scholarly journals A nanobody-functionalized organic electrochemical transistor for the rapid detection of SARS-CoV-2 and MERS antigens at the physical limit

2020 ◽  
Author(s):  
Keying Guo ◽  
Shofarul Wustoni ◽  
Anil Koklu ◽  
Escarlet Díaz-Galicia ◽  
Maximilian Moser ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Point Of Care ◽  
Protein Detection ◽  
High Specificity ◽  
Human Saliva ◽  
Nasopharyngeal Swab ◽  
Transistor Channel ◽  
Electrochemical Transistor ◽  
Detection And Quantification

AbstractThe COVID-19 pandemic highlights the need for rapid protein detection and quantification at the single-molecule level in a format that is simple and robust enough for widespread point-of-care applications. We here introduce a modular nanobody-organic electrochemical transistor architecture that enables the fast and specific detection and quantification of single-molecule to nanomolar protein antigen concentrations in complex bodily fluids. The sensor combines a new solution-processable organic semiconductor material in the transistor channel with the high-density and orientation-controlled bioconjugation of nanobody fusion proteins on disposable gate electrodes. It provides results after a 10 minutes exposure to 5 µL of unprocessed samples, maintains high specificity and single-molecule sensitivity in human saliva or serum, and is rapidly reprogrammed towards any protein target for which nanobodies exist. We demonstrate the use of this highly modular platform for the detection of green fluorescent protein, SARS-CoV-1/2, and MERS-CoV spike proteins and validate the sensor for COVID-19 screening in unprocessed clinical nasopharyngeal swab and saliva samples.

scholarly journals Head-to-head comparison of nasal and nasopharyngeal sampling using SARS-CoV-2 rapid antigen testing in Lesotho

2022 ◽  
Author(s):  
Niklaus Daniel Labhardt ◽  
Lucia Gonzalez Fernandez ◽  
Bulemba Katende ◽  
Josephine Muhairwe ◽  
Moniek Bresser ◽  
...  
Keyword(s):  
Point Of Care ◽  
High Specificity ◽  
Good Alternative ◽  
Nasopharyngeal Swab ◽  
Symptom Duration ◽  
High Agreement ◽  
Minimum Requirement ◽  
History Of ◽  
Antigen Testing ◽  
Or History

Objectives To assess the real-world diagnostic performance of nasal and nasopharyngeal swabs for SD Biosensor STANDARD Q COVID-19 Antigen Rapid Diagnostic Test (Ag-RDT). Methods Individuals ≥5 years with COVID-19 compatible symptoms or history of exposure to SARS-CoV-2 presenting at hospitals in Lesotho received two nasopharyngeal and one nasal swab. Ag-RDT from nasal and nasopharyngeal swabs were performed as point-of-care on site, the second nasopharyngeal swab used for polymerase chain reaction (PCR) as the reference standard. Results Out of 2198 participants enrolled, 2131 had a valid PCR result (61% female, median age 41 years, 8% children), 84.5% were symptomatic. Overall PCR positivity rate was 5.8%. The sensitivity for nasopharyngeal, nasal, and combined nasal and nasopharyngeal Ag-RDT result was 70.2% (95%CI: 61.3-78.0), 67.3% (57.3-76.3) and 74.4% (65.5-82.0), respectively. The respective specificity was 97.9% (97.1-98.4), 97.9% (97.2-98.5) and 97.5% (96.7-98.2). For both sampling modalities, sensitivity was higher in participants with symptom duration ≤ 3days versus ≤ 7days. Agreement between nasal and nasopharyngeal Ag-RDT was 99.4%. Conclusions The STANDARD Q Ag-RDT showed high specificity. Sensitivity was, however, below the WHO recommended minimum requirement of ≥ 80%. The high agreement between nasal and nasopharyngeal sampling suggests that for Ag-RDT nasal sampling is a good alternative to nasopharyngeal sampling.

scholarly journals Spike vs nucleocapsid SARS-CoV-2 antigen detection: application in nasopharyngeal swab specimens

2021 ◽  
Author(s):  
Moria Barlev-Gross ◽  
Shay Weiss ◽  
Amir Ben-Shmuel ◽  
Assa Sittner ◽  
Keren Eden ◽  
...  
Keyword(s):  
Point Of Care ◽  
High Specificity ◽  
Antigen Detection ◽  
Screening Tools ◽  
Nasopharyngeal Swab ◽  
Lower Sensitivity ◽  
Sample Collection ◽  
Time Resolved ◽  
Qrt Pcr ◽  
Optimal Target

AbstractPublic health experts emphasize the need for quick, point-of-care SARS-CoV-2 detection as an effective strategy for controlling virus spread. To this end, many “antigen” detection devices were developed and commercialized. These devices are mostly based on detecting SARS-CoV-2’s nucleocapsid protein. Recently, alerts issued by both the FDA and the CDC raised concerns regarding the devices’ tendency to exhibit false positive results. In this work we developed a novel alternative spike-based antigen assay, comprised of four high-affinity, specific monoclonal antibodies, directed against different epitopes on the spike’s S1 subunit. The assay’s performance was evaluated for COVID-19 detection from nasopharyngeal swabs, compared to an in-house nucleocapsid-based assay, composed of antibodies directed against the nucleocapsid. Detection of COVID-19 was carried out in a cohort of 284 qRT-PCR positive and negative nasopharyngeal swab samples. The time resolved fluorescence (TRF) ELISA spike-assay displayed very high specificity (99%) accompanied with a somewhat lower sensitivity (66% for Ct<25), compared to the nucleocapsid ELISA assay which was more sensitive (85% for Ct<25) while less specific (87% specificity). Despite being out-performed by qRT-PCR, we suggest that there is room for such tests in the clinical setting, as cheap and rapid pre-screening tools. Our results further suggest that when applying antigen detection, one must consider its intended application (sensitivity vs specificity), taking into consideration that the nucleocapsid might not be the optimal target. In this regard, we propose that a combination of both antigens might contribute to the validity of the results.Abstract FigureGraphic abstractSchematic representation of sample collection and analysis. The figure was created using BioRender.com

Site-Specific Protein Covalent Attachment to Nanotubes and Its Electronic Impact on Single Molecule Function

2019 ◽  
Author(s):  
Adam Beachey ◽  
Harley Worthy ◽  
William David Jamieson ◽  
Suzanne Thomas ◽  
Benjamin Bowen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Functional Integration ◽  
Attachment Site ◽  
Covalent Attachment ◽  
Great Promise ◽  
Functional Coupling ◽  
Phenyl Azide ◽  
Electronic Impact ◽  
Protein Attachment

<p>Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of three different proteins, including the fluorescent protein GFP, to carbon nanotube side walls. Single molecule fluorescence revealed that on attachment to SWCNTs GFP’s fluorescence changed in terms of intensity and improved resistance to photobleaching; essentially GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the functional center having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein-CNT hybrid bioconjugates. It can be potentially applied easily to any protein of choice; attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position.</p>

Barcode signal amplifying strategy for sensitive and accurate protein detection on LC-MS/MS

The Analyst ◽  
2021 ◽  
Author(s):  
Chang Shu ◽  
Tengfei Li ◽  
Duo Li ◽  
Zhong-Qiu Li ◽  
Xing-Hua Xia
Keyword(s):  
Side Effects ◽  
Life Science ◽  
Protein Detection ◽  
High Specificity ◽  
Protein Drugs ◽  
P Protein ◽  
Precise Evaluation ◽  
Pharmaceutical Activity ◽  
Low Toxicity

Protein drugs showing strong pharmaceutical activity, high specificity, low toxicity and side effects, have drawn extensive attention from the field of life science and medicine. Precise evaluation of the function...

scholarly journals N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dandan Shan ◽  
Joseph M. Johnson ◽  
Syrena C. Fernandes ◽  
Hannah Suib ◽  
Soyoon Hwang ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Performance ◽  
Point Of Care ◽  
Capillary Blood ◽  
Molecular Testing ◽  
Sample Collection ◽  
Protein Assay ◽  
N Protein ◽  
Protein Levels ◽  
Percent Agreement

AbstractThe COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1–7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

scholarly journals COVID-19 Biomarkers and Advanced Sensing Technologies for Point-of-Care (POC) Diagnosis

2021 ◽  
Vol 8 (7) ◽  
pp. 98
Author(s):  
Ernst Emmanuel Etienne ◽  
Bharath Babu Nunna ◽  
Niladri Talukder ◽  
Yudong Wang ◽  
Eon Soo Lee
Keyword(s):  
Response Times ◽  
Rna Virus ◽  
Point Of Care ◽  
Protein Detection ◽  
Turnaround Time ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Specificity And Sensitivity ◽  
Small Market ◽  
Multiple Biomarkers

COVID-19, also known as SARS-CoV-2 is a novel, respiratory virus currently plaguing humanity. Genetically, at its core, it is a single-strand positive-sense RNA virus. It is a beta-type Coronavirus and is distinct in its structure and binding mechanism compared to other types of coronaviruses. Testing for the virus remains a challenge due to the small market available for at-home detection. Currently, there are three main types of tests for biomarker detection: viral, antigen and antibody. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) remains the gold standard for viral testing. However, the lack of quantitative detection and turnaround time for results are drawbacks. This manuscript focuses on recent advances in COVID-19 detection that have lower limits of detection and faster response times than RT-PCR testing. The advancements in sensing platforms have amplified the detection levels and provided real-time results for SARS-CoV-2 spike protein detection with limits as low as 1 fg/mL in the Graphene Field Effect Transistor (FET) sensor. Additionally, using multiple biomarkers, detection levels can achieve a specificity and sensitivity level comparable to that of PCR testing. Proper biomarker selection coupled with nano sensing detection platforms are key in the widespread use of Point of Care (POC) diagnosis in COVID-19 detection.

scholarly journals Direct diagnostic testing of SARS-CoV-2 without the need for prior RNA extraction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shan Wei ◽  
Esther Kohl ◽  
Alexandre Djandji ◽  
Stephanie Morgan ◽  
Susan Whittier ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Cost Effective ◽  
Clinical Samples ◽  
Nasopharyngeal Swab ◽  
Percentage Agreement ◽  
Testing Method ◽  
Viral Transport

AbstractThe COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/μl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.

scholarly journals Nanopore Technology for the Application of Protein Detection

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1942
Author(s):  
Xiaoqing Zeng ◽  
Yang Xiang ◽  
Qianshan Liu ◽  
Liang Wang ◽  
Qianyun Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Detection ◽  
High Sensitivity ◽  
Single Molecule Detection ◽  
Sequence Detection ◽  
Fast Detection ◽  
Material Basis ◽  
Sensing Technology ◽  
Detection Technology ◽  
Structure Recognition

Protein is an important component of all the cells and tissues of the human body and is the material basis of life. Its content, sequence, and spatial structure have a great impact on proteomics and human biology. It can reflect the important information of normal or pathophysiological processes and promote the development of new diagnoses and treatment methods. However, the current techniques of proteomics for protein analysis are limited by chemical modifications, large sample sizes, or cumbersome operations. Solving this problem requires overcoming huge challenges. Nanopore single molecule detection technology overcomes this shortcoming. As a new sensing technology, it has the advantages of no labeling, high sensitivity, fast detection speed, real-time monitoring, and simple operation. It is widely used in gene sequencing, detection of peptides and proteins, markers and microorganisms, and other biomolecules and metal ions. Therefore, based on the advantages of novel nanopore single-molecule detection technology, its application to protein sequence detection and structure recognition has also been proposed and developed. In this paper, the application of nanopore single-molecule detection technology in protein detection in recent years is reviewed, and its development prospect is investigated.

scholarly journals Diagnosis of SARS-Cov-2 Infection by RT-PCR Using Specimens Other Than Naso- and Oropharyngeal Swabs: A Systematic Review and Meta-Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 363
Author(s):  
Vânia M. Moreira ◽  
Paulo Mascarenhas ◽  
Vanessa Machado ◽  
João Botelho ◽  
José João Mendes ◽  
...  
Keyword(s):  
Systematic Review ◽  
Clinical Performance ◽  
Point Of Care ◽  
Meta Analysis ◽  
High Sensitivity ◽  
Nasopharyngeal Swab ◽  
Sample Collection ◽  
Rt Pcr ◽  
Oropharyngeal Swab ◽  
Nucleic Acid Assays

The rapid and accurate testing of SARS-CoV-2 infection is still crucial to mitigate, and eventually halt, the spread of this disease. Currently, nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the recommended standard sampling techniques, yet, these have some limitations such as the complexity of collection. Hence, several other types of specimens that are easier to obtain are being tested as alternatives to nasal/throat swabs in nucleic acid assays for SARS-CoV-2 detection. This study aims to critically appraise and compare the clinical performance of RT-PCR tests using oral saliva, deep-throat saliva/posterior oropharyngeal saliva (DTS/POS), sputum, urine, feces, and tears/conjunctival swab (CS) against standard specimens (NPS, OPS, or a combination of both). In this systematic review and meta-analysis, five databases (PubMed, Scopus, Web of Science, ClinicalTrial.gov and NIPH Clinical Trial) were searched up to the 30th of December, 2020. Case-control and cohort studies on the detection of SARS-CoV-2 were included. The methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS 2). We identified 1560 entries, 33 of which (1.1%) met all required criteria and were included for the quantitative data analysis. Saliva presented the higher accuracy, 92.1% (95% CI: 70.0–98.3), with an estimated sensitivity of 83.9% (95% CI: 77.4–88.8) and specificity of 96.4% (95% CI: 89.5–98.8). DTS/POS samples had an overall accuracy of 79.7% (95% CI: 43.3–95.3), with an estimated sensitivity of 90.1% (95% CI: 83.3–96.9) and specificity of 63.1% (95% CI: 36.8–89.3). The remaining index specimens could not be adequately assessed given the lack of studies available. Our meta-analysis shows that saliva samples from the oral region provide a high sensitivity and specificity; therefore, these appear to be the best candidates for alternative specimens to NPS/OPS in SARS-CoV-2 detection, with suitable protocols for swab-free sample collection to be determined and validated in the future. The distinction between oral and extra-oral salivary samples will be crucial, since DTS/POS samples may induce a higher rate of false positives. Urine, feces, tears/CS and sputum seem unreliable for diagnosis. Saliva testing may increase testing capacity, ultimately promoting the implementation of truly deployable COVID-19 tests, which could either work at the point-of-care (e.g. hospitals, clinics) or at outbreak control spots (e.g., schools, airports, and nursing homes).

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