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Back to Journal »International Journal of Nanomedicine» Volume 16

Graphene oxide-based electrochemical gene sensor for label-free detection of Mycobacterium tuberculosis from original clinical samples

Author Javed A, Abbas SR, Hashmi MU, Babar NUA, Hussain I 

Published on November 2, 2021, the 2021 volume: 16 pages 7339-7352

DOI https://doi.org/10.2147/IJN.S326480

Single anonymous peer review

Editor approved for publication: Dr. Yan Shen

Aisha Javed,1 Shah Rukh Abbas,1 Muhammad Uzair Hashmi,1 Noor Uzair Hashmi,1 Noor Ul Ain Babar,2 Irshad Hussain2 1Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biological Sciences, National University of Science and Technology, Islamabad, 44000, Pakistan ; 2 Department of Chemistry, Syed Babar Ali School of Science and Engineering, Lahore University of Management Sciences, Lahore, 54792, Pakistan Corresponding author: Shah Rukh Abbas Department of Industrial Biotechnology, Atta-ur-Rahman School of Applied Biological Sciences, National University of Science and Technology, H-12, Islamabad, Pakistan Tel 92 3355449622; 92 51-9085-6125 Email [email protection] Background: Rapid detection of Mycobacterium tuberculosis remains a daunting challenge for the control of deadly diseases. New diagnostic methods, such as LED fluorescence microscopy, Genexpert, interferon gamma release assay (IGRA), are costly, time-intensive, and laborious, so they are limited in their efficacy spectrum. In addition, their low sensitivity hinders their robustness Sex and portability. Electroanalysis methods are now considered to be an excellent alternative method and are currently used to effectively detect analytes with portable potential. The report recommends label-free electrochemical detection of Mycobacterium tuberculosis (Mtb) through its marker insert sequence (IS6110). Method: In this process, the graphene oxide-chitosan nanocomposite (GO-CHI) was fabricated and characterized, which is a biocompatible matrix with a large electroactive area and an overall positively charged surface . Then the obtained GO-CHI nanocomposite material is fixed on the ITO surface to form a positive functional electrochemical sensor for detecting Mtb. The DNA probe specific to IS6110 is electrostatically anchored on the surface of the positively charged electrode, and the charge transfer resistance has been studied by cyclic voltammetry and differential pulse voltammetry techniques for the sensitivity and specificity of Mtb (complementary and non-complementary). Complementary) detection. Result: It was found that cyclic voltammetry is diffusion-controlled, which helps the absorption of the analyte on the electrode surface. When analyzed in the DNA concentration range of 7.86 pM to 94.3 pM, it was found that the label-free "gene sensor" could detect the hybridization efficiency, the detection limit was 3.4 pM, and the correlation coefficient R2=0.99. The genetic sensor can also detect target DNA from the original sputum samples of clinical isolates without the need for DNA purification. Conclusion: The electrochemical gene sensor has high sensitivity and specificity; thus, it provides a promising platform for the clinical diagnosis of tuberculosis and other infectious diseases. Keywords: label-free detection, DNA gene sensor, mycobacterial detection, tuberculosis, graphene oxide nanocomposite, GO nanocomposite electrochemical sensor

According to the World Health Organization Global Tuberculosis Report 2020, an estimated 10 million people are infected with tuberculosis (TB) each year in 2019, of which 1.7 million deaths are reported in developing countries. 1 It calls for the development of a robust, rapid and reliable sensitive method for the detection of Mycobacterium tuberculosis (Mtb) to diagnose tuberculosis at an early stage, which may guarantee its effectiveness. Current tuberculosis diagnosis methods include LED fluorescence microscope, chest X-ray, Genexpert, interferon gamma release assay (IGRA), antigen detection assay, these methods are expensive, time consuming, laborious, low sensitivity, and require highly complex laboratories set up. 2 These limitations and the need for skilled personnel hinder the robustness, portability and accuracy of the test.

In order to overcome these limitations, many new diagnostic tests are being introduced, most of which are based on polymerase chain reaction (PCR), which have higher sensitivity and shorter reaction time, but are limited by their economy. 3 Although DNA hybridization-based methods provide a successful alternative,4,5 when it comes to non-invasive detection procedures, however, electrochemical biosensors are considered ideal candidates due to their robust properties and have Transform into a device with great potential for point-of-care (POC). 6,7 Cell, protein and hormone receptors are commonly used as target ligands/analytes in these biosensors. The rapid and active detection of nucleic acids is very popular in disease diagnosis and forensic identification. 8,9 Electrochemical signal changes based on DNA/analyte hybridization are usually used as the main basis for various electrochemical biosensors and genetic sensors. 10

DNA hybridization usually causes very weak electrochemical signal changes on conventional metal electrodes; however, various nanomaterial-based modifiers are currently being used to enhance them. These modifiers have many electroactive and biologically active sites on the surface. Points can be used to easily adsorb/desorb changes during analysis. 4,11 Among various nanomaterials, graphene oxide (GO) has received unstoppable attention. 12 GO is electrochemically active and can be further functionalized with biopolymers 13 such as chitosan (CHI). Chitosan (CHI) obtained by partial deacetylation of chitin is biocompatible and biodegradable, and has antibacterial properties. It exhibits great film-forming ability, making it a popular choice for immobilizing biologically active molecules on biosensors. It is used as a scaffold for tissue engineering and a drug carrier in wound dressings. Recently, there have been reports that biosensors based on chitosan can be used to measure a variety of analytes from biological, environmental, and chemical sources. 14 However, due to the degradability of CHI in aqueous media, the use of CHI-based biosensors in in vitro measurements is limited. 15 In this regard, recent studies have demonstrated the possibility of improving mechanical properties (under wet and dry conditions) and thermodynamic properties. The stability of CHI is improved by adding graphene oxide (GO) to the CHI matrix. 16 This is due to the cross-linking between the amine (-NH2) and hydroxyl (-OH) groups and the carboxyl (-COOH) group of GO. The CHI-GO substrate can also be made into a super flexible film, which helps the development of sensors17. Due to the ionization of hydroxyl and carboxylic acid, GO shows a negative charge when dispersed in water. The CHI with –OH and –NH2 in the acidic medium is protonated into polycationic material, which helps the interaction and connection of the polymer chain and GO to form a graphene oxide-chitosan (GO-CHI) hybrid Nanocomposite materials. 18

Based on the feasibility, sensitivity and applicability of the transduction principle, two methods have been used to monitor electrochemical biosensors. The first method is a label-free method, in which the signal is generated by an intrinsically electrically active substance containing the analyte. 19 The use of nucleic acid hybridization is an example of the use of label-free methods. The receptors of target ligands in these biosensors can range from cells or proteins or hormones to targeted DNA probes as analytes. However, among the labeling methods, sandwich analysis (redox labeling), nucleic acid intercalators and molecular beacons are used for detection purposes. Although robust, these methods may be limited by electrode contamination and the generation of non-specific signals, which can make accurate measurements difficult. This limitation can be avoided by carefully pre-treating the sample or membrane coating to avoid false positive results.

DNA-based electrochemical biosensors work by detecting changes in electrochemical signals during DNA hybridization. 20 DNA biosensors are tiny single-stranded DNA (ssDNA) or RNA, with about 100-200 nucleotides or less, and provide higher stability. Due to its specificity and robust hybridization, DNA-based biosensors are being studied as potential genetic sensors in various applications. 21,22

In the current research, a DNA-based electrochemical sensor platform has been established for early detection of tuberculosis from sputum and purified DNA samples. DNA probes have been used to detect the IS6110 insert element. IS6110 is a mobile genetic element that has been used for the detection of Mycobacterium tuberculosis complex (Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium micropedis and Mycobacterium canettii) And molecular epidemiology research. Due to its high level of replication throughout the genome, it is considered the gold standard biomarker for tuberculosis detection. 23 IS6110 is a 1354 bp repetitive insert sequence, with 1-20 copies per cell, making it 24 cyclic voltammetry (CV) and differential pulse voltammetry (DPV) are used to evaluate the performance of the sensor platform The main analytical technique. This study aims to meet the need for robust, accurate and portable tuberculosis diagnostic procedures to screen the population and identify missed cases of active tuberculosis for correct treatment.

An improved method has been published called the Improved Hummers method for the preparation of graphene oxide (GO). 25 Previously, the Hummers method (including KMnO4, NaNO3, H2SO4) was most commonly used to prepare graphene oxide. 26 Excluding NaNO3 and increasing the amount of KMnO4, and reacting in a 9:1 mixture of H2SO4/H3PO4 improves the efficiency of the oxidation process. In short, add 3g graphite flakes (150 mm, Sigma-Aldrich) to a mixture of concentrated sulfuric acid (360mL H2SO4, Sigma-Aldrich) and phosphoric acid (40mL H3PO4, Sigma-Aldrich) (mixing ratio is 9:1), The total volume is 60 mL, and the mixture is stirred for 4 hours. Subsequent addition of 180 grams of KMnO4 (Sigma-Aldrich) resulted in an exothermic reaction (35°C-40°C). The resulting solution was placed on a magnetic stirrer (Velp-Scientifica, Europe), heated to 50°C for 6 hours under continuous stirring, then stirred overnight, and then slowly cooled to room temperature. Add 30mL hydrogen peroxide (H2O2, 3%) to stop the oxidation reaction. The resulting mixture was washed several times with deionized (DI) water (200 mL), hydrochloric acid (HCL, 30%) and ethanol (C2H5OH, 70%). Then the mixture was washed several times with an equal amount of deionized water and centrifuged at 6000 rpm for 15 minutes to obtain a viscous gel, which was allowed to dry overnight at 60°C, as shown in Figure 1. This modified Hummers method provides a larger amount of hydrophilic graphene oxide material than the original Hummers method. 27 Figure 1 The GO is synthesized step by step through the improved Hummer method and GO-CHI nanocomposite materials. Figure 2 SEM of graphene oxide (GO) at (A) 50μm, (B) 5μm, (C) 1μm (D) graphene oxide and chitosan nanocomposite (GO-CHI) at (E) 50μm Figure, (F) 5μm, (G) 2μm and (H) GO-CHI EDX spectrum.

Figure 1. GO is synthesized step by step through the improved Hummer method and GO-CHI nanocomposite materials.

Figure 2 SEM of graphene oxide (GO) at (A) 50μm, (B) 5μm, (C) 1μm (D) graphene oxide and chitosan nanocomposite (GO-CHI) at (E) 50μm Figure, (F) 5μm, (G) 2μm and (H) GO-CHI EDX spectrum.

To prepare the GO-CHI nanocomposite, 0.15g CHI ((MW 200,000, Santa Cruz Biotech, Heidelberg, Germany) was slowly added to 15mL 1% v/v acetic acid (Phytotechnology labs, Lenexa, USA) and stirred continuously for 15 minutes. Add 7 mg of GO to 2.5 mL of Millipore ultrapure water (Thermo Fisher Scientific, USA) and sonicate (Cole-Parmer 8890, USA) for 1 minute for complete dispersion. This separately prepared GO dispersion is gradually added to the previously prepared CHI In the solution, the resulting solution was vortexed for 35 minutes, and then sonicated in a sonicator bath at room temperature for 3 hours to obtain a fully dispersed solution, as shown in Figure 1.

In order to manufacture and assemble DNA probes combined with gene sensors for IS6110 sensing, indium tin oxide (ITO) coated glass coverslips were purchased from Sigma-Aldrich (USA), and 70% ethanol (Sigma-Aldrich, USA) ) And Millipore ultrapure water (Thermo Fisher Scientific, USA), and then blow dry to remove microfibers or dust particles. By pouring 100mL of nanocomposite material on the surface of the glass slide, functionalize the ITO glass slide with G0-CHI composite material, and rotate it on the spin coater at 3000 rpm for 20 seconds, so as to be on the surface of the ITO glass slide Get a uniform GO-CHI layer on top. It was then allowed to dry at room temperature for 4 hours. In order to remove contamination, the surface of the sensor (electrode) was cleaned with 0.1M NaOH (Sigma-Aldrich, USA), and then dried overnight in a nitrogen environment.

In order to fix the DNA probe on the electrode surface, the amine group in the GO-CHI composite material is the first exposed surface by using 40 µL 1% glutaraldehyde (Sigma-Aldrich, USA), 30 µL phosphate buffer (0.1 M, pH7) and 0.8 g bovine serum albumin (BSA Sigma-Aldrich, USA) solution for 2 hours. The electrode was then washed with Millipore ultrapure water (Thermo Fisher Scientific, USA) to remove untreated glutaraldehyde and residues. Then, 30μL 1.5mM ssDNA probe (Integrated DNA Technologies, USA) has an amino linker C6( AmMC6) -3ʹ)5 was poured dropwise and covalently connected to the amine activated electrode (GO-CHI coated ITO) by incubating for 1 hour at room temperature. Then the electrode surface was washed several times with Millipore ultrapure-1 (Thermo Fisher Scientific, USA) water (to remove the remaining unbound ssDNA strands on the electrode surface), and stored at 4°C for future use.

Check functionalized ITO glass slides with assembled DNA probes to detect Mtb sensitively and specifically. Mtb purified DNA samples (n=4) collected from the original sputum sample bank (n=4) of tuberculosis patients in the BSL-3 clinical laboratory of the Pakistan Institute of Medical Sciences (PIMS) Islamabad, after obtaining their consent and ethics After approval, it is used to evaluate the hybridization efficiency, thereby evaluating the detection limit of the developed DNA gene sensor (ssDNA probe to GO-CHI/ITO electrode). Non-complementary DNA samples of methicillin-resistant Staphylococcus aureus (MRSA) were taken from the BSL-2 Microbiology Laboratory of the National University of Science and Technology (NUST) in Islamabad. After NUST's Institutional Review Board (IRB) approved the ethical compliance of the research project, PIMS Islamabad's Tuberculosis Center also kindly provided original sputum samples. Purify the original sputum sample with 1% N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH) in the PIMS BSL-3 laboratory. After incubating for 25 minutes, centrifuge the sample and wash the pellet with Tris HCL. Then use GeneJET™ Gel Extraction Kit to purify DNA and store at -20°C. A 10-fold serial dilution method was used to serially dilute the DNA in the purified DNA sample, and the concentration was measured using a Nanodrop spectrophotometer (Thermo Scientific™ NanoDrop 2000). The recorded DNA sample concentrations were 7.86 pM, 15.7 pM, 47.15 pM, and 94.3 pM.

The genomic DNA of purified and original sputum samples were denatured by using a water bath (Thermo Scientific, USA) to heat at 95 °C for 5 minutes and immediately cool in ice to obtain denatured ssDNA.

The purified DNA sample and BSA (0.8g) were dispersed in phosphate buffered saline (PBS, pH7). For each electrochemical experiment, 10 μL samples from TB and non-complementary DNA samples were poured dropwise onto the electrode surface and incubated for 45 minutes to achieve maximum hybridization. It was then washed with Millipore ultrapure water to remove unbound DNA, and the probe-functionalized ITO slides were electrochemically scanned after treatment with sample analytes.

The vacuum-dried samples of GO and GO-CHI were sputtered with aluminum and characterized by FESEM (Nova NanoSEM 650, EU), and scanned at 1500kV with a magnification of 40,000X. The energy dispersive X-ray spectroscopy (EDS) of the sample was completed at 20 eV, the probe current was 50 nA, and the take-off angle was 350.

Use PerkinElmer Spectrum-100 FTIR spectrometer for FTIR analysis and scan the sample between 400–4000 cm-1. Both GO and GO-CHI pallets are prepared using a manual hydraulic press (Specac, USA) using potassium bromide (KBr) as a base. The obtained spectrum was further analyzed by eFTIR (Operant LLC, USA) for peak labeling.

In order to evaluate the crystal structure of graphene oxide, X-ray diffraction (XRD) was performed on a STOE powder diffractometer θ-θ (STOE Inc. Germany) with a working voltage of 40kV, a current of 40mA, and a scan rate of 0.5 min -1. Sample Dry completely at 60°C for 3 hours, and then perform XRD analysis to remove any possible moisture in the sample. The value of the interplanar distance "d" is calculated using the following Bragg's law:

The surface charge of the sample was measured on Malvern Zetasizer Ver. to evaluate the zeta potential change between GO and GO-CHI nanocomposites. 7.10 (Malvern Instruments, UK) At room temperature (25°C). Use a transparent disposable zeta cell to load the sample into the zeta particle size analyzer.

The standard three-electrode configuration glass cell is used for electrochemical characterization by CV and DPV on a computer-controlled electrochemical workstation (Gamry potentiostat, Reference 600™). The GO-CHI coated ITO electrode is used as the working electrode (0.5 cm2), the platinum wire is used as the auxiliary (counter) electrode, and the saturated calomel electrode (SCE) is used as the reference electrode. During the CV study, 3mM ferricyanide [Fe(CN)6]3-containing 0.1 M KCL was used as the electrolyte, and for differential pulse voltammetry, phosphate buffered saline (50mM) was used as the supporting electrolyte. CV is performed at a scan rate of 100 mV s-1. The potential range of the two experiments is between -0.6 to 0.6 V and SCE. Before use, all glassware and electrochemical cells were thoroughly cleaned by boiling in a 1:3 solution of sulfuric acid and nitric acid, and then boiling in Millipore ultrapure water (Thermo Fisher Scientific, USA). They were rinsed several times with Millipore water, methanol, and ethanol, and finally rinsed with acetone, and finally dried in an oven at 100°C for 1 hour. All solutions involved in electrochemical research are freshly prepared with ultrapure water.

A Bio-Rad T100tm thermal cycler was used to amplify purified DNA samples. The forward primer (5ʹ-AGAAGGCGTACTCGACCTGA-3ʹ) and the reverse primer (5ʹ-GATCGTCTCGGCTAGTGCAT-3ʹ) are used to amplify the Mtb insert sequence IS6110. The primer sequence for IS6110 was adapted from Liu et al. 5 The purified DNA is initially denatured at 95°C for 4 minutes, then at 94°C for 30 seconds, then 35 cycles, then at 56°C for 45 seconds, at 72°C for 45 seconds, and finally at 72°C Extend for 7 minutes. The obtained PCR products were run on a 2% agarose gel (Sigma-Aldrich, USA), stained with ethidium bromide (Sigma-Aldrich, USA) and visualized by Gel LOGIC 2200 PRO imaging system.

Scanning electron micrographs (Figure 2) show different flake morphologies, reflecting changes in the diffusion rate of oxidants. The difference in the diffusion rate indicates the change in the degree of oxidation. The size and crystal structure of the flakes play a decisive role in the formation of pristine graphene oxide (PGO), and the more disordered small-sized flakes show a significantly higher oxidation rate. 28 Figure 2A-C shows the exfoliated scanning area (50μm to 1μm) of graphene oxide prepared from graphene oxide, showing appropriately dispersed carbon layers and oxygen molecules, forming clear, interconnected three-dimensional graphene sheets.

In the GO-CHI nanocomposite, the amide bond (the formation of CN bond from the carboxyamine group) causes the exfoliated GO flakes to be properly dispersed in the chitosan matrix, so the SEM micrograph below (Figure 2E-G) reveals The appearance of the embedded structure shows the dispersion of the unidirectional GO nanosheets in the turbid chitosan matrix.

The EDX spectra of graphene oxide (Figure 2D) and its composite material (Figure 2H) show that both graphene oxide and nanocomposite materials are successfully formed with oxygen species. Compared with GO-CHI nanocomposites, the higher oxygen concentration in the EDX spectrum of graphene oxide is due to various functional groups such as -OH, -COOH and -CHO amide (CN), carbamate (CHN) and The glycosidic bond between the amine (–NH2) and the hydroxyl (–OH) group of CHI and the carboxyl (–COOH) group of GO.

Perform FTIR to evaluate the abundance of functional groups present in CHI, GO, and GO-CHI. The appearance of peaks at wavenumbers of 1739 cm-1 and 3325 cm-1 confirmed the presence of graphene oxide. The peak at 1739 cm-1 indicates the presence of a carbonyl moiety (C=O), indicating that the synthesis of graphene oxide was successful. 29 The broad peak at 3325 cm-1 indicates that GO absorbs water, as shown by the OH stretch of water molecules. Characteristic chitosan peaks were seen at 1030 cm-1, 1602 cm-1, and 3450 cm-1. The peaks at 1030 cm-1 and 1602 cm-1 show the presence of glycosidic bonds (CO) and C=O (NHCO), respectively. Graphene oxide-chitosan nanocomposite (GO-CHI) showed characteristic peaks at 1035 cm-1, 1648 cm-1 and 3325 cm-1. The peak at 3325 cm-1 is attributed to the amine stretching of chitosan and hydroxyl (OH) in GO, which leads to the formation of nanocomposites. 15 The conservative peaks of glycosidic bond and C=O in NHCO are stretched by vibration, and NH of NH2 bends. For graphene oxide, the peak shift of nanocomposites ranges from 1739 cm-1 to 1648 cm-1, while that of chitosan ranges from 1624 cm-1 to 1648 cm-1, revealing that the interaction between GO and CHI leads to nanocomposite The formation of the material is shown in Figure 3. Figure 3 FTIR analysis of graphene oxide (GO), chitosan (CHI) and graphene oxide-chitosan nanocomposite (GO-CHI).

Figure 3 FTIR analysis of graphene oxide (GO), chitosan (CHI) and graphene oxide-chitosan nanocomposite (GO-CHI).

The FTIR spectrum of GO-CHI (formed by mixing GO and chitosan) showed a shift around 1602 cm-1, indicating the formation of amide (CN) and carbamate (CHN). The peak near 1035 cm-1 reflects the presence of COC bonds in chitosan glycosidic bonds.

In order to confirm the crystal structure of graphene oxide, powder X-ray diffraction (XRD) was performed, and the XRD pattern of graphene oxide was compared with the XRD pattern of graphite flakes, as shown in Figure 4. Figure 4 (A) XRD of graphite flakes. (B) Graphene oxide (GO).

Figure 4 (A) XRD of graphite flakes. (B) Graphene oxide (GO).

It is found that the diffraction angle of graphite at 2q is 26o, revealing the high structure and layered structure of graphite (Figure 4A). Using the Bragg equation, it is found that the interlayer spacing "d" is 0.34 nm.

The GO diffraction angle obtained by graphite oxide flakes is 10.2°, and the 26° peak in Figure 4B disappears, indicating proper oxidation and exfoliation. It is found that the interlayer spacing has increased by 0.82 nm, which is about 3 times higher than that of graphite (0.34 nm).

The XRD pattern showed a peak of 10.2o at 2q, indicating that the signal originated from the moiré pattern in the graphene double layer. It should be noted that GO is consistent in two randomly distributed domains. Since the complete graphene domain is the main site for signal generation, the oxidized GO shows no signal due to irregular C atoms formed by irregular C atoms. 30 In the last step, PGO is converted to GO by peeling PGO into a single layer sheet. This is achieved by hydrolyzing sulfate as a protective cluster.

In order to evaluate the surface charge of the GO-CHI nanocomposite, zeta potential measurement was performed. It is found that the surface charge on graphene oxide is -20.2 mV, which exhibits a negative charge due to the abundant carboxyl groups. After adding CHI, the potential of the resulting nanocomposite material (GO-CHI) changed from -20.2mV to 34.9mV. This conversion of surface charge occurs due to the addition of chitosan with a large number of positively charged amino (–NH2) groups. The positive charge on the surface of the GO-CHI nanocomposite plays an important role in enhancing the adhesion of negatively charged DNA on the surface of the ITO electrode.

The GO-CHI-coated ITO electrode was used as the working electrode and was characterized by cyclic voltammetry in 3mM potassium ferricyanide K3[Fe(CN)6]3-, which contained 0.1M KCL as the electrolyte solution. Compared with bare ITO, due to smooth electron transmission, when GO-CHI nanocomposite material is coated on the surface of ITO, the obtained voltammogram shows a significant increase in peak current. In the case of GO-CHI, the increase in peak current and peak potential reflects better electron mobility because the high surface area leads to an increase in conductivity.

As shown in Figure 5, the redox peak currents (Ipa, Ipc) at various potentials are recorded. Figure 5 Cyclic voltammogram (CV) of bare ITO and GO-CHI/ITO electrode at 100mV1/2.

Figure 5 Cyclic voltammogram (CV) of bare ITO and GO-CHI/ITO electrode at 100mV1/2.

The anode peak current (Ipa) of bare ITO is -51.43 μA, while the anode peak current (Ipa) of ITO coated with GO-CHI is -179.96 μA, resulting in approximately 3.5 times higher conductivity. Similarly, bare ITO showed a cathode peak current (IPC) of 37.66 μA and increased to 167.88 μA (about 4 times) in the case of ITO coated with GO-CHI (Table 1). Table 1 Redox peak current and peak potential of bare ITO and GO-CHI/ITO

Table 1 Redox peak current and peak potential of bare ITO and GO-CHI/ITO

The peak respiration (Δ Ep) was calculated to evaluate the electron transfer in the chemical process, and it was found that the ITO (0.12) of the GO-CHI coating was larger than that of the bare ITO (0.06), confirming the irreversibility of the reaction. For reversible electron transfer, Δ Ep must be equal to 0.059V, and Ipa/Ipc must be equal to 1. Here, the Δ Ep of the ITO coated with GO-CHI (0.12) is greater than 0.0059V and Ipa/Ipc (1.07) is greater than 1.31

In order to deeply study the kinetics of the electron transfer process on the surface of the GO-CHI electrode, the GO-CHI-coated ITO electrode was subjected to cyclic voltammetry again at different scan rates (20-120 mV), as shown in Figure 6A. As the scan rate increased from 20 (88.3 μA, -84.42 μA) to 120 mV (239.48 μA,-272.18 μA), an increase in Ipc and Ipa was observed. When Ipc and Ipa are plotted against the square root of the scan rate, it is found that the process is diffusion-controlled and quasi-reversible, showing a linear curve as shown in Figure 6B, showing the stability of the sensor, that is, the coating on the ITO electrode GO-CHI transduction film. Figure 6 (A) GO-CHI/ITO electrode in 3mM K3[Fe(CN)6]3- and 0.1 M KCl at 20 mV s-1 to 120 mV s-1 CV at scan rate. (B) Anode and cathode peak currents at different scan rates.

Figure 6 (A) The CV of GO-CHI/ITO electrode in 3mM K3[Fe(CN)6]3- and 0.1 M KCl at various scan rates from 20 mV s-1 to 120 mV s-1. (B) Anode and cathode peak currents at different scan rates.

The diffusion control reaction is considered to have a very fast charge transfer reaction and is kinetically favorable. This is due to the high electroactive surface area of ​​graphene oxide and chitosan films.

In order to prove that the GO-CHI electrode can operate at 3mM K3 [Fe(CN)6]3− and 0.1 M KCl at different scan rates, the electroactive surface area of ​​the bare electrode and the GO-CHI modified electrode is calculated by Randle Sevcik's equation: 32

Ip = 2.69x105n3/2AD1/2V1/2 C

Where Ip corresponds to the peak current, D is the diffusion coefficient of the analyte, that is 4.033×10-6 cm2 s-1, 33 A is the area of ​​the working electrode (0.48cm2), C is the volume concentration, and the unit is mol cm- 3 3mM K3[Fe(CN)6]3− and 0.1 M KCl, n is the number of electrons, that is, 1 and V are the scan rates in V s−1. The electroactive area of ​​the bare electrode is 0.48 cm2, and the electroactive area of ​​the GO-CHI modified electrode is increased to 6.04 cm.2.

When stored at 4°C, the GO-CHI modified ITO electrode showed stable conductance for up to 25 days with minimal conduction loss (data not shown). GO can prevent the degradation of CHI. According to reports, GO-CHI composite materials can form stable electrostatic bonds, which can promote electron migration while maintaining the characteristics of the film. 34

Perform CV and DPV to observe the change of the current value when the DNA probe is immobilized on the GO-CHI/ITO working electrode, and the subsequent hybridization with the purified target DNA sample. When the capture DNA probe (IS6110's ssDNA) was immobilized on the GO-CHI/ITO electrode in 3 mM K3[Fe(CN)6]3, the peak currents (154.31 μA, -157.7 μA) of the cathode and anode were clearly observed Reduce-and 0.1M KCL, as shown in Figure 7. When 10μL of purified target DNA (25pM/μL) was allowed to bind and hybridize with the ssDNA probe, a further decrease in peak current was observed (105.16 μA, -135.56 μA). This is because the analyte covers the surface of the electrode, which may cause Block the active sites of modified GO-CHI/ITO, thereby hindering charge transfer. Figure 7 (A) CV analysis, (B) bare ITO, GO-CHI on ITO, GO-CHI-ITO bound ssDNA probe and target DNA (25pM/μL) ssDNA probe linked to GO-CHI-ITO DPV, all in 3 mM K3 [Fe(CN)6]3- 0.1 M KCl, the scan rate is 100 mV s-1. Figure 8 (A) The specific analysis of the target analyte by the biosensor through DPV. (B) Sensitivity analysis of biosensors by DPV. (C) Calibration curve of serial dilutions of target DNA at different concentrations based on DPV results. (D) The original sputum sample of the biosensor is analyzed by DPV.

Figure 7 (A) CV analysis, (B) bare ITO, GO-CHI on ITO, GO-CHI-ITO bound ssDNA probe and target DNA (25pM/μL) ssDNA probe linked to GO-CHI-ITO DPV, all in 3 mM K3 [Fe(CN)6]3- 0.1 M KCl, the scan rate is 100 mV s-1.

Figure 8 (A) The specific analysis of the target analyte by the biosensor through DPV. (B) Sensitivity analysis of biosensors by DPV. (C) Calibration curve of serial dilutions of target DNA at different concentrations based on DPV results. (D) The original sputum sample of the biosensor is analyzed by DPV.

The CV and DPV voltammograms revealed insights into the sensitivity and specificity of the biosensor, where only the selective hybridization of the complementary strand (dsDNA) showed an increase in charge transfer resistance. Compared with non-specific scaffolds, a very significant increase in charge transfer resistance was observed. Small is specified by the peak current. When hybridized with complementary targets, the current was reduced by 30%, while non-specific targets only showed a 9% reduction in current.

The slight decrease in the peak current of DNA capture probe (ssDNA) binding GO-CHI is due to the residual glutaraldehyde (after washing) that was previously used to cross-link ssDNA to the CHI amino group in the GO-CHI working electrode. When incubated with the ssDNA probe, the target DNA analyte is electrochemically characterized, and a decrease in current is observed due to the formation of dsDNA. The oligonucleotide of the target DNA successfully hybridized with the oligonucleotide of the capture probe. Therefore, no functional group (free oligonucleotide) hinders the flow of electrons, which increases the resistance in the current. The highly selective and specific strands from DNA do not leave any free radicals, and the charge is further reduced, as shown in the DPV voltammogram.

The treated ssDNA probe functionalized GO-CHI electrode with target DNA is reused in an aqueous solution by a simple thermal regeneration method, which removes the bound target DNA through thermal denaturation. By heating to remove the bound probe molecules, the hybridized DNA form also supports the complete regeneration of the GO-CHI composite membrane.

After obtaining satisfactory Mtb detection results on the positively charged functionalized GO-CHI/ITO electrode, further experiments were performed to check the sensitivity and sensitivity of the DNA gene sensor in 25°C neutral PBS (pH≈7) Specificity. As a more sensitive electrical analysis technique, differential pulse voltammetry is used for further analysis.

Purified TB DNA sample: The specificity of the DNA gene sensor was evaluated by alternately exposing the electrode surface to 10μL (25pM/μL) of complementary strand and 10μL (50nM/μL) of non-complementary strand (MRSA). Compared with the non-complementary strand binding, after the ssDNA modified electrode is combined with the complementary DNA strand, a significant change of 30% reduction in the peak current is observed. The non-complementary strand binding shows that the peak current of the ssDNA modified electrode is only reduced by 9%, which is not very obvious. , As shown in Figure 8A. The significant reduction in the peak current of the complementary strand is due to the conformational change from ssDNA to dsDNA during hybridization, as shown in Figure 8A.

In addition, a 10-fold serial dilution method was used to serially dilute 10 μL of 94.3 pM/μL DNA samples, and a Nano drop spectrophotometer was used to record DNA samples of different concentrations (7.86 pM, 15.7 pM, 47.15 pM, 94.3 pM) to evaluate the saturation point and Sensitivity, as shown in Figure 8B. It was observed that 94.3 pM was the saturation point reached as the lowest current, and no obvious peak height was seen at this concentration, and it was observed as the maximum analyte concentration. When the concentration was reduced from 94.3 pM to 47.15 pM and 15.7 pM, a significant increase in peak current was observed. The highest peak current (all concentrations) was observed at 7.86 pM, indicating the lowest concentration of target DNA detected, as shown in Figure 8B. From the DPV analysis, draw a calibration chart for the current generated by four serial dilutions (each 10μL) of different concentrations (target DNA) samples, and calculate the correlation coefficient to be R2=0.9933, as shown in Figure 8C.

The limit of detection (LOD) of the biosensor is calculated by the following formula:

And σ is the standard deviation, and m is the slope. Therefore, the calculated LOD is 3.40 pM, indicating that it is sensitive to very low amounts of target DNA in the sample. When comparing this proposed gene sensor with the recently reported nanomaterial-DNA electrochemical biosensor 35-38 for Mtb detection (Table 2), the synthetic DNA gene sensor in this study showed the lowest LOD and high Specificity. Therefore, the comparison further shows that this method can be successfully applied to Mtb detection. Table 2 Comparison of reported nanoparticle-based electrochemical DNA biosensors for the detection of Mtb and the proposed work

Table 2 Comparison of reported nanoparticle-based electrochemical DNA biosensors for the detection of Mtb and the proposed work

Raw sputum TB sample: Use DPV to test the selectivity of the synthesized ssDNA biosensor (19 nucleotides) against fresh raw sputum samples without using any DNA purification kit to analyze the sensor performance of the original biological sample. Decontaminate and sterilize the three sterilized samples as described in Target DNA Hybridization. The original samples have been tested by Genexpert installed in the TB Center and classified as "medium positive tuberculosis samples" and "highly positive tuberculosis samples". After disinfection, the samples were marked as medium and high according to the Genexpert results, and analyzed with the manufactured DNA gene sensor by recording DPV after 45 minutes of incubation with the sample (different from the 2 hours in the case of Genexpert), as shown in Figure 8D . A significant peak reduction (amount of current generated) was observed in all three samples, indicating the detection performance of the genetic sensor on the original biological sample. In addition, there is a significant difference in peak reduction between high-positive and medium-positive samples, which not only supports and provides sensitivity equivalent to Genexpert, but also shows that the manufactured DNA genetic sensor can distinguish the pathogenic intensity of samples.

Use GeneJET™ Gel Extraction Kit to extract and purify DNA samples, and perform gel electrophoresis on the PCR products. The four tuberculosis types gave IS6110 of the correct size (ie 157bp) in repeated runs, which was also confirmed by Macrogen Korea's Next Generation Sequencing (NSG), and no product was seen in the negative samples. This confirms that the biosensor is sensitive and specifically related to the presence of IS6110 in the sample (Figure 9). Figure 9 Duplicate gel electrophoresis of the four Mtb DNA samples used in the study, confirming the presence of 1S6110.

Figure 9 Duplicate gel electrophoresis of the four Mtb DNA samples used in the study, confirming the presence of 1S6110.

In the current study, ITO glass slides are used as electrode substrates to make DNA genetic sensors, and they are coated with GO-CHI nanocomposite materials for rapid detection of tuberculosis. Nanocomposites are prepared by mixing freshly prepared graphene oxide and chitosan. This graphene oxide-chitosan nanocomposite (GO-CHI) is uniformly coated on the ITO surface to increase conductivity and provide overall positive charge to the electrode surface for effective binding and immobilization of ssDNA probes. The DNA gene sensor shows the specificity of distinguishing the non-complementary DNA of MRSA from the complementary DNA of Mycobacterium tuberculosis (IS6110). Different concentrations of target DNA were used to study the analytical performance of DNA gene sensors. The square of the linear correlation coefficient was 0.99, and the detection limit was 3.40 pM. Among the recently reported electrochemical biosensors for Mtb detection, the detection limit was found to be the lowest, proving the high sensitivity of the biosensor (Table 2). The obtained results also show that the electrochemical DNA genetic sensor based on GO-CHI can distinguish the pathogenic intensity of the original sputum sample. Therefore, it clearly shows that GO-CHI-based electrochemical biosensors can be regarded as successful candidates for immediate medical diagnosis of tuberculosis.

We are grateful for the technical support and convenience provided by the Pakistan Institute of Medical Sciences (PIMS) Hospital for providing us with tuberculosis samples and Genexpert facilities. We further thank the School of Chemistry and Materials Engineering (SCME)-NUST for facilitating FTIR, XRD and SEM.

All authors participated in data analysis, drafting or revising the article, finally approved the version to be published, agreed to the journal to be submitted, and agreed to be responsible for all aspects of the work.

Dr. Shah Rukh Abbas reported a pending utility patent application No. 175/2019; no license has been obtained yet. The author declares that this work has no other potential conflicts of interest.

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