Tuberculosis detection from raw sputum samples using Au-electroplated screen-printed electrodes as E-DNA sensor

Abstract

Tuberculosis (TB) remains a leading cause of death globally, especially in underdeveloped nations. The main impediment to TB eradication is a lack of efficient diagnostic tools for disease diagnosis. In this work, label free and ultrasensitive electrochemical DNA biosensor for detecting Mycobacterium tuberculosis has been developed based on the electrodeposition of gold nanoparticles on the surface of carbon screen-printed carbon electrode (Zensors) for signal amplification. Particularly, screen-printed electrodes were modified by electrochemical deposition of Au to enhance the conductivity and facilitate the immobilization of ssDNA probes via Au-S bonds. The electrochemically modified SPEs were characterized using Scanning electron microscopy/Energy Dispersive X-Ray Analysis (SEM/EDX) and X-Ray Diffraction (XRD). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques were used to investigate the DNA hybridization between single-stranded (ssDNA) probe and target DNA (tDNA). Under the ideal conditions, DPV exhibited a correlation coefficient R2 = 0.97, when analyzed with different tDNA concentrations. The proposed DNA biosensor exhibits a good detection range from 2 to 10 nm with a low detection limit of 1.91 nm, as well as high selectivity that, under ideal conditions, distinguishes non-complementary DNA from perfectly matched tDNA. By eliminating the need for DNA purification, this work paves the path for creating disposable biosensors capable of detecting DNA from raw sputum samples.

Keywords: IS-6110; electrochemical biosensing; mtb detection; screen printed electrode (SPE); tuberculosis.

FIGURE 1

Schematic illustration of stepwise modification of screen-printed electrode for the detection Mtb DNA.

FIGURE 2

SEM and EDX analysis of carbon SPE (A,B) and electrodeposited gold SPE (C,D).

FIGURE 3

XRD analysis of Au-modified SPE.

FIGURE 4

(A) Cyclic voltammetry of bare Carbon SPE and electrodeposited Au-SPE. (B,C) CV and DPV analysis of Au-modified SPE functionalized with ssDNA probe/MCH and tDNA in 5 mm K₃ [Fe (CN)₆] and 0.1 M KCl. (D) DPV analysis of different Mtb-tDNA concentrations ranging (2nm–10 nm) on modified SPE to check the analytical performance of biosensor. (E) Linear correlation of current response against the different concentration of target DNA. (F) Specificity of biosensor against Salmonella, E. coli and Mtb.

FIGURE 5

(A) CV voltammogram of Au-modified SPE in the 5 mm K₃ [Fe (CN)₆] and 0.1 KCl solution at various scan rates 20, 40, 60, 80, 100 mVs⁻¹. (B) Stability investigation of the fabricated biosensor modified with ssDNA probe and MCH. (C) DPV analysis of raw sputum samples divided into sample+ and sample++ based on bacillary loads (D) Histogram of electrochemical response of the clinical sputum samples grouped according to the bacillary load. (E) Reproducibility of biosensor.