Quartz Crystal Microbalance Aptasensor for the Molecular Detection of Dengue Virus
Authors: Sianghio Geminesse S. , Dimaculangan Reyza Kathleen A. , Albano Dharmatov Rahula B. 
Published: May 2020

Introduction

Dengue fever is the major public health concern in the Philippines and is endemic in all regions of the country in which all serotypes are present[1]. Despite the widespread nature of the virus transmitted by mosquitos, molecular diagnostics have significant limitations. An early diagnosis of dengue may prevent severe clinical complications associated with the infection. A novel genosensor using an aptamer for the detection of dengue virus serotypes 1-4 was demonstrated in this study.

Method

This genosensor composed of a thiolated aptamer immobilized on a 10MHz gold electrode QCM targeting the 3’ untranslated region of all complete genome sequences of dengue virus available in GenBank[2]. Aptamer concentration, buffer pH and ionic strength of buffer solutions were optimized to achieve the highest sensitivity of the QCM genosensor. Atomic force microscopy [3], electrochemical impedance spectrum (EIS) measurements and cyclic voltammetry (CV) were used to characterize the surface of the QCM electrode prepared[4]. Evaluation of binding kinetics and affinity study of the Aptamer-dengue genome was determined by surface plasmon resonance (SPR)[5].

Results and Conclusions

Atomic Force Microscopy (AFM), Electrochemical Impedance (EIS) and Cyclic Voltammetry (CV) have been used to characterize the surface of the PQC electrode. The AFM results indicate that the thiolated aptamer onto the electrode has decreased the roughness of gold surface by forming a uniform layer on the electrode surface. The EIS and CV results indicate that the charge transfer resistance is increased after immobilization of the aptamer onto the bare gold electrodes, indicating that direct S-Au bonding via disulfide moieties and the negative charge of protein have blocked the electrode reaction between Fe (II) and Fe (III). The results showed that the developed aptamer QCM sensor has an optimum concentration range of the target dengue genome is 20–100 mg/ml for hybridization. This work demonstrated the possibility of using an immobilized aptamer on a QCM and other sensor platforms for the direct detection of the RT-PCR products of dengue virus serotypes 1-4.

References

[1] A.A. de los Reyes, J.M.L. Escaner, Dengue in the Philippines: Model and analysis of parameters affecting transmission, J. Biol. Dyn. 12 (2018) 894–912. doi:10.1080/17513758.2018.1535096.
[2] E. Alm, B. Lesko, G. Lindegren, C. Ahlm, S. Söderholm, K.I. Falk, N. Lagerqvist, Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections, PLoS Negl. Trop. Dis. 8 (2014) 6–15. doi:10.1371/journal.pntd.0003416.
[3] X. Zhang, V.K. Yadavalli, Surface immobilization of DNA aptamers for biosensing and protein interaction analysis, Biosens. Bioelectron. 26 (2011) 3142–3147. doi:10.1016/j.bios.2010.12.012.
[4] A.-E. Radi, Electrochemical Aptamer-Based Biosensors: Recent Advances and Perspectives, Int. J. Electrochem. 2011 (2011) 1–17. doi:10.4061/2011/863196.
[5] E. González-Fernández, N. De-Los-Santos-Álvarez, A.J. Miranda-Ordieres, M.J. Lobo-Castañón, SPR evaluation of binding kinetics and affinity study of modified RNA aptamers towards small molecules, Talanta. 99 (2012) 767–773. doi:10.1016/j.talanta.2012.07.019.

Figure 1