A microfluidic electrochemical biosensor based on multiwall carbon nanotube/ferrocene for genomic DNA detection of Mycobacterium tuberculosis in clinical isolates

Abstract

Herein we present a microfluidic-multiplexed platform that integrates electrochemical sensors based on carbon nanotubes associated with ferrocene as redox marker (carbon nanotube (CNT)/ferrocene) for direct detection of pathogenic viral DNA from Hepatitis C and genomic DNA from Mycobacterium tuberculosis in clinical isolates. By operating the fluidic device under high flow (150 μl/min), the formation of a very thin depletion layer at the sensor surface (δS = 230 nm) enhances the capture rate up to one DNA strand per second. By comparison, this capture rate is only 0.02 molecule/s in a static regime without flow. This fluidic protocol allows thus enhancing the limit of detection of the electrochemical biosensor from picomolar in bulk solution to femtomolar with a large dynamic range from 0.1 fM to 1 pM. Kinetics analysis also demonstrates an enhancement of the rate constant of electron transfer (kS) of the electrochemical process from 1 s(-1) up to 6 s(-1) thanks to the geometry of the miniaturized fluidic electrochemical cell. This microfluidic device working under high flow allows selective direct detection of a Mycobacterium tuberculosis (H37Rv) rpoB allele from clinical isolate extracted DNA. We envision that a microfluidic approach under high flow associated with a multiwall CNT/ferrocene sensor could find useful applications as the point-of-care for multi-target diagnostics of biomarkers in real samples.

Carbon nanotubes in microfluidic lab-on-a-chip technology: current trends and future perspectives

“…This system resulted in rapid detection of melatonin, tryptophan and serotonin in periods shorted than 150 s (Gomez et al 2015). Furthermore, it was found that an amperometric sensor could be made of CNTs and ferrocene (as a redox-responsive reporter) that was applied in order to detect pathogenic viral DNA from hepatitis C and genomic DNA from Myocobacterium tuberculosis with high sensitivity (Zribi et al 2016). …”

Electrochemical Biosensing for the Diagnosis of Viral Infections and Tropical Diseases

“…Biosensors for detecting the proper antigen or the immunological markers associated with the infections have demonstrated to be an efficient tool for simple and rapid diagnosis in real time and complex samples. Table 2 [72,[99][100][101][102][103][104][105][106][107][108][109][110][111][112] shows some examples of methods using electrochemical DNA sensors or immunosensors characterized by their low limits of detection.…”

Diagnostics Strategies with Electrochemical Affinity Biosensors Using Carbon Nanomaterials as Electrode Modifiers

“…Direct detection of a genomic DNA sample, without pre-analytical and amplification steps, allowed shortening analysis times and minimizing system complexity and other errors issued from contamination occurring in these further steps. Since accurate diagnostics need specific detection of very small amounts of DNA, there is a need to develop simple label-free DNA hybridization platforms able to perform highly sensitive and specific direct analyses [16]. Moreover, DNA sensors are widely used in diagnostic tests for early cancer and mutation detection, analysis of gene sequences, forensic investigation and assessment of medical treatment [17,18].…”

“…They are critically discussed in the following sections with the aim of showing the enormous potential of DNA electrochemical devices in the field of medical and clinical diagnostics. Furthermore, electrochemical detection is fully compatible with cost-effective hand held microfluidic platforms that bring the possibility of manipulating tiny amounts of samples (<µL) for fast analysis [16]. …”

“…Zribi et al [16] developed a microfluidic-multiplexed electrochemical platform for direct impedimetric DNA detection without any amplification of Hepatitis C viral DNA and Mycobacterium tuberculosis genomic DNA in clinical isolates. The developed monolithic chip integrated three independent fluidic polydimethylsiloxanes (PDMSs) channels, each containing one electrochemical chamber with three gold electrodes.…”