Biosensors based on potent miniprotein binder for sensitive testing of SARS-CoV­2 variants of concern.

The miniprotein binder TRI2-2 was employed as an antibody alternative to build a single antibody-coupled TRI2-2 based gold nanoparticle-based lateral flow immunoassay (AT-GLFIA) biosensor. The biosensor provides high specificity and affinity binding between TRI2-2 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) spike antigen receptor binding domain (S-RBD). It also enables rapid testing of wild-type (WT), B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.1 (Gamma), and B.1.1.529 (Omicron) SARS-CoV-2 S-RBD and is at least ~ 16-fold more sensitive than conventional antibody pair-based GLFIA (AP-GLFIA). Besides, we developed a wireless micro-electrochemical assay (WMECA) biosensor based on the TRI2-2, which demonstrates an excellent VOCs testing capability at the pg mL-1 level. Overall, our results demonstrate that integrating miniprotein binders into conventional immunoassay systems is a promising design for improving the testing capabilities of such systems without hard-to-obtain antibody pair, complex reporter design, laborious signal amplification strategies, or specific instrumentation.

Biomimetic Molybdenum Sulfide-Catalyzed Tumor Ferroptosis and Bioimaging.

The chemical generation of singlet oxygen (1 O2 ) by the MoO4 2- -catalyzed disproportionation of hydrogen peroxide (H2 O2 ) has been widely applied in numerous catalytic processes; however, such molybdate ions cannot be administered for redox-based cancer therapeutics. This work reports the albumin-mediated biomimetic synthesis of highly active molybdenum sulfide (denoted MoB) nanocatalysts that mediate the simultaneous generation of 1 O2 and superoxide anion (O2 •- ) from H2 O2 , which is relatively abundant in malignant tumors. The MoB-catalyzed reactive oxygen species (ROS) are able to activate the ferroptosis pathway and cause lipid peroxidation for efficient cancer therapy. Furthermore, for the first time, the catalytic activity of MoB is visualized in situ. Moreover, a catalytic imaging modality based on MoB is developed for specific imaging of inflammation diseases without background interference. Therefore, this study presents a biomimetic strategy toward Mo-based nanocatalysts for ROS-facilitated tumor ferroptosis and catalytic imaging.

Engineering light-initiated afterglow lateral flow immunoassay for infectious disease diagnostics.

The pandemic of highly contagious diseases has put forward urgent requirements for high sensitivity and adaptive capacity of point-of-care testing (POCT). Herein, for the first time, we report an aggregation-induced emission (AIE) dye-energized light-initiated afterglow nanoprobes (named LiAGNPs), implemented onto a lateral flow immunoassay (LFIA) test strip, for diagnosis of two highly contagious diseases, human immunodeficiency virus (HIV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as model validation. The primary working mechanism relies on the cyclically generated singlet oxygen (1O2)-triggered time-resolved luminescent signals of LiAGNPs in which AIE dyes (TTMN) and chemiluminescent substrates (SO) are loaded. The designed LiAGNPs were found 2-fold and 32-fold sensitive than the currently used Eu(III)-based time-resolved fluorescent nanoparticles and gold nanoparticles in lateral flow immunoassay (LFIA), respectively. In addition, the extra optical behaviors of nude color and fluorescence of LiAGNPs enable the LFIA platform with the capability of the naked eye and fluorescent detection to satisfy the applications under varying scenarios. In short, the versatile LiAGNPs have great potential as a novel time-resolved reporter in enhancing detection sensitivity and application flexibility with LFIA platform for rapid but sensitive infectious disease diagnostics.

A Universal Boronate-Affinity Crosslinking-Amplified Dynamic Light Scattering Immunoassay for Point-of-Care Glycoprotein Detection.

Herein, we report a universal boronate-affinity crosslinking-amplified dynamic light scattering (DLS) immunoassay for point-of-care (POC) glycoprotein detection in complex samples. This enhanced DLS immunoassay consists of two elements, i.e., antibody-coated magnetic nanoparticles (MNP@mAb) for target capture and DLS signal transduction, and phenylboronic acid-based boronate-affinity materials as crosslinking amplifiers. Upon the addition of targets, glycoproteins are first captured by MNP@mAb and amplified by target-induced crosslinking stemming from the selective binding between the boronic acid ligand and cis-diol-containing glycoprotein, thereby resulting in a remarkably increased DLS signal in the average nanoparticle size. Benefiting from the multivalent binding and fast boronate-affinity reaction between glycoproteins and crosslinkers, the proposed immunosensing strategy has achieved the ultrasensitive and rapid quantitative assay of glycoproteins at the fM level within 15 min. Overall, this work provides a promising and versatile design strategy for extending the DLS technique to detect glycoproteins even in the field or at POC.

Gold Nanobeads with Enhanced Absorbance for Improved Sensitivity in Competitive Lateral Flow Immunoassays.

BACKGROUND: Colloidal gold based lateral flow immunoassay (LFIA) commonly suffers from relatively low detection sensitivity due to the insufficient brightness of conventional gold nanoparticles (AuNPs) with the size of 20-40 nm. METHODS: Herein, three kinds of gold nanobeads (GNBs) with the size of 94 nm, 129 nm, and 237 nm, were synthesized by encapsulating numerous hydrophobic AuNPs (10 nm) into polymer matrix. The synthesized GNBs exhibited the enhanced colorimetric signal intensity compared with 20-40 nm AuNPs. The effects of the size of GNBs on the sensitivity of LFIA with competitive format were assessed. RESULTS: The results showed that the LFIA using 129 nm GNBs as amplified signal probes exhibits the best sensitivity for fumonisin B1 (FB1) detection with a cut-off limit (for visual qualitative detection) at 125 ng/mL, a half maximal inhibitory concentration at 11.27 ng/mL, and a detection limit at 1.76 ng/mL for detection of real corn samples, which are 8-, 3.82-, and 2.89-fold better than those of conventional AuNP40-based LFIA, respectively. The developed GNB-LFIA exhibited negligible cross-reactions with other common mycotoxins. In addition, the accuracy, precision, reliability, and practicability were demonstrated by determining real corn samples. CONCLUSIONS: All in all, the proposed study provides a promising strategy to enhance the sensitivity of competitive LFIA via using the GNBs as amplified signal probes.

A novel magneto-gold nanohybrid-enhanced lateral flow immunoassay for ultrasensitive and rapid detection of ochratoxin A in grape juice.

Conventional gold nanoparticle-based lateral flow immunoassay (LFIA) usually suffers a huge challenge in measuring target concentration in food matrices with dark color because of its poor resistance to the background matrix and color interference. To address this issue, we first report a novel bifunctional magneto-gold nanohybrid (MGNH) for the simultaneous magnetic separation and colorimetric target sensing by integrating MGNHs into LFIA. Under optimum conditions, an ultrasensitive detection of ochratoxin A (OTA) in grape juice was achieved with a limit of detection at 0.094 ng mL-1. The average recoveries of this MGNH-LFIA ranged from 92.31% to 108.97% with a coefficient of variation of below 12%. The excellent selectivity of our MGNH-LFIA against OTA was demonstrated. Besides, our MGNH-LFIA is comparable to liquid chromatography coupled with mass spectrometry in terms of accuracy, reproducibility, and practicability. The designed MGNH-LFIA platform is readily extended for improving other small molecule detection in food samples.

Self-assembled colloidal gold superparticles to enhance the sensitivity of lateral flow immunoassays with sandwich format.

Background: Traditional lateral flow immunoassay (LFIA) based on 20-40 nm gold nanoparticles (AuNPs) as signal reporter always suffers from relatively low detection sensitivity due to its insufficient brightness, severely restricting its wide-ranging application in the detection of target analytes with trace concentration. Methods: To address this problem, the self-assembled colloidal gold superparticles (GSPs) were synthesized as an improved absorption-dominated labeling probe for improving the sensitivity of sandwich LFIA. Five kinds of GSPs with the size ranging from 100 nm to 400 nm were synthesized by embedding hydrophobic AuNPs of size 12 nm as building blocks into the polymer nanobeads. The as-prepared GSPs were suggested as novel labeling probes of LFIA. The effects of the size of assembled GSPs on the sensitivity of sandwich LFIA was assessed, and the detection performance of GSPs-LFIA was further compared with traditional AuNPs-LFIA. Results: The resultant GSPs showed extremely high light absorption but very low light scattering, which favor the absorption-dominated signal output in LFIA. Among them, the GSP270-LFIA (size 270 nm) exhibits the highest sensitivity for human chorionic gonadotropin and hepatitis B surface antigen detection in real serum sample, which are approximate 39.79- and 13.8-fold higher than that of traditional AuNP40-LFIA. Conclusions: The proposed research demonstrated that the current GSPs can provide an ultrasensitive and quantitative detection for disease biomarkers in real serum samples as promising reporters of sandwich LFIA platform.

Core-Shell-Heterostructured Magnetic-Plasmonic Nanoassemblies with Highly Retained Magnetic-Plasmonic Activities for Ultrasensitive Bioanalysis in Complex Matrix.

Herein, a facile self-assembly strategy for coassembling oleic acid-coated iron oxide nanoparticles (OC-IONPs) with oleylamine-coated gold nanoparticles (OA-AuNPs) to form colloidal magnetic-plasmonic nanoassemblies (MPNAs) is reported. The resultant MPNAs exhibit a typical core-shell heterostructure comprising aggregated OA-AuNPs as a plasmonic core surrounded by an assembled magnetic shell of OC-IONPs. Owing to the high loading of OA-AuNPs and reasonable spatial distribution of OC-IONPs, the resultant MPNAs exhibit highly retained magnetic-plasmonic activities simultaneously. Using the intrinsic dual functionality of MPNAs as a magnetic separator and a plasmonic signal transducer, it is demonstrated that the assembled MPNAs can achieve the simultaneous magnetic manipulation and optical detection on the lateral flow immunoassay platform after surface functionalization with recognition molecules. In conclusion, the core-shell-heterostructured MPNAs can serve as a nanoanalytical platform for the separation and concentration of target compounds from complex biological samples using magnetic properties and simultaneous optical sensing using plasmonic properties.

Integrated magneto-fluorescence nanobeads for ultrasensitive glycoprotein detection using antibody coupled boronate-affinity recognition.

Herein, we report a novel magnet-mediated antibody-boronate sandwich-typed assay (ABSTA) strategy for the ultrasensitive, specific, rapid, and enzyme-free detection of glycoproteins in complex samples. The proposed ABSTA method exhibited ultrahigh sensitivity for HCG with a detection limit of 0.19 mIU mL-1, which is approximately 40-fold lower than that of conventional sandwich enzyme immunoassay.

An amphiphilic-ligand-modified gold nanoflower probe for enhancing the stability of lateral flow immunoassays in dried distillers grains.

An amphiphilic ligand-capped gold nanoflower (AuNF) was proposed as a novel lateral flow immunoassay (LFA) reporter for zearalenone (ZEN) detection in distillers dried grains solubles (DDGS). The amphiphilic ligand consists of a thiol-terminated hydrophobic alkane chain, a tetra (ethylene glycol) unit, and a terminal carboxyl group. The novel AuNF probe (N-AuNF-Abs) was prepared by coupling the amino group of anti-ZEN antibodies with the AuNF carboxyl group via an amido covalent linkage. For comparison, a traditional AuNF probe (Tr-AuNF-Abs) was prepared by labeling antibodies on the surface of citrate capped AuNFs via an electrostatic adsorption method. The detection performance of the two probes in LFA was systematically investigated, including the half maximal inhibitory concentration (IC50), robustness and reproducibility for ZEN quantitative detection in DDGS samples, and shelf life. The N-AuNF-Ab based LFA (N-LFA) had a lower IC50 value (15.97 ng mL-1) for ZEN detection in phosphate buffered saline than that of the Tr-AuNF-mAb based LFA (Tr-LFA, 31.06 ng mL-1). The IC50 value of N-LFA in DDGS extract was 17.46 ng mL-1, whereas the Tr-LFA showed poor robustness and reproducibility in DDGS samples, resulting in a failed determination. The intra- and inter-assays of N-LFA for ZEN-spiked DDGS samples indicated that the average recoveries ranged from 93.0% to 125.9%, with coefficients of variation ranging from 2.8% to 21.9%. These results indicated that the N-LFA strip exhibits good robustness and an acceptable accuracy for ZEN quantitative detection in complex DDGS samples. In accelerated aging studies, N-LFA showed a longer shelf life (5 years) than that of Tr-LFA (1 year). In summary, the proposed method provided a novel strategy to prepare a super-stable probe for enhancing the detection performance of LFA for small molecular detection in complex sample matrices such as DDGS.