Genosensor on gold films with enzymatic electrochemical detection of a SARS virus sequence (original) (raw)
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Detection of SARS-CoV-2 Viral Particles using Direct, Reagent-Free Electrochemical Sensing
This manuscript describes a new method that enables direct analysis of viral particles in unprocessed samples.Using an electrochemical readout method that requires no external reagents, we detect the SARS-CoV-2 virus in the saliva of infected patients.The approach relies on a molecular sensor tethered to the surface of a gold electrode that contains an antibody, specific to the targetof interest, which here is the SARS-CoV-2 S1 spike protein that is displayed on the viral capsule. The antibody is attached to the electrode using a negatively charged linker that is composed of DNA. When a positive potential is applied to the electrode, the sensor complex is attracted to the electrode surface. The kinetics of transport is measured using chronoamperometry and readout is possible based on the absense or precense of virus and its effect on the complex movevment on electrode surface.
Label-based and label-free electrochemical DNA biosensors for the detection of viruses: A review
Current Topics in Electrochemistry
Nowadays, the rapid determination of several viruses is highly important. Most of the rapid detection of human pathogen viruses has been developed by using biosensor technology. The detection layer of the biosensor consists of short single-stranded DNA (probe) able to form a duplex with a complementary target nucleic acid fragment with high efficiency and specificity. The probe is associated with a transducer that translates the hybridization event into a physically measurable value based on electrochemical methods. Electrochemical DNA biosensors offer merits such as rapid response, portability, high sensitivity, ease of use, and low detection limit. This review provides an overview of label-based and label-free electrochemical DNA biosensors for the detection of viruses as well as their application in the past four years.
Designing and fabrication of electrochemical nano-biosensor for the fast detection of SARS-CoV-2-RNA
Scientific Reports, 2023
SARS-CoV-2 caused a global panic among populations. Rapid diagnostic procedures for the virus are crucial for disease control. Thus, the designed signature probe from a highly conserved region of the virus was chemically immobilized onto the nanostructured-AuNPs/WO 3-screen printed electrodes. Different concentrations of the matched oligonucleotides were spiked to test the specificity of the hybridization affinity whereas the electrochemical impedance spectroscopy was used for tracking the electrochemical performance. After a full assay optimization, limits of detection and quantification were calculated based on linear regression and were valued at 298 and 994 fM, respectively. Further, the high performance of the fabricated RNA-sensor chips was confirmed after testing the interference status in the presence of the mismatched oligos in one nucleotide and completely one. Worthy to mention that the single-stranded matched oligos can be hybridized to the immobilized probe in 5 min at room temperature. The designed disposable sensor chips are capable of detecting the virus genome directly. Therefore, the chips are a rapid tool for SARS-CoV-2 detection. Since December 2019, the globe was stricken by a life-threatening respiratory disease caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2). The disease is first disclosed in Wuhan, China by severe respiratory manifestations 1,2. The virus is a non-segmented, positive sense, enveloped RNA virus that belongs to the genus Betacoronavirus of the family Coronaviridae 3. The genome of SARS-CoV-2 is 29.9 Kb having 38% of G/C pairs 4. The virus has high mutation rates that are clearly reflected in the pathogenicity, infectivity, virus transmission, and spread as well 5. The viral genomic codes encrypt 27 proteins including 16 non-structural proteins (NSP) and other structural proteins 6. Its spike protein (S-protein) plays an essential role in the virus binding to the host cell. Mediating by the specific cellular proteases, the S-protein is cleaved into S1 and S2 subunits, whereas, cleavage is essential for the virus entry and integration into the host cell 7. The genomic thread is organized as 5′ UTR ORF 1a/b-S (Spike)-E (envelope)-M (Membrane)-N (Nucleocapsid)-3′ UTR poly (A) tail, the accessory proteins are interspersed within the structural proteins. SARS-CoV-2 has about 80% and 96% nucleotides similar to SARS-CoV and bat-CoV-RaTG13, respectively 8-10. Biosensors are outstanding investigation tools used for the fast detection of infectious microorganisms, especially viruses 11. Recently, most of the scientific research shed light on the COVID-19 crisis. Thus, various studies were carried out for the selective and prompt detection of SARS-CoV-2 using different types of biosensing technologies 12,13. For instance, a fabricated biosensor based on an entropy-driven amplified electrochemiluminescence which conducted to link the Ru(bpy) 3 2+ modified S3 to the linear ssDNA at the vertex of the DNA tetrahedron which was used for detection of SARS-CoV-2-RNA-dependant RNA polymerase gene (RdRp). A DNA tetrahedral structure reduced the cross-reactivity and increased the sensitivity of the ssDNA by enhancing the signal intensity of the fabricated sensor. The limit of detection was 2.67 fM which provided a reliable platform for clinical analysis 14. Moreover, an ultrasensitive colorimetric/luminescence-developed biosensor was used to detect SARS-CoV-2 RNA and its variants in the patient's samples using the PHAsed NASBA-Translation Optical Method (PHANTOM). Isothermal amplification of the viral genome which is coupled with the fabricated biosensor resulted in the conformational switch to translate a nano-lantern (LacZ), a reporter protein which resulted
Label-Free Electrochemical Detection of DNA Hybridization: A Method for COVID-19 Diagnosis
Transactions of the Indian National Academy of Engineering
This paper presents label-free electrochemical transduction as a suitable scheme for COVID-19-specific viral RNA/c-DNA detection, with an aim to facilitate point of care diagnosis. In lieu of this, we discuss the proposed electrochemical biosensing scheme, based on electrodeposited gold nanoparticles as the transducing elements. Specific to this approach, here, the protocols associated with the immobilization of the single-stranded probe nucleotide on to the biosensor, have also been laid out. This paper also discusses the methods of electrochemical analysis, to be used for data acquisition and subsequent calibration, in relation to target analyte detection. Towards facilitating portable diagnosis, development of miniaturized sensors and their integration with readout units have also been discussed.
ACS Nano
A large-scale diagnosis of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is essential to downregulate its spread within as well as across communities and mitigate the current outbreak of the pandemic novel coronavirus disease 2019 (COVID-19). Herein, we report the development of a rapid (less than 5 min), low-cost, easy-toimplement, and quantitative paper-based electrochemical sensor chip to enable the digital detection of SARS-CoV-2 genetic material. The biosensor uses gold nanoparticles (AuNPs), capped with highly specific antisense oligonucleotides (ssDNA) targeting viral nucleocapsid phosphoprotein (N-gene). The sensing probes are immobilized on a paper-based electrochemical platform to yield a nucleic-acid-testing device with a readout that can be recorded with a simple hand-held reader. The biosensor chip has been tested using samples collected from Vero cells infected with SARS-CoV-2 virus and clinical samples. The sensor provides a significant improvement in output signal only in the presence of its targetSARS-CoV-2 RNAwithin less than 5 min of incubation time, with a sensitivity of 231 (copies μL −1) −1 and limit of detection of 6.9 copies/μL without the need for any further amplification. The sensor chip performance has been tested using clinical samples from 22 COVID-19 positive patients and 26 healthy asymptomatic subjects confirmed using the FDA-approved RT-PCR COVID-19 diagnostic kit. The sensor successfully distinguishes the positive COVID-19 samples from the negative ones with almost 100% accuracy, sensitivity, and specificity and exhibits an insignificant change in output signal for the samples lacking a SARS-CoV-2 viral target segment (e.g., SARS-CoV, MERS-CoV, or negative COVID-19 samples collected from healthy subjects). The feasibility of the sensor even during the genomic mutation of the virus is also ensured from the design of the ssDNA-conjugated AuNPs that simultaneously target two separate regions of the same SARS-CoV-2 Ngene.
Analytical and Bioanalytical Chemistry
This paper reports the development of a low-cost (< US$ 0.03 per device) immunosensor based on gold-modified screenprinted carbon electrodes (SPCEs). As a proof of concept, the immunosensor was tested for a fast and sensitive determination of S proteins from both SARS-CoV and SARS-CoV-2, by a single disposable device. Gold nanoparticles were electrochemically deposited via direct reduction of gold ions on the electrode using amperometry. Capture antibodies from spike (S) protein were covalently immobilized on carboxylic groups of self-assembled monolayers (SAM) of mercaptoacetic acid (MAA) attached to the gold nanoparticles. Label-free detection of S proteins from both SARS-CoV and SARS-CoV-2 was performed with electrochemical impedance spectroscopy (EIS). The immunosensor fabricated with 9 s gold deposition had a high performance in terms of selectivity, sensitivity, and low limit of detection (LOD) (3.16 pmol L −1), thus permitting the direct determination of the target proteins in spiked saliva samples. The complete analysis can be carried out within 35 min using a simple one-step assay protocol with small sample volumes (10 µL). With such features, the immunoplatform presented here can be deployed for mass testing in point-of-care settings.
Biosensors and Bioelectronics, 2015
In this work, a simple and label-free electrochemical biosensor with duel amplification strategy was developed for DNA detection based on isothermal exponential amplification (EXPAR) coupled with hybridization chain reaction (HCR) of DNAzymes nanowires. Through rational design, neither the primer nor the DNAzymes containing molecular beacons (MBs) could react with the duplex probe which were fixed on the electrode surface. Once challenged with target, the duplex probe cleaved and triggered the EXPAR mediated target recycle and regeneration circles as well as the HCR process. As a result, a greater amount of targets were generated to cleave the duplex probes. Subsequently, the nanowires consisting of the G-quadruplex units were self-assembled through hybridization with the strand fixed on the electrode surface. In the presence of hemin, the resulting catalytic G-quadruplex-hemin HRP-mimicking DNAzymes were formed. Electrochemical signals can be obtained by measuring the increase in reduction current of oxidized 3.3′,5.5′-tetramethylbenzidine sulfate (TMB), which was generated by DNAzyme in the presence of H 2 O 2 . This method exhibited ultrahigh sensitivity towards avian influenza A (H7N9) virus DNA sequence with detection limits of 9.4 fM and a detection range of 4 orders of magnitude. The biosensor was also capable of discriminating single-nucleotide difference among concomitant DNA sequences and performed well in spiked cell lysates.
DNA hybridization biosensors using polylysine modified SPCEs
Biosensors and Bioelectronics, 2008
Two electrochemical DNA hybridization biosensors (genosensors) for the detection of a 30-mer sequence unique to severe acute respiratory syndrome (SARS) virus are described in this work. Both genosensors rely on the hybridization of the oligonucleotide target with its complementary probe, which is immobilized on positively charged polylysine modified screen-printed carbon electrodes (SPCEs), through electrostatic interactions. In one design, a biotinylated target is used and the detection of the hybridization reaction is monitored using alkaline phosphatase labeled streptavidin (S-AP). This enzyme catalyzes the hydrolysis of the substrate 3-indoxyl phosphate (3-IP) to indigo, which is then solubilized to indigo carmine and detected by means of cyclic voltammetry (CV). In the other design, the target is labeled using an Au(I) complex, sodium aurothiomalate, and the duplex formation is detected by measuring, for first time, the current generated by the hydrogen evolution catalyzed by the gold label. Using 30 min of hybridization time, a detection limit of 8 pM is calculated for the enzymatic genosensor. Although this good sensitivity cannot be reached with the metal label (0.5 nM), the use of this label allows a considerable decrease of the analysis time. Both genosensors do not require the modification of the oligonucleotide probe and using stringent experimental conditions (60 min of hybridization time and 50% formamide in the hybridization buffer) can discriminate between a complementary oligonucleotide and an oligonucleotide with a three-base mismatch.
Biosensing strategies for the detection of SARS-CoV-2 nucleic acids
Journal of Pharmaceutical and Biomedical Analysis, 2023
The COVID-19 pandemic had devastating effects throughout the world, producing a severe crisis in the health systems and in the economy of a long list of countries, even developed ones. Therefore, highly sensitive and selective analytical bioplatforms that allow the descentralized and fast detection of the severe acute respiratory síndrome coronavirus 2 (SARS-CoV-2), are extremely necessary. Since 2020, several reviews have been published, most of them focused on the different strategies to detect the SARS-CoV-2, either from RNA, viral proteins or host antibodies produced due to the presence of the virus. In this review, the most relevant biosensors for the detection of SARS-CoV-2 RNA are particularly addressed, with special emphasis on the discussion of the biorecognition layers and the different schemes for transducing the hybridization event.Open in a separate window