphical abstract Carbon nanotubes are used as black tags in an ultrasensitive lateral flow immunoassay (LFI). The LFIwas applied to the determination of rabbit IgG. The detection limit is more than 3 orders of magnitudelower than that of the conventional gold nanopaticle-based LFI.

Clostridium difficileis one of the primary causative agents of nosocomial antibiotic-associated diseases. Early detection and prevention are effective strategies to curb disease transmission, which often requires highly sensitive point-of-care testing (POCT) methods. Although lateral flow immunoassay (LFIA) technology-known for its convenience, speed, and cost-effectiveness-has gained prominence in POCT, its application in early screening remains limited due to relatively low sensitivity. Herein, a two-stage cascading enhancement strategy based on reductase-like and peroxidase-like activities of nanozyme to construct a colorimetrically enhanced LFIA platform is proposed. Specifically, Zn2+-doped WOX (Zn/WOX) loaded with Au nanoparticles (Zn/WOX@Au) first generates an initial colorimetric signal through immunochromatography. Subsequently, in situ light-induced deposition of Pt creates Zn/WOX@Au@Pt, which exhibits enhanced peroxidase-like activity based on optimal band structure between the components for colorimetric signal amplification. As a result, a 500-fold enhancement in the visual limit of detection (LOD) is achieved. Moreover, the fitted LOD based on grayscale analysis forClostridium difficiletoxin B (Tcd B) reaches 0.01ngmL-1. This strategy not only maintains the operational simplicity of LFIA but also considerably strengthens the colorimetric signal, offering a novel approach for highly sensitive colorimetric-enhanced LFIA detection.

Pt-Ru bimetallic nanoclusters with super peroxidase-like activity for ultra-sensitive lateral flow immunoassay

Developing ultrasensitive lateral flow immunoassays (LFIAs) has garnered significant attention in the field of point-of-care testing. In this study, a trimetallic dendritic nanozyme (Pd@Pt-Ru) was synthesized through Ru deposition on a Pd@Pt core and utilized to enhancing the sensitivity of LFIAs. Pd@Pt-Ru exhibited a Km value of 5.23 mM for detecting H2O2, which indicates an H2O2 affinity comparable with that of horseradish peroxidase. The Ru surface layer reduces the activation energy barrier, which increases the maximum reaction rate. As a proof of concept, the proposed Pd@Pt-Ru nanozyme was incorporated into LFIAs (A-Pd@Pt-Ru-LFIAs) for detecting human chorionic gonadotropin (hCG). Compared with conventional gold nanoparticle (AuNP)-LFIAs, A-Pd@Pt-Ru-LFIAs demonstrated 250-fold increased sensitivity, thereby enabling a visible detection limit as low as 0.1 IU/L. True positive and negative rates both reached 100%, which renders the proposed Pd@Pt-Ru nanozyme suitable for detecting hCG in clinical samples.

Herein, an ultrasensitive lateral flow immunoassay (LFIA), based on metal-organic framework-decorated polydopamine (PCN-224@PDA) was first established to detect multiple sulfonylureas (SUs) in functional foods. The PCN-224@PDA was synthesized using the one-pot hydrothermal method and covalently coupled with SUs antibodies, and the coupling rate was up to 91.8%. The detection limits of the developed PCN-224@PDA-LFIA for multiple SUs in functional teas and capsules were 0.22-3.72 μg/kg and 0.40-3.71 μg/kg, and quantification limits were 0.75-8.19 μg/kg and 1.03-9.08 μg/kg, respectively. The analytical sensitivity was 128-fold higher than that of similar methods reported so far. The recovery rates ranged from 83.8 to 119.0%, with coefficients of variation of 7.6-14.4%. The parallel analysis of 20 real samples by LC-MS/MS confirmed the reliability of the proposed method. Therefore, our work offers novel, ultrasensitive, and rapid technical support for on-site monitoring of SUs in functional foods.

The coronavirus disease 2019 (COVID-19) pandemic highlighted the need for rapid and accurate viral detection at the point-of-care testing (POCT). Compared with nucleic acid detection, lateral flow immunoassay (LFIA) is a rapid and flexible method for POCT detection. However, the sensitivity of LFIA limits its use for early identification of patients with COVID-19. Here, an innovative surface-enhanced Raman scattering (SERS)-LFIA platform based on two-dimensional black phosphorus decorated with Ag nanoparticles as important antigen-capturing and Raman-signal-amplification unit was developed for detection of SARS-CoV-2 variants within 5-20 min. The novel SERS-LFIA platform realized a limit of detection of 0.5 pg/mL and 100 copies/mL for N protein and SARS-CoV-2, demonstrating 1000 times more sensitivity than the commercial LFIA strips. It could reliably detect seven different SARS-CoV-2 variants with cycle threshold (Ct) < 38, with sensitivity and specificity of 97 and 100%, respectively, exhibiting the same sensitivity with q-PCR. Furthermore, the detection results for 48 SARS-CoV-2-positive nasopharyngeal swabs (Ct = 19.8-38.95) and 96 negative nasopharyngeal swabs proved the reliability of the strips in clinical application. The method also had good specificity in double-blind experiments involving several other coronaviruses, respiratory viruses, and respiratory medications. The results showed that the innovative SERS-LFIA platform is expected to be the next-generation antigen detection technology. The inexpensive amplification-free assay combines the advantages of rapid low-cost POCT and highly sensitive nucleic acid detection, and it is suitable for rapid detection of SARS-CoV-2 variants and other pathogens. Thus, it could replace existing antigens and nucleic acids to some extent.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy

Lateral flow immunoassays (LFIAs) are popular because they are rapid, convenient, stable, low cost, and easy to read. However, conventional LFIAs based on gold nanoparticles lack sensitivity, which hinders their widespread use. Here, we prepared durian-like gold nanoparticles (GNDs) and labeled them with staphylococcal protein A to detect brucella antibody. Then, the analytical performances of GNDs and gold nanospheres (GNSs) with the same diameter were compared. It was found that the sensitivity of GNDs was five to ten times higher than that of GNSs. The nonspherical morphologies of the nanoparticles greatly increased the sensitivity of the LFIA. On the basis of GNDs and GNSs, we developed an ultrasensitive dual-color brucellosis LFIA. GNSs labeled with streptavidin were used to demonstrate the control line. This dual-color LFIA had a diagnostic sensitivity and specificity of 100%. Human standard Brucella-positive serum (containing brucella antibody at 4000 IU/mL) could be detected in this system even for a dilution factor of 10-5. The detection limit was 0.04 IU/mL. This is two orders of magnitude better than conventional LFIA strips (detection limit 4 IU/mL). This dual-color LFIA contains all components of a conventional LFIA with no additional processing steps or reagents. It can detect antibodies in serum, plasma, and even whole blood without sample pretreatment or blood filtration pads. Both types of nanoparticles were synthesized in a simple and low-cost manner. This suggests that it will have utility for the early diagnosis of brucellosis and other diseases. Graphical abstract.

A dual-mode lateral flow immunoassay by ultrahigh signal-to background ratio SERS probes for nitrofurazone metabolites ultrasensitive detection

An ultrasensitive lateral flow immunoassay platform for foodborne biotoxins and pathogenic bacteria based on carbon-dots embedded mesoporous silicon nanoparticles fluorescent reporter probes

Lateral flow immunoassay (LFIA) based on fluorescent microbeads has attracted much attention for its use in rapid and accurate food safety monitoring. However, conventional fluorescent microbeads are limited by the aggregation-caused quenching effect of the loaded fluorophores, thus resulting in low signal intensity and insufficient sensitivity of fluorescent LFIA. In this study, a green-emitting fluorophore with an aggregation-induced emission (AIE) characteristic was encapsulated in polymer nanoparticles via an emulsification technique to form ultrabright fluorescent microbeads (denoted as AIEMBs). The prepared AIEMBs were then applied in a competitive LFIA (AIE-LFIA) as signal reporters for the rapid and highly sensitive screening of fumonisin B1 (FB1) in real corn samples. High sensitivity with a detection limit of 0.024 ng/mL for FB1 was achieved by the developed AIE-LFIA. Excellent selectivity, good accuracy, and high reliability of the AIE-LFIA were demonstrated, indicating a promising platform for FB1 screening.

Gatifloxacin (GAT), an antibiotic belonging to the fluoroquinolone (FQ) class, is a toxicant that may contaminate food products. In this study, a method of ultrasensitive immunochromatographic detection of GAT was developed for the first time. An indirect format of the lateral flow immunoassay (LFIA) was performed. GAT-specific monoclonal antibodies and labeled anti-species antibodies were used in the LFIA. Bimetallic core@shell Au@Ag nanoparticles (Au@Ag NPs) were synthesized as a new label. Peroxidase-mimic properties of Au@Ag NPs allowed for the catalytic enhancement of the signal on test strips, increasing the assay sensitivity. A mechanism of Au@Ag NPs-mediated catalysis was deduced. Signal amplification was achieved through the oxidative etching of Au@Ag NPs by hydrogen peroxide. This resulted in the formation of gold nanoparticles and Ag+ ions, which catalyzed the oxidation of the peroxidase substrate. Such "chemical enhancement" allowed for reaching the instrumental limit of detection (LOD, calculated by Three Sigma approach) and cutoff of 0.8 and 20 pg/mL, respectively. The enhanced assay procedure can be completed in 21 min. The enhanced LFIA was tested for GAT detection in raw meat samples, and the recoveries from meat were 78.1-114.8%. This method can be recommended as a promising instrument for the sensitive detection of various toxicants.

Conventional lateral flow immunoassay (LFIA) usually suffers from poor antimatrix interference, unsatisfactory sensitivity, and lack of quantitative ability for target analyte detection in food matrices. In response to these limits, here, multifunctional nanomaterial ZnFe2O4 nanoparticles (ZFOs) were developed and integrated into LFIA for powerful magnetic separation/enrichment and colorimetric/photothermal target sensing. Under optimum conditions, the detection for clenbuterol (CL) with magnetic enrichment achieves 9-fold higher sensitivity compared to that without enrichment and 162-fold higher sensitivity compared to that based on traditional colloidal golds. Attributing the improved performances of ZFOs, CL can be detected at ultralow levels in pork and milk with 10 min of immunoreaction time. The vLODs were 0.01 μg kg-1 for two modes, and the cutoff values of CL were about 5 and 3 μg kg-1, respectively. More importantly, the enrichment ZFO-mediated LFIA (ZE-LFIA) exhibits a similar limit of detection (LOD) in both buffer solution and food matrix, demonstrating a universal resistance to the food matrix. The multitudinous performance merits of this ZE-LFIA with high sensitivity, matrix tolerance, accuracy, and specificity have ensured a broad application potential for target detection of clenbuterol and can serve as an experience for other veterinary drug residues' detection.

SERS-based lateral flow immunoassay for sensitive and simultaneous detection of anti-SARS-CoV-2 IgM and IgG antibodies by using gap-enhanced Raman nanotags

Strong plasmon coupled gold nanoantennas with closed hot-spots enabled ultrasensitive lateral flow immunoassay of porcine epidemic diarrhea virus.