Surface-Enhanced Carboxyphenyl Diazonium Functionalized Screen-Printed Carbon Electrode for the Screening of Tuberculosis in Sputum Samples

Abstract

Curbing tuberculosis (TB) requires a combination of good strategies, including a proper prevention measure, diagnosis, and treatment. This study proposes an improvised tuberculosis diagnosis based on an amperometry approach for the sensitive detection of MPT64 antigen in clinical samples. An MPT64 aptamer specific to the target antigen was covalently attached to the carboxyphenyl diazonium-functionalized carbon electrode via carbodiimide chemistry. The electrochemical detection assay was adapted from a sandwich assay format to trap the antigen between the immobilized aptamer and horseradish peroxidase (HRP) tagged polyclonal anti-MPT64 antibody. The amperometric current was measured from the catalytic reaction response between HRP, hydrogen peroxide, and hydroquinone, which is used as an electron mediator. From the analysis, the detection limit in the measurement buffer was 1.11 ng mL−1. Additionally, the developed aptasensor exhibited a linear relationship between the current signal and the MPT64 antigen-spiked serum concentration ranging from 10 to 150 ng mL−1 with a 1.38 ng mL−1 detection limit. Finally, an evaluation using the clinical sputum samples from both TB (+) and TB (−) individuals revealed a sensitivity and specificity of 88% and 100%, respectively. Based on the analysis, the developed aptasensor was found to be simple in its fabrication, sensitive, and allowed for the efficient detection and diagnosis of TB in sputum samples.

Simultaneous Amperometric Aptasensor Based on Diazonium Grafted Screen-Printed Carbon Electrode for Detection of CFP10 and MPT64 Biomarkers for Early Tuberculosis Diagnosis

“…As described in the methodology section, the dual aptasensor was based on a sandwich enzyme-linked assay utilizing both aptamer and antibody as biorecognition molecules. Adapted from our previous works on the individual detection of each tuberculosis antigen [ 8 , 27 ], the dual aptasensor was prepared by covalent attachment of the respective CFP10 and MPT64 aminated aptamers on the diazonium-modified carbon surfaces, which are rich with carboxyl terminal. The blocking step with ethanolamine and BSA introduced afterward helped to lower the background signal from the non-specific molecule binding.…”

“…The presence of the HQ in the detection system can improve the detection response since the reduction process of the enzymatic reaction is generally slow due to slow electron transfer. Thus, HQ can accelerate the electron transfer process between the surface and the HRP redox center [ 27 , 31 , 32 ].…”

“…As for CFP10 aptamer conditions, Figure 5 a,b indicates that the most significant S/B current ratios were at 0.8 µM and 60 min for the concentration and incubation time, respectively. The amperometric current dropped when more than 0.8 µM CFP10 aptamer was loaded on the electrode surface, probably because of the growth of a crowd and densely packed surface that limited the binding efficiency between the aptamer and the CFP10 target antigen [ 17 , 27 ]. A slightly higher aptamer concentration (1 µM) was chosen as the best condition for the MPT64 sensor since it produced the highest S/B current ratio ( Figure 5 c).…”

“…The in situ grafting of the 4-aminobenzoic diazonium salt was performed exactly as reported in our previous study [ 27 ]. The grafted diazonium film was then incubated with 100 mM EDC and 25 mM NHS prepared in MES buffer for 1 h at room temperature to activate the carboxylic group.…”