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1.
Anal Chem ; 94(47): 16436-16442, 2022 Nov 29.
Article in English | MEDLINE | ID: covidwho-2116713

ABSTRACT

Label-free electrochemiluminescence (ECL) immunoassays (lf-ECLIA), based on biomarker-induced ECL signal changes, have attracted increasing attention due to the simple, rapid, and low-cost detection of biomarkers without secondary antibodies and complicated labeling procedures. However, the interaction rule and mechanism between analytical interfaces and biomarkers have rarely been explored. Herein, the interactions between biomarkers and analytical interfaces constructed by assembly of a nanoluminophore and antibody-functionalized gold nanoparticles on an indium tin oxide electrode were studied. The nanoluminophore was synthesized by mixing Cu2+/l-cysteine chelate and N-(4-Aminobutyl)-N-ethylisoluminol-bifunctionalized gold nanoparticles with chitosan. It was found that positively charged biomarkers increased the ECL intensity, whereas negatively charged biomarkers decreased the ECL intensity. The assembly pH influenced the biomarker charges, which determined the ECL enhancement or inhibition. The detection pH only affected the ECL intensity but not the ECL changing trends. Based on the ECL signal changes, a charge-dependent lf-ECLIA was established, which exhibited inhibition responses to negatively charged human immunoglobulin G and copeptin and enhancement responses to positively charged cardiac troponin I, heart-type fatty acid binding protein, brain natriuretic peptide, and SARS-CoV-2 N protein. The linear range was 0.1-1000 pg/mL, and the detection limits were distributed in 0.024-0.091 pg/mL. Besides, a mechanism of the charge-dependent ECL enhancement and inhibition effects is proposed, which is very important for the development of new lf-ECLIA methodologies.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Humans , Gold , Luminescent Measurements/methods , Biosensing Techniques/methods , SARS-CoV-2 , Immunoassay/methods , Biomarkers , Electrochemical Techniques/methods , Limit of Detection
2.
Anal Chim Acta ; 1232: 340442, 2022 Nov 01.
Article in English | MEDLINE | ID: covidwho-2104217

ABSTRACT

In the present work, we report an innovative approach for immunosensors construction. The experimental strategy is based on the anchoring of biological material at screen-printed carbon electrode (SPE) modified with electrodeposited Graphene Quantum Dots (GQD) and polyhydroxybutyric acid (PHB). It was used as functional substract basis for the recognition site receptor-binding domain (RBD) from coronavirus spike protein (SARS-CoV-2), for the detection of Anti-S antibodies (AbS). SEM images and EDS spectra suggest an interaction of the protein with GQD-PHB sites at the electrode surface. Differential pulse voltametric (DPV) measurements were performed before and after incubation, in presence of the target, shown a decrease in voltametric signal of an electrochemical probe ([Fe(CN)6]3/4-). Using the optimal experimental conditions, analytical curves were performed in PBS and human serum spiked with AbS showing a slight matrix effect and a relationship between voltametric signal and AbS concentration in the range of 100 ng mL-1 and 10 µg mL-1. The selectivity of the proposed sensor was tested against yellow fever antibodies (YF) and the selective layer on the electrode surface did not interact with these unspecific antibodies. Eight samples of blood serum were analyzed and 87.5% of these total investigated provided adequate results. In addition, the present approach showed better results against traditional EDC/NHS reaction with enhancements in time and the possibility to develop an immunosensor in a single drop, since the proteins can be anchored prior to the electrode modification step.


Subject(s)
Biosensing Techniques , COVID-19 , Graphite , Quantum Dots , Humans , Graphite/chemistry , Quantum Dots/chemistry , SARS-CoV-2 , Electrochemical Techniques/methods , Spike Glycoprotein, Coronavirus , Limit of Detection , Immunoassay , Electrodes , Carbon/chemistry , Antibodies
3.
Int J Mol Sci ; 23(21)2022 Oct 28.
Article in English | MEDLINE | ID: covidwho-2090210

ABSTRACT

The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a threat to public health and a worldwide crisis. This raised the need for quick, effective, and sensitive detection tools to prevent the rapid transmission rate of the infection. Therefore, this study aimed to develop an electrochemical impedance spectroscopy (EIS)-based aptasensor employing an interdigitated gold electrode (IDE) to detect SARS-CoV-2 Spike (S) glycoprotein and viral particles. This allowed us to sensitively detect SARS-CoV-2 S glycoprotein with a limit of detection (LOD) of 0.4 pg/mL in a buffer solution and to obtain a linear increase for concentrations between 0.2 to 0.8 pg/mL with high specificity. The proposed aptasensor also showed a good sensitivity towards the heat-inactivated SARS-CoV-2 variants in a buffer solution, where the Delta, Wuhan, and Alpha variants were captured at a viral titer of 6.45 ± 0.16 × 103 TCID50/mL, 6.20 × 104 TCID50/mL, and 5.32 ± 0.13 × 102 TCID50/mL, respectively. Furthermore, the detection of SARS-CoV-2 performed in a spiked human nasal fluid provided an LOD of 6.45 ± 0.16 × 103 TCID50/mL for the Delta variant in a 50 µL sample and a detection time of less than 25 min. Atomic force microscopy images complemented the EIS results in this study, revealing that the surface roughness of the IDE after each modification step increased, which indicates that the target was successfully captured. This label-free EIS-based aptasensor has promising potential for the rapid detection of SARS-CoV-2 in complex clinical samples.


Subject(s)
Biosensing Techniques , COVID-19 , Humans , SARS-CoV-2/genetics , Dielectric Spectroscopy , Biosensing Techniques/methods , COVID-19/diagnosis , Limit of Detection , Gold/chemistry , Electrodes , Electrochemical Techniques/methods
4.
Biosensors (Basel) ; 12(10)2022 Oct 17.
Article in English | MEDLINE | ID: covidwho-2071229

ABSTRACT

The development of immunosensors to detect antibodies or antigens has stood out in the face of traditional methods for diagnosing emerging diseases such as the one caused by the SARS-CoV-2 virus. The present study reports the construction of a simplified electrochemical immunosensor using a graphene-binding peptide applied as a recognition site to detect SARS-CoV-2 antibodies. A screen-printed electrode was used for sensor preparation by adding a solution of peptide and reduced graphene oxide (rGO). The peptide-rGO suspension was characterized by scanning electron microscopy (SEM), Raman spectroscopy, and Fourier transform infrared spectroscopy (FT-IR). The electrochemical characterization (electrochemical impedance spectroscopy-EIS, cyclic voltammetry-CV and differential pulse voltammetry-DPV) was performed on the modified electrode. The immunosensor response is based on the decrease in the faradaic signal of an electrochemical probe resulting from immunocomplex formation. Using the best set of experimental conditions, the analytic curve obtained showed a good linear regression (r2 = 0.913) and a limit of detection (LOD) of 0.77 µg mL-1 for antibody detection. The CV and EIS results proved the efficiency of device assembly. The high selectivity of the platform, which can be attributed to the peptide, was demonstrated by the decrease in the current percentage for samples with antibody against the SARS-CoV-2 S protein and the increase in the other antibodies tested. Additionally, the DPV measurements showed a clearly distinguishable response in assays against human serum samples, with sera with a response above 95% being considered negative, whereas responses below this value were considered positive. The diagnostic platform developed with specific peptides is promising and has the potential for application in the diagnosis of other infections that lead to high antibody titers.


Subject(s)
Biosensing Techniques , COVID-19 , Graphite , Humans , Graphite/chemistry , Biosensing Techniques/methods , Electrochemical Techniques/methods , SARS-CoV-2 , Spectroscopy, Fourier Transform Infrared , Immunoassay , COVID-19/diagnosis , Electrodes , Limit of Detection , Peptides
5.
PLoS One ; 17(10): e0275838, 2022.
Article in English | MEDLINE | ID: covidwho-2065150

ABSTRACT

The World Health Organization (WHO) emphasizes that tuberculosis (TB) in children and adolescents is often overlooked by healthcare providers and difficult to diagnose. As childhood TB cases rise, finding a diagnostic high in sensitivity and specificity is critical. In this study 91 urine samples from children aged 1-10 years were analyzed for tuberculostearic acid (TBSA) by gas chromatography/mass spectrometry (GC/MS) and capture ELISA (C-ELISA). In C-ELISA the CS35/A194-01 antibody performed very poorly with both curve-based and model-based cutoffs. The area under the ROC curve (AUC) of the CS35 OD450 values was only 0.60. Replacing the capture antibody with BJ76 gave a better performance in both sensitivity and specificity (AUC = 0.95). When these samples were analyzed by GC/MS, 41 classified as 'probable/possible' for TB were distinctly TBSA positive with ten samples having <3 ng/mL LAM. However, from the 50 samples with 'unlikely' TB classification, 36 were negative but 7 had >3 ng/mL and were designated as LAM positive. This experimental assay assessment study signifies that i) the antibody pair CS35/A194-01 that has been successful for adult active TB diagnosis is not adequate when LAM level is low as in pediatric TB; ii) no one mAb appears to recognize all TB-specific LAM epitopes.


Subject(s)
HIV Infections , Mycobacterium tuberculosis , Tuberculosis , Adolescent , Adult , Antibodies , Child , Epitopes , Humans , Limit of Detection , Lipopolysaccharides , Sensitivity and Specificity , Tuberculosis/diagnosis
6.
Anal Biochem ; 658: 114928, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-2041444

ABSTRACT

In the present work, an aptasensing method based on integration of RNA on Cu-MOF was developed for detection of C-Reactive Protein (CRP). Cu-MOF showed stimulated fluorescence and mimetic peroxidase enzymatic activity at the time and can be used as dual-signal transduction. CRP binding RNA was used as a highly selective recognition element and immobilized on the Cu-MOF. The immobilized RNA can block the peroxidase activity and fluorescence of the signal traducer probe. Adding CRP to the RNA/Cu-MOF will release RNA from the surface of Cu-MOF and recover both the stimulated fluorescence and peroxidase activity. A biosensor was built for detection of CRP using the two modes of transduction, either colorimetry or fluorometry. A dynamic linear range was obtained from 0.1 to 50 ng mL -1with a limit of detection (LOD) as small as 40 pg mL -1was calculated in fluorescence mode and 240 pg mL -1 as LOD in colorimetry mode. The LODs are lower than the LOD of nephelometric techniques used in clinical practice and is comparable to the normal clinical cutoff value in high-sensitivity CRP assays (1 µg/mL). The aptasensor was successfully applied for detection of CRP in Covid-19 patients with spike recoveries between 84 and 102% and RSD from 0.94% to 2.05%.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , Humans , C-Reactive Protein , Immobilized Nucleic Acids , Biosensing Techniques/methods , Limit of Detection , Peroxidase , RNA
7.
J Pharm Biomed Anal ; 221: 115076, 2022 Nov 30.
Article in English | MEDLINE | ID: covidwho-2041967

ABSTRACT

To protect critical injury from blood clots with side effects in severe COVID-19, a highly selective and sensitive biosensor was developed for the quantification of trace levels of thrombin using the combination of a DNA aptamer (TBA) of thrombin and a complementary strand of TBA. TBA rapidly binds with thrombin, whereas it slowly binds with the complementary strand to form a double stranded DNA (dsDNA). SFC green intercalated into dsDNA cannot emit light in 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction because high-energy intermediates formed from ODI-CL reaction cannot transfer energy to SFC trapped in dsDNA. However, SFC freely existing with the formation of G-quadruplex from the reaction of thrombin and TBA emits bright chemiluminescence because the high-energy intermediates can transfer energy to SFC (or camel) in solution. Thus, the brightness of light emitted in ODI-CL reaction was proportionally enhanced with the increase of thrombin in a sample due to the increase of G-quadruplex and reduction of dsDNA. The limit of detection (LOD) of the label free aptasensor operated with good linear calibration curve (10-320 mU/ml) was as low as 3 mU/ml (or 43 pM). Also, the biosensor was quantified trace levels of thrombin with good accuracy, precision, and reliability.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , COVID-19/diagnosis , Humans , Limit of Detection , Reproducibility of Results , Thrombin
8.
Biosens Bioelectron ; 217: 114714, 2022 Dec 01.
Article in English | MEDLINE | ID: covidwho-2031161

ABSTRACT

Timely and accurate detection of virus is crucial for preventing spread of disease and early treatment of the infected cases. Herein we design an integrated logic-operated three-dimensional DNA walker for colorimetric detection of viral RNA fragments, by taking SARS-CoV-2 as an example. The DNA walker is composed of small amounts of dually-blocked walking strands and large amounts of dual-stem-loop track strands on gold nanoparticles. The walking strand contains a swing arm domain and a DNAzyme domain blocked at both sides of catalytic core, while the track strand contains a substrate domain located at the peripheral larger loop. Only the presence of both ORF1ab and N RNA fragments can fully de-block the walking strand, which then continuously hybridizes with track strands and cleaves them by DNAzyme-catalyzed hydrolysis. As the cleavage of track strands from long-stranded, double stem-loop structure to short-stranded, linear sequence, the DNA walker shows much lowered stability due to decreased negative charge density and diminished steric repulsion, which then gets aggregated at high salt concentration, accompanied by a visible color change. The colorimetric DNA walker detects RNA fragments down to 1 nM, responds dual viral genes in a "AND" logic way, and shows high specificity to target sequence. It can further detect large nucleic acids containing ORF1ab and N sequences, and reach 200 copies/mL detection limit by coupling a simple upstream amplification of sample. The method may provide a convenient way for reliable detection of viral RNA.


Subject(s)
Biosensing Techniques , COVID-19 , DNA, Catalytic , Metal Nanoparticles , Biosensing Techniques/methods , Colorimetry/methods , DNA/chemistry , DNA, Catalytic/chemistry , Gold/chemistry , Humans , Limit of Detection , Logic , Metal Nanoparticles/chemistry , RNA, Viral/genetics , SARS-CoV-2
9.
Int J Mol Sci ; 23(17)2022 Sep 01.
Article in English | MEDLINE | ID: covidwho-2023754

ABSTRACT

Carbohydrate antigen 199 (CA199) is a serum biomarker which has certain value and significance in the diagnosis, prognosis, treatment, and postoperative monitoring of cancer. In this study, a lateral flow immunoassay based on europium (III) polystyrene time-resolved fluorescence microspheres (TRFM-based LFIA), integrated with a portable fluorescence reader, has been successfully establish for rapid and quantitative analysis of CA199 in human serum. Briefly, time-resolved fluorescence microspheres (TRFMs) were conjugated with antibody I (Ab1) against CA199 as detection probes, and antibody II (Ab2) was coated as capture element, and a "TRFMs-Ab1-CA199-Ab2" sandwich format would form when CA199 was detected by the TRFM-based LFIA. Under the optimal parameters, the detection limit of the TRFM-based LFIA for visible quantitation with the help of an ultraviolet light was 4.125 U/mL, which was four times lower than that of LFIA based on gold nanoparticles. Additionally, the fluorescence ratio is well linearly correlated with the CA199 concentration (0.00-66.0 U/mL) and logarithmic concentration (66.0-264.0 U/mL) for quantitative detection. Serum samples from 10 healthy people and 10 liver cancer patients were tested to confirm the performances of the point-of-care application of the TRFM-based LFIA, 20.0 U/mL of CA199 in human serum was defined as the threshold for distinguishing healthy people from liver cancer patients with an accuracy of about 60%. The establishment of TRFM-based LFIA will provide a sensitive, convenient, and efficient technical support for rapid screening of CA199 in cancer diagnosis and prognosis.


Subject(s)
Liver Neoplasms , Metal Nanoparticles , Biomarkers, Tumor , Gold , Humans , Immunoassay , Limit of Detection , Microspheres
10.
Biosensors (Basel) ; 12(8)2022 Jul 25.
Article in English | MEDLINE | ID: covidwho-2023152

ABSTRACT

Alzheimer's disease (AD) is a long-term neurodegenerative disease that poses a serious threat to human life and health. It is very important to develop a portable quantitative device for AD diagnosis and personal healthcare. Herein, we develop a portable electrochemical sensing platform for the point-of-care detection of AD biomarkers in the blood. Such a portable platform integrates nanoAu-modified vertical graphene (VG@Au) into a working electrode, which can significantly improve sensitivity and reduce detection limit due to the large specific surface, excellent electrical conductivity, high stability, and good biocompatibility. The tau protein, as an important factor in the course of AD, is selected as a key AD biomarker. The results show that the linear range of this sensing platform is 0.1 pg/mL to 1 ng/mL, with a detection limit of 0.034 pg/mL (S/N = 3), indicating that this portable sensing platform meets the demand for the detection of the tau protein in the blood. This work offers great potential for AD diagnosis and personal healthcare.


Subject(s)
Alzheimer Disease , Biosensing Techniques , Graphite , Neurodegenerative Diseases , Alzheimer Disease/diagnosis , Biosensing Techniques/methods , Electrochemical Techniques/methods , Electrodes , Gold , Humans , Limit of Detection , Point-of-Care Testing , tau Proteins
11.
Anal Chem ; 94(36): 12500-12506, 2022 09 13.
Article in English | MEDLINE | ID: covidwho-2016507

ABSTRACT

Screening high-performance anodic electrochemiluminescence (ECL) systems with low triggering potential is a promising way to broaden their applications. In addition to electrochemiluminophore, co-reactant also plays an important role in the ECL process, since the oxidation of co-reactants is one of the most important steps in the anodic ECL process. Herein, a novel co-reactant-mediated high-performance low-potential Au nanocluster (AuNC)-based ECL system has been successfully developed. Benefiting from the isopropyl substitution and hydroxyl addition to the triethylamine (TEA), the BSA-AuNC/2-(diisopropylamino)ethanol (DIPEA-OH) ECL system achieved higher energy efficiency at a lower potential of 0.75 V. In addition, compared with the BSA-AuNC/TEA system, the ECL intensity and quantum yield (ΦECL) with DIPEA-OH as a co-reactant increased 22.34-fold and 13-fold (as high as 68.17%), respectively. Based on the low potential, high ΦECL of the AuNC/DIPEA-OH ECL system, a sandwich-type immunosensor has been constructed for a highly selective SARS-CoV-2 N protein assay. In the absence of any complex signal amplification strategies, the ECL immunosensor for the SARS-CoV-2 N protein detection showed a linear range of 0.001-100 ng/mL and a detection limit of 0.35 pg/mL. Moreover, the ECL platform had good reproducibility and stability and exhibited acceptable detection performance in the detection of actual serum samples. This work established a framework for in-depth design and study of anode ECL co-reactants for AuNCs and other luminophores, and expanded the potential application of ECL sensors in the clinical diagnosis of COVID-19.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , COVID-19/diagnosis , Electrochemical Techniques , Electrodes , Humans , Immunoassay , Limit of Detection , Luminescent Measurements , Reproducibility of Results , SARS-CoV-2
12.
Biosensors (Basel) ; 12(8)2022 Aug 03.
Article in English | MEDLINE | ID: covidwho-1969093

ABSTRACT

In this research, we assessed the applicability of electrochemical sensing techniques for detecting specific antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike proteins in the blood serum of patient samples following coronavirus disease 2019 (COVID-19). Herein, screen-printed carbon electrodes (SPCE) with electrodeposited gold nanostructures (AuNS) were modified with L-Cysteine for further covalent immobilization of recombinant SARS-CoV-2 spike proteins (rSpike). The affinity interactions of the rSpike protein with specific antibodies against this protein (anti-rSpike) were assessed using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. It was revealed that the SPCE electroactive surface area increased from 1.49 ± 0.02 cm2 to 1.82 ± 0.01 cm2 when AuNS were electrodeposited, and the value of the heterogeneous electron transfer rate constant (k0) changed from 6.30 × 10-5 to 14.56 × 10-5. The performance of the developed electrochemical immunosensor was evaluated by calculating the limit of detection and limit of quantification, giving values of 0.27 nM and 0.81 nM for CV and 0.14 nM and 0.42 nM for DPV. Furthermore, a specificity test was performed with a solution of antibodies against bovine serum albumin as the control aliquot, which was used to assess nonspecific binding, and this evaluation revealed that the developed rSpike-based sensor exhibits low nonspecific binding towards anti-rSpike antibodies.


Subject(s)
Biosensing Techniques , COVID-19 , Nanostructures , Antibodies , Biosensing Techniques/methods , COVID-19/diagnosis , Carbon/chemistry , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Humans , Immunoassay/methods , Limit of Detection , SARS-CoV-2 , Spike Glycoprotein, Coronavirus
13.
Biosensors (Basel) ; 12(7)2022 Jul 12.
Article in English | MEDLINE | ID: covidwho-1963724

ABSTRACT

A new sensing platform based on long-period fiber gratings (LPFGs) for direct, fast, and selective detection of human immunoglobulin G (IgG; Mw = 150 KDa) was developed and characterized. The transducer's high selectivity is based on the specific interaction of a molecularly imprinted polymer (MIPs) design for IgG detection. The sensing scheme is based on differential refractometric measurements, including a correction system based on a non-imprinted polymer (NIP)-coated LPFG, allowing reliable and more sensitive measurements, improving the rejection of false positives in around 30%. The molecular imprinted binding sites were performed on the surface of a LPFG with a sensitivity of about 130 nm/RIU and a FOM of 16 RIU-1. The low-cost and easy to build device was tested in a working range from 1 to 100 nmol/L, revealing a limit of detection (LOD) and a sensitivity of 0.25 nmol/L (0.037 µg/mL) and 0.057 nm.L/nmol, respectively. The sensor also successfully differentiates the target analyte from the other abundant elements that are present in the human blood plasma.


Subject(s)
Biosensing Techniques , Molecular Imprinting , Humans , Immunoglobulin G , Limit of Detection , Polymers/chemistry
14.
Biosens Bioelectron ; 213: 114449, 2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-1944326

ABSTRACT

Currently, vaccination is the most effective medical measure to improve group immunity and prevent the rapid spread of COVID-19. Since the individual difference of vaccine effectiveness is inevitable, it is necessary to evaluate the vaccine effectiveness of every vaccinated person to ensure the appearance of herd immunity. Here, we developed an artificial intelligent (AI)-assisted colorimetric polydopamine nanoparticle (PDA)-based lateral flow immunoassay (LFIA) platform for the sensitive and accurate quantification of neutralizing antibodies produced from vaccinations. The platform integrates PDA-based LFIA and a smartphone-based reader to test the neutralizing antibodies in serum, where an AI algorithm is also developed to accurately and quantitatively analyze the results. The developed platform achieved a quantitative detection with 160 ng/mL of detection limit and 625-10000 ng/mL of detection range. Moreover, it also successfully detected totally 50 clinical serum samples, revealing a great consistency with the commercial ELISA kit. Comparing with commercial gold nanoparticle-based LFIA, our PDA-based LFIA platform showed more accurate quantification ability for the clinical serum. Therefore, we envision that the AI-assisted PDA-based LFIA platform with sensitive and accurate quantification ability is of great significance for large-scale evaluation of vaccine effectiveness and other point-of-care immunoassays.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Antibodies, Neutralizing , Artificial Intelligence , COVID-19/diagnosis , Colorimetry , Gold , Humans , Immunoassay/methods , Limit of Detection
15.
Biosens Bioelectron ; 213: 114436, 2022 Oct 01.
Article in English | MEDLINE | ID: covidwho-1944325

ABSTRACT

The emergence of the COVID-19 epidemic has affected the lives of hundreds of millions of people globally. There is no doubt that the development of fast and sensitive detection methods is crucial while the worldwide effective vaccination programs are miles away from actualization. In this study, we have reported an electrochemical N protein aptamer sensor with complementary oligonucleotide as probe for the specific detection of COVID-19. The electrochemical aptasensor was prepared by fixing the double-stranded DNA hybrid obtained by the hybridization of N protein aptamer and its Fc-labeled complementary strand on the surface of a gold electrode. After incubation with the target, the aptamer dissociated from the labeled complementary DNA oligonucleotide hybrid to preferentially bind with N protein in the solution. The concentration of N protein was measured by detecting the changes in electrochemical current signals induced by the conformational transformation of the complementary DNA oligonucleotide left on the electrode surface. The sensor had a linear relationship between the logarithm of the N protein concentration from 10 fM to 100 nM (ΔIp = 0.098 log CN protein/fM - 0.08433, R2 = 0.99), and the detection limitation was 1 fM (S/N = 3). The electrochemical aptamer sensor was applied to test the spiked concentrations of throat swabs and blood samples from three volunteers, and the obtained results proved that the sensor has great potentials for the early detection of COVID-19 in patients.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , COVID-19/diagnosis , DNA, Complementary , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Humans , Limit of Detection , Protein Binding
16.
Methods Mol Biol ; 2511: 79-88, 2022.
Article in English | MEDLINE | ID: covidwho-1941368

ABSTRACT

Detection and mutation surveillance of SARS-CoV-2 are crucial for combating the COVID-19 pandemic. Here we describe a lab-based method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes. It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. The method proved to be rapid and sensitive (limit of detection: 29 viral RNA copies/µL of extracted nucleic acid) in detecting SARS-CoV-2 in clinical samples. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses.


Subject(s)
COVID-19 , Coinfection , Nucleic Acid Amplification Techniques , SARS-CoV-2 , Adenoviruses, Human/genetics , COVID-19/diagnosis , Coinfection/diagnosis , Humans , Influenza A virus/genetics , Limit of Detection , Mutation , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
17.
Biosens Bioelectron ; 215: 114580, 2022 Nov 01.
Article in English | MEDLINE | ID: covidwho-1936100

ABSTRACT

Rational detection of syndrome coronavirus 2 (SARS-CoV-2) is crucial to prevention, control, and treatment of disease. Herein, a dual-wavelength ratiometric electrochemiluminescence (ECL) biosensor based on resonance energy transfer (RET) between g-C3N4 nanosheets and Ru-SiO2@folic acid (FA) nanomaterials was designed to realize ultrasensitive detection of SARS-CoV-2 virus (RdRp gene). Firstly, the unique g-C3N4 nanosheets displayed very intense and stable ECL at 460 nm, then the triple helix DNA was stably and vertically bound to g-C3N4 on electrode by high binding affinity between ssDNA and g-C3N4. Meanwhile, trace amounts of target genes were converted to a large number of output by three-dimensional (3D) DNA walker multiple amplification, and the output bridged a multifunctional probe Ru-SiO2@FA to electrode. Ru-SiO2@FA not only showed high ECL at 620 nm, but also effectively quenched g-C3N4 ECL. As a result, ECL decreased at 460 nm and increased at 620 nm, which was used to design a rational ECL biosensor for detection of SARS gene. The results show that the biosensor has excellent detection sensitivity for RdRp gene with a dynamic detection range of 1 fM to 10 nM and a limit of detection (LOD) of 0.18 fM. The dual-wavelength ratio ECL biosensor has inestimable value and application prospects in the fields of biosensing and clinical diagnosis.


Subject(s)
Biosensing Techniques , COVID-19 , Biosensing Techniques/methods , COVID-19/diagnosis , DNA , Electrochemical Techniques/methods , Energy Transfer , Folic Acid , Humans , Limit of Detection , Luminescent Measurements/methods , Nanostructures , RNA-Dependent RNA Polymerase , Ruthenium , SARS-CoV-2/genetics , Silicon Dioxide
18.
Biosens Bioelectron ; 215: 114556, 2022 Nov 01.
Article in English | MEDLINE | ID: covidwho-1936098

ABSTRACT

Herein, an aptasensor was designed to detect the receptor-binding domain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2-RBD) based on the encapsulation of the methylene blue (MB) inside the mesoporous silica film (MPSF), and an aptamer as an electrochemical probe, a porous matrix, and a bio-gatekeeper, respectively. The signal analysis of the proposed aptasensor indicated that the surface coverage of the encapsulated MB inside the MPSF (MB@MPSF) was 1.9 nmol/cm2. Aptamers were capped the MB@MPSF, avoiding the release of MB into the solution via the electrostatic attraction between the positively charged amino groups of the MPSF and negatively charged phosphate groups of the aptamers. Therefore, the electrochemical signal of the encapsulated MB in the absence of the SARS-CoV-2-RBD was high. In the presence of SARS-CoV-2-RBD, the aptamers that had a high affinity to the SARS-CoV-2-RBD molecules were removed from the electrode surface to interact with SARS-CoV-2-RBD. It gave rise to the release of the MB from the MPSF to the solution and washed away on the electrode surface. Therefore, the electrochemical signal of the aptasensor decreased. The electrochemical signal was recorded with a square wave voltammetry technical in the range of 0.5-250 ng/mL of SARS-CoV-2-RBD in a saliva sample. The limit of detection was found to be 0.36 ng/mL. Furthermore, the selectivity factor values of the proposed aptasensor to 32 ng/mL SARS-CoV-2-RBD in the presence of C-reactive protein, hemagglutinin, and neuraminidase of influenza A virus were 35.9, 11.7, and 17.37, respectively, indicating the high selectivity of the proposed aptasensor.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , COVID-19 , Graphite , Aptamers, Nucleotide/chemistry , COVID-19/diagnosis , Electrochemical Techniques , Electrodes , Gold/chemistry , Graphite/chemistry , Humans , Lasers , Limit of Detection , Methylene Blue/chemistry , SARS-CoV-2 , Silicon Dioxide
19.
Biosensors (Basel) ; 12(7)2022 Jul 13.
Article in English | MEDLINE | ID: covidwho-1938693

ABSTRACT

The severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) has spread globally and there is still a lack of rapid detection techniques for SARS-CoV-2 surveillance in indoor air. In this work, two test rigs were developed that enable continuous air monitoring for the detection of SARS-CoV-2 by sample collection and testing. The collected samples from simulated SARS-CoV-2 contaminated air were analyzed using an ultra-fast COVID-19 diagnostic sensor (UFC-19). The test rigs utilized two air sampling methods: cyclone-based collection and internal impaction. The former achieved a limit of detection (LoD) of 0.004 cp/L in the air (which translates to 0.5 cp/mL when tested in aqueous solution), lower than the latter with a limit of 0.029 cp/L in the air. The LoD of 0.5 cp/mL using the UFC-19 sensor in aqueous solution is significantly lower than the best-in-class assays (100 cp/mL) and FDA EUA RT-PCR test (6250 cp/mL). In addition, the developed test rig provides an ultra-fast method to detect airborne SARS-CoV-2. The required time to test 250 L air is less than 5 min. While most of the time is consumed by the air collection process, the sensing is completed in less than 2 s using the UFC-19 sensor. This method is much faster than both the rapid antigen (<20 min) and RT-PCR test (<90 min).


Subject(s)
Air Pollution, Indoor , COVID-19 , COVID-19/diagnosis , Humans , Limit of Detection , SARS-CoV-2 , Sensitivity and Specificity
20.
Biosensors (Basel) ; 12(7)2022 Jun 22.
Article in English | MEDLINE | ID: covidwho-1911186

ABSTRACT

The pandemic of new coronary pneumonia caused by the COVID-19 virus continues to ravage the world. Large-scale population testing is the key to controlling infection and related mortality worldwide. Lateral flow immunochromatographic assay (LFIA) is fast, inexpensive, simple to operate, and easy to carry, very suitable for detection sites. This study developed a COVID-19 N protein detect strip based on p-toluenesulfonyl modified rare earth fluorescent microspheres. The p-toluenesulfonyl-activated nanomaterials provide reactive sulfonyl esters to covalently attach antibodies or other ligands containing primary amino or sulfhydryl groups to the nanomaterial surface. Antibodies are immobilized on these nanomaterials through the Fc region, which ensures optimal orientation of the antibody, thereby increasing the capture rate of the target analyte. The use of buffers with high ionic strength can promote hydrophobic binding; in addition, higher pH could promote the reactivity of the tosyl group. The detection limit of the prepared COVID-19 N protein strips can reach 0.01 ng/mL, so it has great application potential in large-scale population screening.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies , COVID-19/diagnosis , Humans , Immunoassay/methods , Limit of Detection , Microspheres
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