PdPtRu trimetallic nanozymes and application to electrochemical immunosensor for sensitive SARS-COV-2 antigen detection.

Herein, a ternary PdPtRu nanodendrite as novel trimetallic nanozyme was reported, which possessed excellent peroxidase-like activity as well as electro-catalytic activity on account of the synergistic effect between the three metals. Based on the excellent electro-catalytic activity of trimetallic PdPtRu nanozyme toward the reduction of H2O2, the trimetallic nanozyme was applied to construct a brief electrochemical immunosensor for SARS-COV-2 antigen detection. Concretely, trimetallic PdPtRu nanodendrite was used to modify electrode surface, which not only generated high reduction current of H2O2 for signal amplification, but also provided massive active sites for capture antibody (Ab1) immobilization to construct immunosensor. In the presence of target SARS-COV-2 antigen, SiO2 nanosphere labeled detection antibody (Ab2) composites were introduced on the electrode surface according sandwich immuno-reaction. Due to the inhibitory effect of SiO2 nanosphere on the current signal, the current signal was decreased with the increasing target SARS-COV-2 antigen concentration. As a result, the proposed electrochemical immunosensor presented sensitive detection of SARS-COV-2 antigen with linear range from 1.0 pg/mL to 1.0 µg/mL and limit of detection down to 51.74 fg/mL. The proposed immunosensor provide a brief but sensitive antigen detection tool for rapid diagnosis of COVID-19.

3D-printed capillaric ELISA-on-a-chip with aliquoting.

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction (Yafia et al. Nature, 2022, 605, 464-469), but the need for precise pipetting and intolerance to commonly used surfactant concentrations limit the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that simplifies chip loading and pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high-sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 µL), enzyme conjugate (50 µL), substrate (80 µL), and sample (210 µL), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 µL. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions to complete the assay in 1.5 h. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg mL-1 for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

Highly Stable Buffer-Based Zinc Oxide/Reduced Graphene Oxide Nanosurface Chemistry for Rapid Immunosensing of SARS-CoV-2 Antigens.

The widespread and long-lasting effect of the COVID-19 pandemic has called attention to the significance of technological advances in the rapid diagnosis of SARS-CoV-2 virus. This study reports the use of a highly stable buffer-based zinc oxide/reduced graphene oxide (bbZnO/rGO) nanocomposite coated on carbon screen-printed electrodes for electrochemical immuno-biosensing of SARS-CoV-2 nuelocapsid (N-) protein antigens in spiked and clinical samples. The incorporation of a salt-based (ionic) matrix for uniform dispersion of the nanomixture eliminates multistep nanomaterial synthesis on the surface of the electrode and enables a stable single-step sensor nanocoating. The immuno-biosensor provides a limit of detection of 21 fg/mL over a linear range of 1-10 000 pg/mL and exhibits a sensitivity of 32.07 ohms·mL/pg·mm2 for detection of N-protein in spiked samples. The N-protein biosensor is successful in discriminating positive and negative clinical samples within 15 min, demonstrating its proof of concept used as a COVID-19 rapid antigen test.

A Tri-Channel Oxide Transistor Concept for the Rapid Detection of Biomolecules Including the SARS-CoV-2 Spike Protein.

Solid-state transistor sensors that can detect biomolecules in real time are highly attractive for emerging bioanalytical applications. However, combining upscalable manufacturing with the required performance remains challenging. Here, an alternative biosensor transistor concept is developed, which relies on a solution-processed In2 O3 /ZnO semiconducting heterojunction featuring a geometrically engineered tri-channel architecture for the rapid, real-time detection of important biomolecules. The sensor combines a high electron mobility channel, attributed to the electronic properties of the In2 O3 /ZnO heterointerface, in close proximity to a sensing surface featuring tethered analyte receptors. The unusual tri-channel design enables strong coupling between the buried electron channel and electrostatic perturbations occurring during receptor-analyte interactions allowing for robust, real-time detection of biomolecules down to attomolar (am) concentrations. The experimental findings are corroborated by extensive device simulations, highlighting the unique advantages of the heterojunction tri-channel design. By functionalizing the surface of the geometrically engineered channel with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody receptors, real-time detection of the SARS-CoV-2 spike S1 protein down to am concentrations is demonstrated in under 2 min in physiological relevant conditions.

Nanostructured sensor platform based on organic polymer conjugated to metallic nanoparticle for the impedimetric detection of SARS-CoV-2 at various stages of viral infection.

The projection of new biosensing technologies for genetic identification of SARS-COV-2 is essential in the face of a pandemic scenario. For this reason, the current research aims to develop a label-free flexible biodevice applicable to COVID-19. A nanostructured platform made of polypyrrole (PPy) and gold nanoparticles (GNP) was designed for interfacing the electrochemical signal in miniaturized electrodes of tin-doped indium oxide (ITO). Oligonucleotide primer was chemically immobilized on the flexible transducers for the biorecognition of the nucleocapsid protein (N) gene. Methodological protocols based on cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) were used to characterize the nanotechnological apparatus. The biosensor's electrochemical performance was evaluated using the SARS-CoV-2 genome and biological samples of cDNA from patients infected with retrovirus at various disease stages. It is inferred that the analytical tool was able to distinguish the expression of SARS-CoV-2 in patients diagnosed with COVID-19 in the early, intermediate and late stages. The biosensor exhibited high selectivity by not recognizing the biological target in samples from patients not infected with SARS-CoV-2. The proposed sensor obtained a linear response range estimated from 800 to 4000 copies µL-1 with a regression coefficient of 0.99, and a detection limit of 258.01 copies µL-1. Therefore, the electrochemical biosensor based on flexible electrode technology represents a promising trend for sensitive molecular analysis of etiologic agent with fast and simple operationalization. In addition to early genetic diagnosis, the biomolecular assay may help to monitor the progression of COVID-19 infection in a novel manner.

Antibody Binding to SARS-CoV-2 S Glycoprotein Correlates with but Does Not Predict Neutralization.

Convalescent plasma from SARS-CoV-2 infected individuals and monoclonal antibodies were shown to potently neutralize viral and pseudoviral particles carrying the S glycoprotein. However, a non-negligent proportion of plasma samples from infected individuals, as well as S-specific monoclonal antibodies, were reported to be non-neutralizing despite efficient interaction with the S glycoprotein in different biochemical assays using soluble recombinant forms of S or when expressed at the cell surface. How neutralization relates to the binding of S glycoprotein in the context of viral particles remains to be established. Here, we developed a pseudovirus capture assay (VCA) to measure the capacity of plasma samples or antibodies immobilized on ELISA plates to bind to membrane-bound S glycoproteins from SARS-CoV-2 expressed at the surface of lentiviral particles. By performing VCA, ELISA, and neutralization assays, we observed a strong correlation between these parameters. However, while we found that plasma samples unable to capture viral particles did not neutralize, capture did not guarantee neutralization, indicating that the capacity of antibodies to bind to the S glycoprotein at the surface of pseudoviral particles is required but not sufficient to mediate neutralization. Altogether, our results highlight the importance of better understanding the inactivation of S by plasma and neutralizing antibodies.

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.

Oriented immobilization of antibodies onto sensing platforms - A critical review.

The immunosensor has been proven a versatile tool to detect various analytes, such as food contaminants, pathogenic bacteria, antibiotics and biomarkers related to cancer. To fabricate robust and reproducible immunosensors with high sensitivity, the covalent immobilization of immunoglobulins (IgGs) in a site-specific manner contributes to better performance. Instead of the random IgG orientations result from the direct yet non-selective immobilization techniques, this review for the first time introduces the advances of stepwise yet site-selective conjugation strategies to give better biosensing efficiency. Noncovalently adsorbing IgGs is the first but decisive step to interact specifically with the Fc fragment, then following covalent conjugate can fix this uniform and antigens-favorable orientation irreversibly. In this review, we first categorized this stepwise strategy into two parts based on the different noncovalent interactions, namely adhesive layer-mediated interaction onto homofunctional support and layer-free interaction onto heterofunctional support (which displays several different functionalities on its surface that are capable to interact with IgGs). Further, the influence of ligands characteristics (synthesis strategies, spacer requirements and matrices selection) on the heterofunctional support has also been discussed. Finally, conclusions and future perspectives for the real-world application of stepwise covalent conjugation are discussed. This review provides more insights into the fabrication of high-efficiency immunosensor, and special attention has been devoted to the well-orientation of full-length IgGs onto the sensing platform.

New Impedimetric Sandwich Immunosensor for Ultrasensitive and Highly Specific Detection of Spike Receptor Binding Domain Protein of SARS-CoV-2.

An impedance sensing platform-combined conducting nanocomposite layer was fabricated to develop an effective and rapid method for detection of coronavirus infection (COVID-19) specific spike receptor binding domain (RBD) protein, a precious antigen marker of COVID-19 disease. Coronavirus infection has spread globally and swiftly with major impacts on health, economy, and quality of life of communities. Fast and reliable detection of COVID-19 is a very significant issue for the effective treatment of this bad illness. For this aim, first, an Epoxy functional group-substituted thiophene monomer was synthesized and electrodeposited on a single-use indium tin oxide (ITO) platform in the presence of acetylene black by employing a cyclic voltammetry technique; thus, a conducting nanocomposite (C-NC) layer with high conductivity was obtained. This composite was electrodeposited for the first time on the ITO surface to generate a facile and cost-effective impedimetric biosensor. In addition, this composite provided proper attachment points for antibody binding and also supported the biosensor construction. The immuno-specific biointeractions between anti-RBD and RBD proteins hampered the electron transfer between the ITO substrate surface and electrolyte, and this reaction caused variations in impedance signals, and these signals were proportional to the immobilized RBD antigen amounts. The as-prepared immunosensor showed a wide linear dynamic range (0.0012-120 pg/mL), an ultra-low detection limit of 0.58 fg/mL with added superiorities of great selectivity, suitable repeatability, multiple reusability, and excellent reproducibility.

Voltammetric-based immunosensor for the detection of SARS-CoV-2 nucleocapsid antigen.

Since the COVID-19 disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) was declared a pandemic, it has spread rapidly, causing one of the most serious outbreaks in the last century. Reliable and rapid diagnostic tests for COVID-19 are crucial to control and manage the outbreak. Here, a label-free square wave voltammetry-based biosensing platform for the detection of SARS-CoV-2 in nasopharyngeal samples is reported. The sensor was constructed on screen-printed carbon electrodes coated with gold nanoparticles. The electrodes were functionalized using 11-mercaptoundecanoic acid (MUA) which was used for the immobilization of an antibody against SARS-CoV-2 nucleocapsid protein (N protein). The binding of the immunosensor with the N protein caused a change in the electrochemical signal. The detection was realised by measuring the change in reduction peak current of a redox couple using square wave voltammetry at 0.04 V versus Ag ref. electrode on the immunosensor upon binding with the N protein. The electrochemical immunosensor showed high sensitivity with a linear range from 1.0 pg.mL-1 to 100 ng.mL-1 and a limit of detection of 0.4 pg.mL-1 for the N protein in PBS buffer pH 7.4. Moreover, the immunosensor did not exhibit significant response with other viruses such as HCoV, MERS-CoV, Flu A and Flu B, indicating the high selectivity of the sensor for SARS-CoV-2. However, cross reactivity of the biosensor with SARS-CoV is indicated, which gives ability of the sensor to detect both SARS-CoV and SARS-CoV-2. The biosensor was successfully applied to detect the SARS-CoV-2 virus in clinical samples showing good correlation between the biosensor response and the RT-PCR cycle threshold values. We believe that the capability of miniaturization, low-cost and fast response of the proposed label-free electrochemical immunosensor will facilitate the point-of-care diagnosis of COVID 19 and help prevent further spread of infection.

SERS Based Lateral Flow Immunoassay for Point-of-Care Detection of SARS-CoV-2 in Clinical Samples.

The current scenario, an ongoing pandemic of COVID-19, places a dreadful burden on the healthcare system worldwide. Subsequently, there is a need for a rapid, user-friendly, and inexpensive on-site monitoring system for diagnosis. The early and rapid diagnosis of SARS-CoV-2 plays an important role in combating the outbreak. Although conventional methods such as PCR, RT-PCR, and ELISA, etc., offer a gold-standard solution to manage the pandemic, they cannot be implemented as a point-of-care (POC) testing arrangement. Moreover, surface-enhanced Raman spectroscopy (SERS) having a high enhancement factor provides quantitative results with high specificity, sensitivity, and multiplex detection ability but lacks in POC setup. In contrast, POC devices such as lateral flow immunoassay (LFIA) offer rapid, simple-to-use, cost-effective, reliable platform. However, LFIA has limitations in quantitative and sensitive analyses of SARS-CoV-2 detection. To resolve these concerns, herein we discuss a unique modality that is an integration of SERS with LFIA for quantitative analyses of SARS-CoV-2. The miniaturization ability of SERS-based devices makes them promising in biosensor application and has the potential to make a better alternative of conventional diagnostic methods. This review also demonstrates the commercially available and FDA/ICMR approved LFIA kits for on-site diagnosis of SARS-CoV-2.

Conventional and Nanotechnology-Based Sensing Methods for SARS Coronavirus (2019-nCoV).

Ongoing pandemic coronavirus (COVID-19) has affected over 218 countries and infected 88,512,243 and 1,906,853 deaths reported by Jan. 8, 2021. At present, vaccines are being developed in Europe, Russia, USA, and China, although some of these are in phase III of trials, which are waiting to be available for the general public. The only option available now is by vigorous testing, isolation of the infected cases, and maintaining physical and social distances. Numerous methods are now available or being developed for testing the suspected cases, which may act as carriers of the virus. In this review, efforts have been made to discuss the conventional as well as fast, rapid, and efficient testing methods developed for the diagnosis of 2019-nCoV.Testing methods can be based on the sensing of targets, which include RNA, spike proteins and antibodies such as IgG and IgM. Apart from the development of RNA targeted PCR, antibody and VSV pseudovirus neutralization assay along with several other diagnostic techniques have been developed. Additionally, nanotechnology-based sensors are being developed for the diagnosis of the virus, and these are also discussed.

Anti-SARS-CoV-2 IgG and IgM detection with a GMR based LFIA system.

Since December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused millions of deaths and seriously threatened the safety of human life; indeed, this situation is worsening and many people are infected with the new coronavirus every day. Therefore, it is very important to understand patients' degree of infection and infection history through antibody testing. Such information is useful also for the government and hospitals to formulate reasonable prevention policies and treatment plans. In this paper, we develop a lateral flow immunoassay (LFIA) method based on superparamagnetic nanoparticles (SMNPs) and a giant magnetoresistance (GMR) sensing system for the simultaneously quantitative detection of anti-SARS-CoV-2 immunoglobulin M (IgM) and G (IgG). A simple and time-effective co-precipitation method was utilized to prepare the SMNPs, which have good dispersibility and magnetic property, with an average diameter of 68 nm. The Internet of Medical Things-supported GMR could transmit medical data to a smartphone through the Bluetooth protocol, making patient information available for medical staff. The proposed GMR system, based on SMNP-supported LFIA, has an outstanding advantage in cost-effectiveness and time-efficiency, and is easy to operate. We believe that the suggested GMR based LFIA system will be very useful for medical staff to analyze and to preserve as a record of infection in COVID-19 patients.

Surface Biochemical Modification of Poly(dimethylsiloxane) for Specific Immune Cytokine Response.

Recent evidence suggests that proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), play a pivotal role in the development of inflammatory-related pathologies (covid-19, depressive disorders, sepsis, cancer, etc.,). More importantly, the development of TNF-α biosensors applied to biological fluids (e.g. sweat) could offer non-invasive solutions for the continuous monitoring of these disorders, in particular, polydimethylsiloxane (PDMS)-based biosensors. We have therefore investigated the biofunctionalization of PDMS surfaces using a silanization reaction with 3-aminopropyltriethoxysilane, for the development of a human TNF-α biosensor. The silanization conditions for 50 µm PDMS surfaces were extensively studied by using water contact angle measurements, electron dispersive X-ray and Fourier transform infrared spectroscopies, and fluorescamine detection. Evaluation of the wettability of the silanized surfaces and the Si/C ratio pointed out to the optimal silanization conditions supporting the formation of a stable and reproducible aminosilane layer, necessary for further bioconjugation. An ELISA-type immunoassay was then successfully performed for the detection and quantification of human TNF-α through fluorescent microscopy, reaching a limit of detection of 0.55 µg/mL (31.6 nM). Finally, this study reports for the first time a promising method for the development of PDMS-based biosensors for the detection of TNF-α, using a quick, stable, and simple biofunctionalization process.

Graphene: A Disruptive Opportunity for COVID-19 and Future Pandemics?

The graphene revolution, which has taken place during the last 15 years, has represented a paradigm shift for science. The extraordinary properties possessed by this unique material have paved the road to a number of applications in materials science, optoelectronics, energy, and sensing. Graphene-related materials (GRMs) are now produced in large scale and have found niche applications also in the biomedical technologies, defining new standards for drug delivery and biosensing. Such advances position GRMs as novel tools to fight against the current COVID-19 and future pandemics. In this regard, GRMs can play a major role in sensing, as an active component in antiviral surfaces or in virucidal formulations. Herein, the most promising strategies reported in the literature on the use of GRM-based materials against the COVID-19 pandemic and other types of viruses are showcased, with a strong focus on the impact of functionalization, deposition techniques, and integration into devices and surface coatings.

3D-printed electrode as a new platform for electrochemical immunosensors for virus detection.

Simple, low-cost, and sensitive new platforms for electrochemical immunosensors for virus detection have been attracted attention due to the recent pandemic caused by a new type of coronavirus (SARS-CoV-2). In the present work, we report for the first time the construction of an immunosensor using a commercial 3D conductive filament of carbon black and polylactic acid (PLA) to detect Hantavirus Araucaria nucleoprotein (Np) as a proof-of-concept. The recognition biomolecule was anchored directly at the filament surface by using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-Hydroxysuccinimide (EDC/NHS). Conductive and non-conductive composites of PLA were characterized using thermal gravimetric analysis (TGA), revealing around 30% w/w of carbon in the filament. Morphological features of composites were obtained from SEM and TEM measurements. FTIR measurement revealed that crosslinking agents were covalently bonded at the filament surface. Electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for the evaluation of each step involved in the construction of the proposed immunosensor. The results showed the potentiality of the device for the quantitative detection of Hantavirus Araucaria nucleoprotein (Np) from 30 µg mL-1 to 240 µg mL-1 with a limit of detection of 22 µg mL-1. Also, the proposed immunosensor was applied with success for virus detection in 100x diluted human serum samples. Therefore, the PLA conductive filament with carbon black is a simple and excellent platform for immunosensing, which offers naturally carboxylic groups able to anchor covalently biomolecules.

Functionalized terahertz plasmonic metasensors: Femtomolar-level detection of SARS-CoV-2 spike proteins.

Effective and efficient management of human betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 virus infection i.e., COVID-19 pandemic, required sensitive and selective sensors with short sample-to-result durations for performing desired diagnostics. In this direction, one appropriate alternative approach to detect SARS-CoV-2 virus protein at low level i.e., femtomolar (fM) is exploring plasmonic metasensor technology for COVID-19 diagnostics, which offers exquisite opportunities in advanced healthcare programs, and modern clinical diagnostics. The intrinsic merits of plasmonic metasensors stem from their capability to squeeze electromagnetic fields, simultaneously in frequency, time, and space. However, the detection of low-molecular weight biomolecules at low densities is a typical drawback of conventional metasensors that has recently been addressed using toroidal metasurface technology. This research is focused on the fabrication of a miniaturized plasmonic immunosensor based on toroidal electrodynamics concept that can sustain robustly confined plasmonic modes with ultranarrow lineshapes in the terahertz (THz) frequencies. By exciting toroidal dipole mode using our quasi-infinite metasurface and a judiciously optimized protocol based on functionalized gold nanoparticles (AuNPs) conjugated with the specific monoclonal antibody specific to spike protein (S1) of SARS-CoV-2 virus onto the metasurface, the resonance shifts for diverse concentrations of the spike protein are monitored. Possessing molecular weight around ~76 kDa allowed to detect the presence of SARS-CoV-2 virus protein with significantly low as limit of detection (LoD) was achieved as ~4.2 fM. We envisage that outcomes of this research will pave the way toward the use of toroidal metasensors as practical technologies for rapid and precise screening of SARS-CoV-2 virus carriers, symptomatic or asymptomatic, and spike proteins in hospitals, clinics, laboratories, and site of infection.

Ultrasensitive Detection Platform of Disease Biomarkers Based on Recombinase Polymerase Amplification with H-Sandwich Aptamers.

The detection of trace protein biomarkers is essential in the diagnostic field. Protein detection systems ranging from widely used enzyme-linked immunosorbent assays to simple, inexpensive approaches, such as lateral flow immunoassays, play critical roles in medical and drug research. Despite continuous progress, current systems are insufficient for the diagnosis of diseases that require high sensitivity. In this study, we developed a heterogeneous sandwich-type sensing platform based on recombinase polymerase amplification using DNA aptamers specific to the target biomarker. Only the DNA bound to the target in the form of a heterogeneous sandwich was selectively amplified, and the fluorescence signal of an intercalating dye added before the amplification reaction was detected, thereby enabling high specificity and sensitivity. We applied this method for the detection of protein biomarkers for various infectious diseases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and observed attomolar-level detection of biomarkers and low cross-reactivity between different viruses. We also confirmed detection efficiency of the proposed method using clinical samples. These results demonstrate that the proposed sensing platform can be used to diagnose various diseases requiring high sensitivity, specificity, and accuracy.

One-step rapid quantification of SARS-CoV-2 virus particles via low-cost nanoplasmonic sensors in generic microplate reader and point-of-care device.

The spread of SARS-CoV-2 virus in the ongoing global pandemic has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemic. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. As low as 370 vp/mL were detected in one step within 15 min and the virus concentration can be quantified linearly in the range of 0 to 107 vp/mL. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.

Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.