Development of a portable and disposable NS1 based electrochemical immunosensor for early diagnosis of dengue virus (original) (raw)
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Biomedical microdevices, 2018
Dengue is an infectious viral disease transmitted by the Aedes aegypti mosquito, the control of which is complex. In addition, the clinical diagnosis is difficult to perform since it resembles other febrile infections; thus, the development of more effective methods to detect dengue virus (DV) has drawn increasing attention. The present study aimed to develop an impedimetric immunosensor for dengue diagnosis using a screen-printed electrode (SPE) functionalized with polymer films derived from 4-aminophenylacetic acid (4-APA). Data obtained from scanning electron microscopy (SEM) showed the deposition of a uniformly distributed material over the electrode surface. The immunosensor was based on the specific interaction between dengue antigen, NS1 protein, and anti-NS1 antibodies, IgG and IgM. In a characterization study using cyclic voltammetry (CV), the polymer film showed two oxidation peaks at +0.17 and + 0.35 V in 0.50 M sulfuric acid solution, indicating its adsorption and electr...
Dengue is an arbovirus disease transmitted mainly by Aedes mosquitoes. As dengue shares similar clinical symptoms with other infectious diseases, prompt and accurate diagnosis is pivotal to clinicians' decisions on appropriate management. Conventional diagnostic tests to detect the dengue-specific IgM antibody are limited in their performance and ease of use. To address these issues, we developed and evaluated a biosensor based on screen-printed carbon electrodes (SPCEs) for the detection of dengue-specific immunoglobulin M (IgM) antibodies. Various optimisations were performed in order to increase the sensitivity and specificity of the biosensor. For optimal and proper orientation of the paratope sites of goat anti-human IgM capture antibodies (GAHICA), various antibody techniques, including passive, covalent, protein A, protein G and streptavidin/biotin systems, were tested on the SPCEs. The assay reagents for the biosensor were also optimised prior to its evaluation. Analytical sensitivity evaluation was carried out using pooled sera, while analytical specificity evaluation was conducted on a panel of six non-dengue serum samples. Subsequently, diagnostic sensitivity and specificity evaluation were performed using 144 reference samples. Electrochemical current signals generated from H 2 O 2 catalysed by HRP-labelled anti-dengue detection antibodies were measured using the chronoamperometric technique. With a limit of detection (LOD) of 10 6 serum dilution, the analytical sensitivity of the developed biosensor was 10 times higher than commercial ELISA. The analytical specificity of this dengue IgM biosensor was 100%. Similarly, the biosensor's diagnostic performance was 100% for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). These findings suggest that the developed biosensor has a great potential to be used to diagnose dengue after seroconversion.
Dengue virus infection is a very deadly disease that threatens human lives in subtropical and tropical regions. Thus, for early and reliable diagnosis of Dengue virus infection, a sensitive, specific, and label-free electrochemical immunosensor was developed for the direct detection of the unstructured protein NS1. The NS1 biosensor was designed with a biosensing surface consisting of antifouling moieties and biorecognition molecules to enhance the specificity of the immunosensor for target analyte detection in complicated biological samples such as human sera. The immunosensor exhibited a wide detection range (5–4000 ng mL −1) based on electrochemical impedance spectroscopy (EIS) measurements with a coefficient of determination (R 2) of 0.94, correlation coefficient (R) of 0.95, high reproducibility, and good stability for 21 days at 4 • C. The developed immunosensor was able to detect the NS1 antigen in actual serum specimens from patients infected with Dengue virus. Moreover, the immunosensor is not only highly selective for the NS1 antigen, but also did not cross-react with human sera infected with malaria parasites.
Biochemical Engineering Journal, 2012
The non-structural protein 1 (NS1) of dengue virus is abundantly circulating in the blood during the acute phase of the dengue infection being correlated with viremia levels and hence can be used to early diagnostic of the dengue hemorrhagic fever. An electrochemical immunosensor based on gold film electrode obtained from a recordable compact disk (CD-trode) was developed for NS1 protein. Anti-NS1 monoclonal antibodies were immobilized on the CD-trode via protein A. The stepwise immobilization of the anti-NS1 was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The analytical response to the NS1 interaction with anti-NS1 immobilized on CD-trode was detected by applying the differential pulse voltammetry technique. The immunosensor showed a linear response from 1 to 100 ng/mL of NS1 and a detection limit of 0.33 ng/mL. This label-free immunosensor exhibited a good reliability for NS1 detection in serum samples presuming an early diagnostic of the dengue virus.
An impedimetric biosensor to test neat serum for dengue diagnosis
Sensors and Actuators B: Chemical, 2015
NS1, a non-structural dengue protein, has been used as a biomarker for the detection of viremia in dengue diagnosis. In this work, an impedimetric label-free immunosensor based on an anti-NS1 modified gold electrode was developed. To immobilize anti-NS1, a mixed self-assembled monolayer consisting of 11mercaptoundecanoic acid (for covalent anti-NS1 attachment) and 6-mercaptohexanol (as a spacer) was prepared. The impedance spectra were recorded in the presence of a redox probe ([Fe(CN) 6 ] 3−/4−) to monitor changes in charge-transfer resistance associated with target binding both in PBS and neat serum. A calibration curve was constructed in order to obtain analytical parameters. NS1 was diluted in PBS to final concentrations in the linear range of 0.01-2.00 g mL −1 with a sensitivity of 14.1 percentage decade −1 (R ∼ 0.99) and a limit of detection (LOD) of 3 ng mL −1. A linear range 0.01-1.00 g mL −1 of NS1 diluted in neat serum was also obtained (R ∼ 0.98) with a sensitivity of 10.4 percentage decade −1 and a LOD of 30 ng mL −1. The results open new insights into the potential use of this biosensor for point-of-care and bedside applications for dengue diagnosis.
Sensors International, 2020
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Electrochemical lateral flow immunosensor for detection and quantification of dengue NS1 protein
Biosensors and Bioelectronics, 2016
An Electrochemical Lateral Flow Immunosensor (ELFI) is developed combining screen-printed gold electrodes (SPGE) enabling quantification together with the convenience of a lateral flow test strip. A cellulose glassy fiber paper conjugate pad retains the marker immunoelectroactive nanobeads which will bind to the target analyte of interest. The specific immunorecognition event continues to occur along the lateral flow bed until reaching the SPGE-capture antibodies at the end of the cellulosic lateral flow strip. The rationale of the immunoassay consists in the analyte antigen NS1 protein being captured selectively and specifically by the dengue NS1 antibody conjugated onto the immunonanobeads thus forming an immunocomplex. With the aid of a running buffer, the immunocomplexes flow and reach the immuno-conjugated electrode surface and form specific sandwich-type detection due to specific, molecular recognition, while unbound beads move along past the electrodes. The successful sandwich immunocomplex formation is then recorded electrochemically. Specific detection of NS1 is translated into an electrochemical signal contributed by a redox label present on the bead-immobilized detection dengue NS1 antibody while a proportional increase of faradic current is observed with increase in analyte NS1 protein concentration. The first generation ELFI prototype is simply assembled in a cassette and successfully demonstrates wide linear range over a concentration range of 1-25ng/mL with an ultrasensitive detection limit of 0.5ng/mL for the qualitative and quantitative detection of analyte dengue NS1 protein.
Sensors, 2011
A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing
Scientific reports, 2015
Nonstructural protein 1 (NS1) is secreted by dengue virus in the first days of infection and acts as an excellent dengue biomarker. Here, the direct electrical detection of NS1 from dengue type 2 virus has been achieved by the measurement of variations in open circuit potential (OCP) between a reference electrode and a disposable Au electrode containing immobilized anti-NS1 antibodies acting as immunosensor. Egg yolk immunoglobulin (IgY) was utilized for the first time as the biological recognition element alternatively to conventional mammalian antibodies in the detection of dengue virus NS1 protein. NS1 protein was detected in standard samples in a 0.1 to 10 µg.mL(-1) concentration range with (3.2 ± 0.3) mV/µg.mL(-1) of sensitivity and 0.09 µg.mL(-1) of detection limit. Therefore, the proposed system can be extended to detect NS1 in real samples and provide an early diagnosis of dengue.
A thiophene-modified screen printed electrode for detection of dengue virus NS1 protein
Talanta, 2014
A thiophene-modified screen printed electrode (SPE) for detection of the Dengue virus non-structural protein 1 (NS1), an important marker for acute phase diagnosis, is described. A sulfur-containing heterocyclic compound, the thiophene was incorporated to a carbon ink to prepare reproducible screen printed electrodes. After cured, the thiophene SPE was coated by gold nanoparticles conjugated to Protein A to form a nanostrutured surface. The Anti-NS1 antibodies immobilized via their Fc portions via Protein A, leaving their antigen specific sites free circumventing the problem of a random antibodies immobilization. Amperometric responses to the NS1 protein of dengue virus were obtained by cyclic voltammetries performed in presence of ferrocyanide/ferricyanide as redox probe. The calibration curve of immunosensor showed a linear response from 0.04 mg mL À 1 to 0.6 mg mL À 1 of NS1 with a good linear correlation (r ¼0.991, po0.05). The detection limit (0.015 mg mL À 1 NS1) was lower than conventional analytical methods. In this work, thiophene monomers incorporated in the carbon ink enhanced the electroanalytical properties of the SPEs, increasing their reproducibility and sensitivity. This point-ofcare testing represents a great potential for use in epidemic situations, facilitating the early diagnosis in acute phase of dengue virus.