Development Of An Electrochemical Immunosensor On Screen Printed Carbon Electrodes For The Detection Of Dengue Ns1 Antigen And Igm Antibody

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Date
2016-02
Authors
Parkash, Om
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Publisher
Universiti Sains Malaysia
Abstract
Dengue is a major public serious health issue in the tropical and subtropical regions and about 2.5 billion lives are at risk in more than 112 countries. Due to the unavailability of effective antiviral drugs, early diagnosis is very crucial for patient management and vector control. However, laboratory diagnosis of dengue still remains a problem, as conventional diagnosis methods have their own pitfalls. Herein, an electrochemical immunosensor exploiting the screen printed carbon electrodes (SPCEs) as biosensing surface and enzyme labelled detection antibody as electrochemical signal amplifier developed to detect dengue non-structural 1 (NS1) protein and immunoglobulin M (IgM) as markers for the acute and late infection phase, respectively. Initially, the SPCEs were electrochemically characterised for their reproducibility, stability and conductivity. Subsequently, recombinant dengue NS1 antigen was sandwich hybridised between anti-NS1 capture antibody and anti-NS1 detection antibody to develop dengue NS1 electrochemical immunosensor assay, whereas dengue IgM electrochemical immunosensor assay was developed based on IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) principle. Chronoamperometric responses resulting from the catalysis 3,5,3‘,5‘-tetramethylbenzidine (TMB)/hydrogen peroxide (H2O2) were generated at -200 mV by horseradish peroxidase (HRP) labelled detection antibody. To enhance the sensitivity of the electrochemical immunosensor assay, various immobilisation techniques were investigated. In addition, various parameters were also optimised such as immunoreagents, concentration, incubation time, washing frequency; suitable blocking agent and washing buffer were also investigated. Analytical sensitivity and specificity, diagnostic capability and stability of the electrochemical immunosensor assay were evaluated to use the immunosensor empirically. The SPCEs used in this study demonstrated good electrochemical properties with good stability, conductivity and reproducibility. Protein A based immobilisation technique for anti-NS1 capture antibody technique found to be the best in dengue NS1 electrochemical immunosensor assay. Dengue NS1 electrochemical sensor showed excellent sensitivity using recombinant NS1 with a limit of detection of 0.03 μg/mL, suggesting that the developed immunosensor could detect NS1 in real serum samples. Development of a matrix buffer made it possible to detect NS1 in spiked and real serum samples. Similarly, the developed immunosensor showed excellent sensitivity using NS1 spiked serum samples with a limit of detection of 0.03 μg/mL, which was lower than 0.12 μg/mL detected by ELISA while analytical sensitivity using real serum samples was found to be 1:256 serum dilution. For dengue IgM electrochemical immunosensor assay, streptavidin/biotin based immobilisation technique was the best immobilisation technique for goat anti-human IgM antibody. The dengue IgM electrochemical immunosensor also showed excellent sensitivity with a limit of detection 106 serum dilution which was lower than 105 serum dilution detected by ELISA, suggesting that the developed immunosensor was highly sensitive. Cross-reactivity analysis showed that both dengue NS1 and IgM electrochemical immunosensor could discriminate dengue from non-dengue samples. Dengue NS1 and IgM electrochemical immunosensor also successfully discriminated the 150 dengue NS1 and dengue IgM real serum samples, respectively. The overall diagnostic performance of the dengue NS1 and IgM immunosensor regarding diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV) and negative predictive value (NPV) was found to be 100%, indicating that the developed immunosensor is fully capable of detecting dengue NS1 and IgM from real serum samples with high sensitivity, specificity. The assay time of electrochemical immunosensor was comparable to ELISA. However, the advantage using immunosensor is that it is more sensitive than ELISA. Apart from these features, the developed immunosensor found to be stable for one year at ambient temperature with the help of preservative trehalose. In conclusion, the developed immunosensor can be used for the diagnosis of dengue during acute and late phase with high sensitivity and specificity and has the potential to be commercialised. However, further modifications are needed to ensure the assay can be a point of care test.
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Keywords
Development of an electrochemical Immunosensor , Detection of dengue ns1 antigen.
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