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1.
Mikrochim Acta ; 189(5): 176, 2022 Apr 05.
Article in English | MEDLINE | ID: covidwho-1777733

ABSTRACT

A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Colorimetry/methods , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques/methods , Point-of-Care Systems , SARS-CoV-2/genetics
2.
Int J Mol Sci ; 23(6)2022 Mar 11.
Article in English | MEDLINE | ID: covidwho-1742488

ABSTRACT

In 2019, the new coronavirus disease (COVID-19), related to the severe acute respiratory syndrome coronavirus (SARS-CoV-2), started spreading around the word, giving rise to the world pandemic we are still facing. Since then, many strategies for the prevention and control of COVID-19 have been studied and implemented. In addition to pharmacological treatments and vaccines, it is mandatory to ensure the cleaning and disinfection of the skin and inanimate surfaces, especially in those contexts where the contagion could spread quickly, such as hospitals and clinical laboratories, schools, transport, and public places in general. Here, we report the efficacy of ZnO nanoparticles (ZnONPs) against SARS-CoV-2. NPs were produced using an ecofriendly method and fully characterized; their antiviral activity was tested in vitro against SARS-CoV-2, showing a decrease in viral load between 70% and 90%, as a function of the material's composition. Application of these nano-antimicrobials as coatings for commonly touched surfaces is envisaged.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/prevention & control , Nanostructures/chemistry , SARS-CoV-2/drug effects , Zinc Oxide/pharmacology , Antiviral Agents/chemistry , COVID-19/chemically induced , COVID-19/epidemiology , Colorimetry , Humans , Microbial Sensitivity Tests/methods , Microscopy, Electron, Transmission , Nanostructures/ultrastructure , Pandemics/prevention & control , Photoelectron Spectroscopy , SARS-CoV-2/physiology , Spectroscopy, Fourier Transform Infrared , Treatment Outcome , Viral Load/drug effects , X-Ray Diffraction , Zinc Oxide/chemistry
3.
Talanta ; 243: 123393, 2022 Jun 01.
Article in English | MEDLINE | ID: covidwho-1740208

ABSTRACT

We present a fast, reliable and easy to scale-up colorimetric sensor based on gold nanoparticles (AuNPs) to detect the sequences coding for the RdRp, E, and S proteins of SARS-CoV-2. The optimization of the system (so-called "the sensor") includes the evaluation of different sizes of nanoparticles, sequences of oligonucleotides and buffers. It is stable for months without any noticeable decrease in its activity, allowing the detection of SARS-CoV-2 sequences by the naked eye in 15 min. The efficiency and selectivity of detection, in terms of significative colorimetric changes in the solution upon target recognition, are qualitatively (visually) and quantitatively (absorbance measurements) assessed using synthetic samples and samples derived from infected cells and patients. Furthermore, an easy and affordable amplification approach is implemented to increase the system's sensitivity for detecting high and medium viral loads (≥103 - 104 viral RNA copies/µl) in patient samples. The whole process (amplification and detection) takes 2.5 h. Due to the ease of use, stability and minimum equipment requirements, the proposed approach can be a valuable tool for the detection of SARS-CoV-2 at facilities with limited resources.


Subject(s)
COVID-19 , Metal Nanoparticles , COVID-19/diagnosis , Colorimetry , Gold , Humans , RNA, Viral/genetics , RNA-Dependent RNA Polymerase , SARS-CoV-2/genetics
4.
Sci Rep ; 12(1): 3775, 2022 03 08.
Article in English | MEDLINE | ID: covidwho-1735272

ABSTRACT

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing/instrumentation , Colorimetry , Humans , Limit of Detection , Molecular Diagnostic Techniques , Nasopharynx/virology , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/pathology , Nucleic Acid Amplification Techniques , Point-of-Care Testing , Proto-Oncogene Proteins B-raf/genetics , RNA, Viral/analysis , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Smartphone
5.
Sensors (Basel) ; 22(5)2022 Feb 27.
Article in English | MEDLINE | ID: covidwho-1715645

ABSTRACT

Measuring the colorimetric signals produced by the biospecific accumulation of colorimetric probes and recording the results is a key feature for next-generation paper-based rapid tests. Manual processing of these tests is time-consuming and prone to a loss of accuracy when interpreting faint and patchy signals. Proprietary, closed-source readers and software companies offering automated smartphone-based assay readings have both been criticized for interoperability issues. Here, we introduce a minimal reader prototype composed of open-source hardware and open-source software that has the benefits of automatic assay quantification while avoiding the interoperability issues associated with closed-source readers. An image-processing algorithm was developed to automate the selection of an optimal region of interest and measure the average pixel intensity. When used to quantify signals produced by lateral flow immunoassays for detecting antibodies against SARS-CoV-2, results obtained with the proposed algorithm were comparable to those obtained with a manual method but with the advantage of improving the precision and accuracy when quantifying small spots or faint and patchy signals.


Subject(s)
Biosensing Techniques , COVID-19 , COVID-19/diagnosis , Colorimetry/methods , Humans , Immunoassay/methods , SARS-CoV-2
6.
Biosens Bioelectron ; 200: 113909, 2022 Mar 15.
Article in English | MEDLINE | ID: covidwho-1670212

ABSTRACT

Coronavirus disease 2019 (COVID-19) has been recognized as a global pandemic outbreak, opening the most severe socio-economic crisis since World War II. Different scientific activities have been emerged in this global scenario, including the development of innovative analytical tools to measure nucleic acid, antibodies, and antigens in the nasopharyngeal swab, serum, and saliva for prompt identification of COVID-19 patients and to evaluate the immune response to the vaccine. The detection of SARS-CoV-2 in saliva remains a challenge for the lack of sufficient sensitivity. To address this issue, we developed a novel paper-based immunoassay using magnetic beads to support the immunological chain and 96-well wax-printed paper plate as a platform for color visualization by using a smartphone combined with Spotxel free-charge app. To assess the reliability of the measurement of SARS-CoV-2 in saliva, untreated saliva was used as a specimen and the calibration curve demonstrated a dynamic range up to 10 µg/mL, with a detection limit equal to 0.1 µg/mL. The effectiveness of this sustainable analytical tool in saliva was evaluated by comparing the data with the nasopharyngeal swab specimens sampled by the same patients and tested with Real-Time PCR reference method, founding 100% of agreement, even in the case of high Cycle Threshold (CT) numbers (low viral load). Furthermore, the positive saliva samples were characterized by the next-generation sequencing method, demonstrating the capability to detect the Delta variant, which is actually (July 2021) the most relevant variant of concern.


Subject(s)
Biosensing Techniques , COVID-19 , Colorimetry , Humans , Immunoassay , Magnetic Phenomena , Nasopharynx , Reproducibility of Results , SARS-CoV-2 , Saliva , Smartphone , Specimen Handling
7.
PLoS One ; 17(1): e0259886, 2022.
Article in English | MEDLINE | ID: covidwho-1666744

ABSTRACT

COVID-19 has exposed stark inequalities between resource-rich and resource-poor countries. International UN- and WHO-led efforts, such as COVAX, have provided SARS-CoV-2 vaccines but half of African countries have less than 2% vaccinated in their population, and only 15 have reached 10% by October 2021, further disadvantaging local economic recovery. Key for this implementation and preventing further mutation and spread is the frequency of voluntary [asymptomatic] testing. It is limited by expensive PCR and LAMP tests, uncomfortable probes deep in the throat or nose, and the availability of hardware to administer in remote locations. There is an urgent need for an inexpensive "end-to-end" system to deliver sensitive and reliable, non-invasive tests in resource-poor and field-test conditions. We introduce a non-invasive saliva-based LAMP colorimetric test kit and a $51 lab-in-a-backpack system that detects as few as 4 viral RNA copies per µL. It consists of eight chemicals, a thermometer, a thermos bottle, two micropipettes and a 1000-4000 rcf electronically operated centrifuge made from recycled computer hard drives (CentriDrive). The centrifuge includes a 3D-printed rotor and a 12 V rechargeable Li-ion battery, and its 12 V standard also allows wiring directly to automobile batteries, to enable field-use of this and other tests in low infrastructure settings. The test takes 90 minutes to process 6 samples and has reagent costs of $3.5 per sample. The non-invasive nature of saliva testing would allow higher penetration of testing and wider adoption of the test across cultures and settings (including refugee camps and disaster zones). The attached graphical procedure would make the test suitable for self-testing at home, performing it in the field, or in mobile testing centers by minimally trained staff.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , COVID-19/virology , COVID-19 Nucleic Acid Testing/economics , COVID-19 Nucleic Acid Testing/methods , Colorimetry , Humans , Molecular Diagnostic Techniques/economics , Molecular Diagnostic Techniques/instrumentation , Nucleic Acid Amplification Techniques/economics , Nucleic Acid Amplification Techniques/instrumentation , Point-of-Care Systems , RNA, Viral/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology
8.
Int J Infect Dis ; 116: 133-137, 2022 Mar.
Article in English | MEDLINE | ID: covidwho-1650171

ABSTRACT

BACKGROUND: COVID-19, which is caused by SARS-CoV-2 and its variants, poses an ongoing global threat, particularly in low-immunization coverage regions. Thus, rapid, accurate, and easy-to-perform diagnostic methods are in urgent demand to halt the spread of the virus. OBJECTIVES: We aimed to validate the clinical performance of the FastProof 30 min-TTR SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP) method using leftover RNA samples extracted from 315 nasopharyngeal swabs. The sensitivity and specificity of RT-LAMP were determined in comparison with reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Of 315 nasopharyngeal swabs, viral RNA was detected in 154 samples (48.9%) by RT-PCR assay. Compared with RT-PCR, overall sensitivity and specificity of RT-LAMP were 81.82% (95% CI: 74.81-87.57) and 100% (95% CI: 97.73-100), respectively. A 100% positivity rate was achieved in samples with cycle threshold (Ct) <31 for RT-PCR targeting the ORF1ab gene. However, samples with Ct >31 accounted for false-negative results by RT-LAMP in 28 samples. CONCLUSIONS: RT-LAMP reliably detected viral RNA with high sensitivity and specificity and has potential application for mass screening of patients with acute COVID-19 infection when viral load is high.


Subject(s)
COVID-19 , COVID-19/diagnosis , Colorimetry/methods , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcription , SARS-CoV-2/genetics , Sensitivity and Specificity , Thailand/epidemiology
9.
Angew Chem Int Ed Engl ; 61(9): e202112995, 2022 02 21.
Article in English | MEDLINE | ID: covidwho-1633678

ABSTRACT

The transmission of SARS-CoV-2 coronavirus has led to the COVID-19 pandemic. Nucleic acid testing while specific has limitations for mass surveillance. One alternative is the main protease (Mpro ) due to its functional importance in mediating the viral life cycle. Here, we describe a combination of modular substrate and gold colloids to detect Mpro via visual readout. The strategy involves zwitterionic peptide that carries opposite charges at the C-/N-terminus to exploit the specific recognition by Mpro . Autolytic cleavage releases a positively charged moiety that assembles the nanoparticles with rapid color changes (t<10 min). We determine a limit of detection for Mpro in breath condensate matrices <10 nM. We further assayed ten COVID-negative subjects and found no false-positive result. In the light of simplicity, our test for viral protease is not limited to an equipped laboratory, but also is amenable to integrating as portable point-of-care devices including those on face-coverings.


Subject(s)
COVID-19/diagnosis , Coronavirus 3C Proteases/metabolism , Peptides/metabolism , SARS-CoV-2/metabolism , Biomarkers/metabolism , Breath Tests , COVID-19/virology , Colorimetry/methods , Humans , Limit of Detection , Proteolysis
10.
Biosensors (Basel) ; 12(1)2022 Jan 07.
Article in English | MEDLINE | ID: covidwho-1632763

ABSTRACT

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited.


Subject(s)
Magnetite Nanoparticles , Mycobacterium tuberculosis , Tuberculosis, Pulmonary , Colorimetry , Diagnostic Tests, Routine , Humans , Polysorbates , Sensitivity and Specificity
11.
J Biomol Tech ; 32(3): 134-136, 2021 09.
Article in English | MEDLINE | ID: covidwho-1625529

ABSTRACT

At this writing, over 100 million people have tested positive for Corona Virus Disease-19 (COVID-19), and the global death toll from this disease has reached nearly 3 million. Despite the many tests currently available, we have not yet achieved the testing capacity needed to limit the spread of the virus and mitigate suffering worldwide. We have developed the One Hour COVID Test to address this challenge. Our test leverages an easy-to-use, commercially available oral swab kit for sample collection paired with a novel RNA processing protocol and a simple colorimetric assay that requires minimal equipment. The test can be easily scaled via automation and takes 1 h from sample collection to result.


Subject(s)
COVID-19 , Colorimetry , COVID-19 Testing , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Prothrombin Time , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
12.
Molecules ; 27(3)2022 Jan 18.
Article in English | MEDLINE | ID: covidwho-1625180

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) has been regarded as one of the ideal targets for the development of antiviral drugs. The currently used methods for the probing of Mpro activity and the screening of its inhibitors require the use of a double-labeled peptide substrate. In this work, we suggested that the label-free peptide substrate could induce the aggregation of AuNPs through the electrostatic interactions, and the cleavage of the peptide by the Mpro inhibited the aggregation of AuNPs. This fact allowed for the visual analysis of Mpro activity by observing the color change of the AuNPs suspension. Furthermore, the co-assembly of AuNPs and peptide was achieved on the peptide-covered electrode surface. Cleavage of the peptide substrate by the Mpro limited the formation of AuNPs/peptide assembles, thus allowing for the development of a simple and sensitive electrochemical method for Mpro detection in serum samples. The change of the electrochemical signal was easily monitored by electrochemical impedance spectroscopy (EIS). The detection limits of the colorimetric and electrochemical methods are 10 and 0.1 pM, respectively. This work should be valuable for the development of effective antiviral drugs and the design of novel optical and electrical biosensors.


Subject(s)
Colorimetry/methods , Coronavirus 3C Proteases/analysis , Electrochemical Techniques , Gold , Metal Nanoparticles , SARS-CoV-2/enzymology , Feasibility Studies , Humans , Sensitivity and Specificity
13.
Anal Methods ; 14(4): 378-382, 2022 01 27.
Article in English | MEDLINE | ID: covidwho-1617007

ABSTRACT

Improved diagnostics are needed to manage the ongoing COVID-19 pandemic. In this study, we enhanced the color changes and sensitivity of colorimetric SARS-CoV-2 RT-LAMP assays based on triarylmethane dyes. We determined a mechanism for the color changes and obtained sensitivities of 10 RNA copies per microliter.


Subject(s)
COVID-19 , SARS-CoV-2 , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Pandemics , RNA, Viral/genetics , Reproducibility of Results
14.
ACS Synth Biol ; 11(1): 383-396, 2022 01 21.
Article in English | MEDLINE | ID: covidwho-1599309

ABSTRACT

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.


Subject(s)
African Swine Fever Virus/genetics , African Swine Fever , COVID-19 Nucleic Acid Testing , COVID-19 , CRISPR-Cas Systems , SARS-CoV-2/genetics , African Swine Fever/diagnosis , African Swine Fever/genetics , Animals , COVID-19/diagnosis , COVID-19/genetics , Colorimetry , Humans , Swine
15.
Exp Biol Med (Maywood) ; 247(3): 276-281, 2022 02.
Article in English | MEDLINE | ID: covidwho-1571696

ABSTRACT

The global SARS-CoV-2 pandemic requires a rapid, reliable, and user-friendly diagnostic test to help control the spread of the virus. Reverse transcription and quantitative PCR (RT-qPCR) is currently the gold standard method for SARS-CoV-2 detection. Here, we develop a protocol based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) and demonstrate increased sensitivity of this technique using fresh RNA extracts compared to RNA samples subjected to freezing/thawing cycles. We further compare RT-LAMP to RT-qPCR and demonstrate that the RT-LAMP approach has high sensitivity in fresh RNA extracts and can detect positive samples with Ct values between 8 and 35.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/isolation & purification , Colorimetry/methods , DNA Primers , Humans , Nasopharynx/virology , Reverse Transcription , SARS-CoV-2/genetics , Sensitivity and Specificity
16.
Analyst ; 146(19): 6026-6034, 2021 Sep 27.
Article in English | MEDLINE | ID: covidwho-1545657

ABSTRACT

The ongoing outbreaks of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in unprecedented challenges to global health. To effectively contain the COVID-19 transmission, rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. To address the huge need for ever-increasing tests, we developed a facile all-in-one nucleic acid testing assay by combining Si-OH activated glass bead (aGB)-based viral RNA fast extraction and in situ colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection in a single tube. aGBs demonstrate a strong ability to capture viral RNA in a guanidinium-based lysis buffer, and the purified aGBs/RNA composite, without RNA elution step, could be directly used to perform RT-LAMP assay. The assay was well characterized by using a novel SARS-CoV-2-like coronavirus GX/P2V, and showed a limit of detection (LOD) of 15 copies per µL in simulated clinical samples within 50 min. We further demonstrated our assay by testing simulated SARS-CoV-2 pseudovirus samples, showing an LOD of 32 copies per µL and high specificity without cross-reactivity with the most closely related GX/P2V or host DNA/RNA. The all-in-one approach developed in this study has the potential as a simple, scalable, and time-saving alternative for point-of-care testing of SARS-CoV-2 in low-income regions, as well as a promising tool for at-home testing.


Subject(s)
COVID-19 , SARS-CoV-2 , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Point-of-Care Testing , RNA, Viral/genetics , Sensitivity and Specificity
17.
J Hazard Mater ; 425: 127923, 2022 03 05.
Article in English | MEDLINE | ID: covidwho-1536650

ABSTRACT

The metallopeptidase angiotensin-converting enzyme 2 (ACE2) is the SARS-CoV-2 receptor required for viral entry based on its specific recognition of the spike protein receptor binding domain (S_RBD) on SARS-CoV-2. We constructed a human ACE2 (hACE2)-based peptide pair by ligating discontinuous key residues involved in the hACE2-S_RBD interaction. We firstly performed in silico simulations to identify a 12-mer and 15-mer peptide pair with capability to bind to the SARS-CoV-2 S_RBD via different binding sites. Then, the bio-layer interferometry validated the specific interactions between the peptides and S_RBD, with affinities at the nanomolar level. Lastly, we developed a colorimetric sandwich-type bioassay based on S_RBD-specific peptide-modified gold nanoparticles and found the colorimetric bioassay offered fast (<30 min), simple, and sensitive detection of S_RBD protein at levels as low as 0.01 nM (0.26 ng mL-1) in SARS-CoV-2. The linear signals ranging from 105 to 107 virus copies mL-1 were achieved in typical types of environmental waters spiked with lysed SARS-CoV-2 pseudovirus. The technology can serve as a beneficial supplement to the routine virus nucleic acid detection in environment media and wastewater treatment.


Subject(s)
Colorimetry , Metal Nanoparticles , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2 , Biological Assay , COVID-19/diagnosis , Gold , Humans , Peptides , Protein Binding , Spike Glycoprotein, Coronavirus/metabolism
18.
Lab Chip ; 21(21): 4249-4261, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1450297

ABSTRACT

Microfluidic paper-based analytical devices (µPADs) are foundational devices for point-of-care testing, yet suffer from limitations in regards to their sensitivity and capability in handling complex assays. Here, we demonstrate an airflow-based, evaporative method that is capable of manipulating fluid flows within paper membranes to offer new functionalities for multistep delivery of reagents and improve the sensitivity of µPADs by 100-1000 times. This method applies an air-jet to a pre-wetted membrane, generating an evaporative gradient such that any solutes become enriched underneath the air-jet spot. By controlling the lateral position of this spot, the solutes in the paper strip are enriched and follow the air jet trajectory, driving the reactions and enhancing visualization for colorimetric readout in multistep assays. The technique has been successfully applied to drive the sequential delivery in multistep immunoassays as well as improve sensitivity for colorimetric detection assays for nucleic acids and proteins via loop-mediated isothermal amplification (LAMP) and ELISA. For colorimetric LAMP detection of the COVID-19 genome, enrichment of the solution on paper could enhance the contrast of the dye in order to more clearly distinguish between the positive and negative results to achieve a sensitivity of 3 copies of SARS-Cov-2 RNAs. For ELISA, enrichment of the oxidized TMB substrate yielded a sensitivity increase of two-to-three orders of magnitude when compared to non-enriched samples - having a limit of detection of around 200 fM for IgG. Therefore, this enrichment method represents a simple process that can be easily integrated into existing detection assays for controlling fluid flows and improving detection of biomarkers on paper.


Subject(s)
COVID-19 , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Sensitivity and Specificity
19.
Mikrochim Acta ; 188(10): 335, 2021 Sep 09.
Article in English | MEDLINE | ID: covidwho-1411927

ABSTRACT

A practical colorimetric assay was developed for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For this purpose, magnetic γ Fe2O3 nanoparticles were synthesized and used as a peroxidase-like mimic activity molecule. In the presence of γ Fe2O3 nanoparticles, the color change of H2O2 included 3,3',5,5'-tetramethylbenzidine was monitored at the wavelength of 654 nm when spike protein interacted with angiotensin-converting enzyme 2 receptor. This oxidation-reduction reaction was examined both spectroscopically and by using electrochemical techniques. The experimental parameters were optimized and the analytical characteristics investigated. The developed assay was applied to real SARS-CoV-2 samples, and very good results that were in accordance with the real time polymerase chain reaction were obtained.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Colorimetry/methods , Magnetic Iron Oxide Nanoparticles/chemistry , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Benzidines/chemistry , Biosensing Techniques/methods , COVID-19 Testing/instrumentation , Catalysis , Chromogenic Compounds/chemistry , Cysteine/chemistry , Humans , Hydrogen Peroxide/chemistry , Limit of Detection , Nasopharynx/virology , Oropharynx/virology , Oxidation-Reduction , Peroxidase/chemistry , Spike Glycoprotein, Coronavirus/metabolism
20.
Analyst ; 146(19): 6026-6034, 2021 Sep 27.
Article in English | MEDLINE | ID: covidwho-1402132

ABSTRACT

The ongoing outbreaks of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in unprecedented challenges to global health. To effectively contain the COVID-19 transmission, rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. To address the huge need for ever-increasing tests, we developed a facile all-in-one nucleic acid testing assay by combining Si-OH activated glass bead (aGB)-based viral RNA fast extraction and in situ colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) detection in a single tube. aGBs demonstrate a strong ability to capture viral RNA in a guanidinium-based lysis buffer, and the purified aGBs/RNA composite, without RNA elution step, could be directly used to perform RT-LAMP assay. The assay was well characterized by using a novel SARS-CoV-2-like coronavirus GX/P2V, and showed a limit of detection (LOD) of 15 copies per µL in simulated clinical samples within 50 min. We further demonstrated our assay by testing simulated SARS-CoV-2 pseudovirus samples, showing an LOD of 32 copies per µL and high specificity without cross-reactivity with the most closely related GX/P2V or host DNA/RNA. The all-in-one approach developed in this study has the potential as a simple, scalable, and time-saving alternative for point-of-care testing of SARS-CoV-2 in low-income regions, as well as a promising tool for at-home testing.


Subject(s)
COVID-19 , SARS-CoV-2 , Colorimetry , Humans , Molecular Diagnostic Techniques , Nucleic Acid Amplification Techniques , Point-of-Care Testing , RNA, Viral/genetics , Sensitivity and Specificity
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