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1.
ACS Appl Mater Interfaces ; 14(8): 10844-10855, 2022 Mar 02.
Article in English | MEDLINE | ID: covidwho-1692677

ABSTRACT

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.


Subject(s)
Antigens, Viral/analysis , COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/analysis , Graphite/chemistry , Nanocomposites/chemistry , Zinc Oxide/chemistry , Antibodies, Immobilized/immunology , Antigens, Viral/immunology , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Coronavirus Nucleocapsid Proteins/immunology , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Electrodes , Humans , Immunoassay/instrumentation , Immunoassay/methods , Limit of Detection , Phosphoproteins/analysis , Phosphoproteins/immunology , Proof of Concept Study , SARS-CoV-2/chemistry
2.
Viruses ; 12(11)2020 10 26.
Article in English | MEDLINE | ID: covidwho-1389519

ABSTRACT

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.


Subject(s)
Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , COVID-19 , Cell Line , Convalescence , HEK293 Cells , Humans , Neutralization Tests , Pandemics , SARS-CoV-2 , Time Factors
3.
ACS Appl Mater Interfaces ; 13(34): 40342-40353, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1366784

ABSTRACT

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.


Subject(s)
Antigens, Viral/analysis , Biosensing Techniques/methods , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Antibodies, Immobilized/immunology , Antibodies, Monoclonal/immunology , Antigens, Viral/immunology , Coronavirus Nucleocapsid Proteins/immunology , Fluorescence , Fluorescent Dyes/chemistry , Humans , Immunoassay/methods , Limit of Detection , Magnetite Nanoparticles/chemistry , Nasopharynx/virology , Phosphoproteins/analysis , Phosphoproteins/immunology , Quantum Dots/chemistry , Saliva/virology , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
4.
ACS Appl Bio Mater ; 4(4): 2974-2995, 2021 04 19.
Article in English | MEDLINE | ID: covidwho-1157888

ABSTRACT

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.


Subject(s)
COVID-19/diagnosis , Immunoassay/methods , Point-of-Care Systems , Spectrum Analysis, Raman , Viral Proteins/immunology , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Biomarkers/blood , Biomarkers/metabolism , COVID-19/virology , Humans , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Viral Proteins/metabolism
5.
ACS Appl Bio Mater ; 4(2): 1178-1190, 2021 02 15.
Article in English | MEDLINE | ID: covidwho-1091529

ABSTRACT

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.


Subject(s)
Biosensing Techniques/methods , COVID-19/diagnosis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Animals , Antibodies, Immobilized/immunology , Antibodies, Neutralizing/analysis , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Graphite/chemistry , Humans , Metal Nanoparticles/chemistry , Nanotechnology/methods , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
6.
Talanta ; 227: 122207, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1078201

ABSTRACT

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.


Subject(s)
Antibodies, Viral/blood , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin M/blood , SARS-CoV-2/immunology , Animals , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Viral/immunology , Cattle , Cell Phone , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Internet of Things , Limit of Detection , Magnetic Iron Oxide Nanoparticles/chemistry , Magnetic Phenomena
7.
ACS Appl Bio Mater ; 4(2): 1307-1318, 2021 02 15.
Article in English | MEDLINE | ID: covidwho-1069089

ABSTRACT

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.


Subject(s)
Dimethylpolysiloxanes/chemistry , Immunoassay/methods , Tumor Necrosis Factor-alpha/analysis , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , COVID-19/metabolism , COVID-19/pathology , COVID-19/virology , Carbon/chemistry , Humans , Immunoassay/instrumentation , Limit of Detection , Microfluidics , Microscopy, Fluorescence , SARS-CoV-2/isolation & purification , Silicon/chemistry , Tumor Necrosis Factor-alpha/immunology , Wettability
8.
Adv Mater ; 33(10): e2007847, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1062095

ABSTRACT

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.


Subject(s)
COVID-19/diagnosis , Graphite/chemistry , Nanostructures/chemistry , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Biosensing Techniques/methods , COVID-19/prevention & control , COVID-19/virology , Electrochemical Techniques , Electrodes , Humans , Limit of Detection , Nanostructures/toxicity , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Surface Properties , Viral Proteins/analysis , Viral Proteins/immunology
9.
Anal Chim Acta ; 1147: 30-37, 2021 Feb 22.
Article in English | MEDLINE | ID: covidwho-1012278

ABSTRACT

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.


Subject(s)
Antibodies, Viral/immunology , Immunoassay/methods , Nucleocapsid Proteins/immunology , Printing, Three-Dimensional , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , COVID-19/diagnosis , COVID-19/virology , Dielectric Spectroscopy , Electrodes , Hantavirus/isolation & purification , Hantavirus/metabolism , Hantavirus Infections/diagnosis , Hantavirus Infections/virology , Humans , Immunoassay/instrumentation , Limit of Detection , Nucleocapsid Proteins/blood , SARS-CoV-2/isolation & purification , Soot/chemistry
10.
Anal Chem ; 93(2): 992-1000, 2021 01 19.
Article in English | MEDLINE | ID: covidwho-967361

ABSTRACT

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.


Subject(s)
Aptamers, Nucleotide/metabolism , Biomarkers/metabolism , Nucleic Acid Amplification Techniques/methods , Recombinases/metabolism , Antibodies, Immobilized/immunology , Antigens, Viral/chemistry , Antigens, Viral/immunology , COVID-19/diagnosis , COVID-19/virology , Communicable Diseases/diagnosis , Fluorescent Dyes/chemistry , Humans , Influenza A virus/metabolism , Influenza B virus/metabolism , Influenza, Human/diagnosis , Point-of-Care Systems , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , SELEX Aptamer Technique
11.
Sci Adv ; 6(42)2020 10.
Article in English | MEDLINE | ID: covidwho-781066

ABSTRACT

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.


Subject(s)
Betacoronavirus/immunology , Peptidyl-Dipeptidase A/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Sequence , Angiotensin-Converting Enzyme 2 , Antibodies, Immobilized/chemistry , Antibodies, Immobilized/immunology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , Cell Surface Display Techniques/methods , Coronavirus Infections/pathology , Coronavirus Infections/virology , Dimerization , Humans , Kinetics , Pandemics , Peptides/chemistry , Peptides/immunology , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Domains/immunology , Protein Subunits/chemistry , Protein Subunits/immunology , Protein Subunits/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/chemistry
12.
ACS Sens ; 5(9): 2747-2752, 2020 09 25.
Article in English | MEDLINE | ID: covidwho-740029

ABSTRACT

With the current intense need for rapid and accurate detection of viruses due to COVID-19, we report on a platform technology that is well suited for this purpose, using intact measles virus for a demonstration. Cases of infection due to the measles virus are rapidly increasing, yet current diagnostic tools used to monitor for the virus rely on slow (>1 h) technologies. Here, we demonstrate the first biosensor capable of detecting the measles virus in minutes with no preprocessing steps. The key sensing element is an electrode coated with a self-assembled monolayer containing the measles antibody, immobilized through an N-heterocyclic carbene (NHC). The intact virus is detected by changes in resistance, giving a linear response to 10-100 µg/mL of the intact measles virus without the need to label or process the sample. The limit of detection is 6 µg/mL, which is at the lower limit of concentrations that can cause infections in primates. The NHC-based biosensors are shown to be superior to thiol-based systems, producing an approximately 10× larger response and significantly greater stability toward repeated measurements and long-term storage. This NHC-based biosensor thus represents an important development for both the rapid detection of the measles virus and as a platform technology for the detection of other biological targets of interest.


Subject(s)
Antibodies, Immobilized/immunology , Benzimidazoles/chemistry , Biosensing Techniques/methods , Electrochemical Techniques/methods , Measles virus/isolation & purification , Antibodies, Immobilized/chemistry , Electrochemical Techniques/instrumentation , Electrodes , Gold/chemistry , Limit of Detection , Measles virus/immunology
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