Rapid Detection of SARS-CoV-2 in Clinical and Environmental Samples via a Resonant Cavity SERS Platform within 20 min.

The coronavirus disease 2019 (COVID-19) epidemic has given a warning that it is important to explore the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical specimens or environmental samples for public health strategies and future variants. The surface-enhanced Raman spectroscopy (SERS) technique was demonstrated to achieve this goal. However, the consistency of signals originating from the poor compatibility of virions with SERS hotspots remains a key scientific challenge for the practical applications of SERS. Herein, we develop a SERS platform for the ultrasensitive and rapid detection of SARS-CoV-2 antigen within 20 min by the combination of a highly consistent SERS substrate and a supervised deep learning algorithm. A V-shaped resonant cavity array (VRC) substrate was fabricated to trap SARS-CoV-2 virions in the periodic V cavity array and stimulate the integral SERS signal of the virus via a resonance coupling effect. Benefiting from the unique architecture of the VRC substrate, we were able to directly detect the SARS-CoV-2 virus with high sensitivity and high consistency. These excellent performances enabled us to identify five different kinds of SARS-CoV-2 variants and detect SARS-CoV-2 from clinical and environmental samples with high accuracies.

Correlation coefficient-directed label-free characterization of native proteins by surface-enhanced Raman spectroscopy.

Investigation of proteins in their native state is the core of proteomics towards better understanding of their structures and functions. Surface-enhanced Raman spectroscopy (SERS) has shown its unique advantages in protein characterization with fingerprint information and high sensitivity, which makes it a promising tool for proteomics. It is still challenging to obtain SERS spectra of proteins in the native state and evaluate the native degree. Here, we constructed 3D physiological hotspots for a label-free dynamic SERS characterization of a native protein with iodide-modified 140 nm Au nanoparticles. We further introduced the correlation coefficient to quantitatively evaluate the variation of the native degree, whose quantitative nature allows us to explicitly investigate the Hofmeister effect on the protein structure. We realized the classification of a protein of SARS-CoV-2 variants in 15 min, which has not been achieved before. This study offers an effective tool for tracking the dynamic structure of proteins and biomedical research.

Revealing protein binding affinity on metal surfaces: an electrochemical approach.

Revealing the binding affinity between viruses and surfaces of environmental matrices is crucial to evaluate the bioactivity of an immobilized virus and accompanying indirect virus-related infection pathways. The understanding for SARS-CoV-2 remaining infective for even days on stainless steel but only hours on copper is still unclear. Electrochemical chronoamperometry, ultrasensitive to interfacial capacitance on surface species, was used to investigate the binding affinity of SARS-CoV-2 on metal surfaces. SRBD, the surrogate of SARS-CoV-2, shows the highest adsorption capacity on a gold surface, followed by Cu, but lowest on a stainless steel surface. The strong binding of SRBD on copper is a result of the naturally grown Cu2O under ambient conditions. Measurement of electrochemical capacitance provides a simple strategy to explore and evaluate the potential risk of an indirect virus-related infection pathway through conductive environmental matrices.

Flow microdialysis sampling-chemiluminescent detection coupled with molecular docking for the investigation of binding behavior between salbutamol and DNA.

The investigation of the binding behavior between drug and DNA provides basic information for understanding pharmacological and toxicologic mechanisms of many drugs. Herein, a facile chemiluminescent (CL) method for investigating the binding behavior between salbutamol and calf thymus DNA (ct-DNA) was established by utilizing flow microdialysis sampling technique. In a reaction equilibrium solution of salbutamol and ct-DNA, free salbutamol was extracted by a microdialysis probe, and then injected into a flow-injection CL detection system to quantitate its concentration. The binding constants of salbutamol acquired by Klotz analysis and Scatchard analysis were 2.97 × 104 M-1and 2.99 × 104 M-1, respectively. Salbutamol showed one sort of binding site on ct-DNA. Meanwhile, the three-dimensional spatial structure of the binding mode was investigated by molecular docking. The results indicate that the binding mode of salbutamol to ct-DNA was groove binding. The hydrogen bonds were primary driving force for the direct recognition of salbutamol by ct-DNA. This proof-of-principle method paves a pathway to investigate the binding behavior between small-molecular drug and DNA, and provides a theoretical guidance for designing DNA-targeting drugs.

Chemiluminescent detection integrated with microdialysis sampling for label-free measuring the affinity of ractopamine monoclonal antibody.

A novel label-free protocol was developed for measuring the affinity between ractopamine and its monoclonal antibody (McAb) based on microdialysis (MD) on-line sampling integrated with flow injection chemiluminescent detection. In this study, unbound ractopamine was sampled by MD probe from homogeneous immunoreaction equilibrious systems, and then real-time quantified using flow injection chemiluminescent detection. The quantified concentrations of unbound ractopamine in the immunoreaction equilibrious systems were treated with Scatchard analysis and Klotz analysis to obtain the affinity constant. The mean recovery of MD probe for sampling ractopamine was found to be 24.2%. The affinity constants calculated by Scatchard analysis and Klotz analysis both were 1.0 × 106 M-1, indicating that the investigated ractopamine mouse McAb was a medium-affinity antibody. The result showed good agreement with that obtained from thiocyanate elution test. This protocol for measuring antibody affinity is free of protein conjugation of hapten and enzyme labeling of McAb. Therefore it avoids affinity decrease resulting from steric hindrance, occupancy of the antigenic determinants, and deactivation of antibody, which has been frequently encountered in the reported conventional approaches. It opens up a new pathway for direct measurement of antibody affinity with a facile, rapid, accurate and low-cost approach.

A protocol for studying the interaction between small-molecular drug and DNA using microdialysis sampling integrated with chemiluminescent detection.

It is of great significance to understand how drug molecules interact with DNA, which is one of the most important aspects of biological investigations in drug discovery at molecular level. Herein, with the model of ractopamine and calf thymus DNA (ct-DNA), a protocol using microdialysis (MD) sampling integrated with flow injection (FI)-chemiluminescent (CL) detection was developed for studying the interaction between small-molecular drug and DNA. After incubating ractopamine with ct-DNA, unbound ractopamine was on-line sampled using a MD probe, followed by being introduced into a FI-CL system for quantitation. The detected concentrations of unbound ractopamine were calibrated with the recovery of the MD probe, and then treated with Klotz analysis and Scatchard analysis to acquire the binding parameters. The MD probe exhibited a mean recovery of 27.3% for ractopamine sampling under the optimal conditions. The binding constants obtained by Klotz analysis and Scatchard analysis were 3.8 × 106 M-1 and 3.9 × 106 M-1, respectively, showing negligible difference. Ractopamine was estimated to have only one type of binding site on ct-DNA. The obtained results demonstrated that the protocol using on-line MD sampling integrated with FI-CL detection is a simple and reliable technique platform for studying the interaction between small-molecular drug and DNA.

A label-free strategy for measuring the affinity between monoclonal antibody and hapten using microdialysis sampling combined with chemiluminescent detection.

It is of great importance to measure antibody affinity in the course of screening monoclonal antibody (McAb) for immunotherapy, immunoassay and immunological purification. Herein, by using terbutaline mouse McAb as a model, a novel label-free strategy based on on-line microdialysis (MD) sampling combined with flow injection chemiluminescent detection was designed for measuring antibody affinity to hapten in a homogeneous system. After this McAb incubated with its hapten, the unbound hapten was sampled on-line by the MD probe and injected into the chemiluminescent detection system for quantification. The obtained concentrations of the unbound hapten were treated with Scatchard analysis and Klotz analysis to calculate the affinity constant. The MD probe showed a recovery of 26.2% for terbutaline under the chosen conditions. The affinity constants obtained using Scatchard analysis and Klotz analysis were 4.9×106M-1 and 4.9×106M-1, respectively, showing negligible difference. The obtained affinity constants indicated that the investigated McAb was an antibody with medium affinity. The designed strategy provided a simple, rapid and low-cost approach for direct measurement of antibody affinity. Furthermore, it avoided the decrease of affinity, which was encountered frequently in the conventional approaches based on probe labeling of McAb and protein conjugation of hapten.

A facile label-free electrochemiluminescent biosensor for specific detection of Staphylococcus aureus utilizing the binding between immunoglobulin G and protein A.

A facile label-free electrochemiluminescent (ECL) biosensor was developed for detection of Staphylococcus aureus (S. aureus) based on the specific binding between Fc region of immunoglobulin G (IgG) and S. aureus protein A (SPA) in the cell wall. Carboxyl graphene, with large surface and excellent electron transfer ability, was used as the carrier of IgG for fabrication of ECL biosensor. This biosensor was constructed by depositing carboxyl graphene/porcin IgG composite on the surface of a glassy carbon electrode. The specific reaction between SPA and IgG resulted in a decrease of ECL signal because the bound S. aureus interrupted the interfacial electron transfer and hindered the diffusion of the ECL active substances. The ECL intensity decreased linearly with S. aureus concentrations in the range of 1.0×10(3)-1.0×10(9) colony-forming units (CFU)mL(-1), with a detection limit of 3.1×10(2)CFUmL(-1). The whole assay could be accomplished within 70min when a ready-for-use biosensor was applied. The recovery test for food, environmental and biological samples showed recoveries between 75.0% and 116.7%. This developed biosensor displayed ideal specificity, high sensitivity, facile manipulation, simple fabrication and short assay time, thus provided a new pathway for pathogenic bacteria rapid screening.