Aptasensor for the Detection of Mycobacterium tuberculosis in Sputum Utilising CFP10-ESAT6 Protein as a Selective Biomarker.

Umi Zulaikha Mohd Azmi,Shahrul Ainliah Alang Ahmad,Nor Azah Yusof,Fatin Nabilah Mohd Faudzi,Jaafar Abdullah,Nurul Hanun Ahmad Raston,Sachin K Khiste,Faruq Mohammad,Hamad A Al-Lohedan,Siti Suraiya

doi:10.3390/nano11092446

Abstract

A portable electrochemical aptamer-antibody based sandwich biosensor has been designed and successfully developed using an aptamer bioreceptor immobilized onto a screen-printed electrode surface for Mycobacterium tuberculosis (M. tuberculosis) detection in clinical sputum samples. In the sensing strategy, a CFP10-ESAT6 binding aptamer was immobilized onto a graphene/polyaniline (GP/PANI)-modified gold working electrode by covalent binding via glutaraldehyde linkage. Upon interaction with the CFP10-ESAT6 antigen target, the aptamer will capture the target where the nano-labelled Fe3O4/Au MNPs conjugated antibody is used to complete the sandwich format and enhance the signal produced from the aptamer–antigen interaction. Using this strategy, the detection of CFP10-ESAT6 antigen was conducted in the concentration range of 5 to 500 ng/mL. From the analysis, the detection limit was found to be 1.5 ng/mL, thereby demonstrating the efficiency of the aptamer as a bioreceptor. The specificity study was carried out using bovine serum albumin (BSA), MPT64, and human serum, and the result demonstrated good specificity that is 7% higher than the antibody–antigen interaction reported in a previous study. The fabricated aptasensor for M. tuberculosis analysis shows good reproducibility with an relative standard deviation (RSD) of 2.5%. Further analysis of M. tuberculosis in sputum samples have shown good correlation with the culture method with 100% specificity and sensitivity, thus making the aptasensor a promising candidate for M. tuberculosis detection considering its high specificity and sensitivity with clinical samples.

Highlights

Licensee MDPI, Basel, Switzerland.Biosensors play a vital role in a plethora of applications, especially in the medical and food industrires, which comprise of the detection and quantification of proteins in clinical and food samples
We proposed a sandwich-type electrochemical aptasensor for the detection of M. tuberculosis with modifications on the screen-printed gold electrode (SPGE) using iron/gold magnetic NPs (MNPs) (Fe3 O4 /Au MNPs) as a label, while the GP/PANI nanocomposite serves as a signal amplification layer
The selection of aptamer is based on a previous study [9], which developed the sequence of an aptamer for a CFP10-ESAT6 antigen

Summary

Introduction

Licensee MDPI, Basel, Switzerland.Biosensors play a vital role in a plethora of applications, especially in the medical and food industrires, which comprise of the detection and quantification of proteins in clinical and food samples. Biosensors are described as the analytical devices used to detect the presence or quantify the concentration of a biological analyte by transducing the biochemical reactions of a bioreceptor with a specific target analyte into an optical, thermal or electrical signal [1]. Aptamers are single-stranded DNA or RNA sequences (most recently, peptides) that are generally less than hundred bases long [5]. Since their discovery in the 1990s, aptamers have received extensive interest for their application in biosensor development as an alternative to antibodies, which act as bioreceptors [6]. A number of aptamer-based sensors have been developed using different transducer techniques and have demonstrated their excellent performance, which validates the promising potential of aptasensors [12,13,14]

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Results

Conclusion