ABSTRACT
Electrochemical detection methods hold many advantages over their optical counterparts, such as operation in complex sample matrices, low-cost and high volume manufacture and possible equipment miniaturisation. Despite these advantages, the use of electrochemical detection is currently limited in the clinical setting. There is a wide range of potential electrode materials, selected for optimal signal-to-noise ratios and reproducibility when detecting target analytes. The use of carbon paste electrodes (CPEs) for electrochemical detection can be limited by their analytical performance, however they remain very attractive due to their low cost and biocompatibility. This paper presents the fabrication of an easy-to-make and use graphite powder/paraffin wax paste combined with a substrate produced via additive manufacturing and confirms its functionality for both direct and indirect electrochemical measurements. The produced CPEs enable the direct voltammetric detection of hexaammineruthenium(III) chloride and dopamine at an experimental limit of detection (ELoD) of 62.5 µM. The key inflammatory biomarker Interleukin-6 through an enzyme-linked immunosorbant assay (ELISA) was also quantified, yielding a clinically-relevant ELoD of 150 pg ml-1 in 10% human serum. The performance of low-cost and easy-to-use CPEs obtained in 0.5 hours is showcased in this study, demonstrating the platform's potential uses for point-of-need electroanalytical applications.
ABSTRACT
Earlier access to patients' biomarker status could transform disease management. However, gold-standard techniques such as enzyme-linked immunosorbent assays (ELISAs) are typically not deployed at the point-of-care due to their cumbersome instrumentation and complexity. Electrochemical immunosensors can be disruptive in this sector with their small size and lower cost but, without further modifications, the performance of these sensors in complex media (e.g., blood) has been limited. This paper presents a low-cost fluidic accessory fabricated using widely accessible materials and processes for boosting sensor sensitivity through confinement of the detection media next to the electrode surface. Liquid confinement first highlighted a spontaneous reaction between the pseudoreference electrode and ELISA detection substrate 3,3',5,5'-tetramethylbenzidine (TMB) that decreases the amount of oxTMB available for detection. Different strategies are investigated to limit this and maximize reliability. Next, flow cell integration during the signal amplification step of sensor preparation was shown to substantially enhance the detection of cytokine interleukin-6 (IL-6) with the best sensitivity boost recorded for fresh human plasma (x7 increase compared to x5.8 in purified serum and x5.5 in PBS). The flow cell requires no specialized equipment and can be seamlessly integrated with commercial sensors, making an ideal companion for electrochemical signal enhancement.
Subject(s)
Electrochemical Techniques , Humans , Electrochemical Techniques/methods , Electrochemical Techniques/instrumentation , Immunoassay/methods , Immunoassay/instrumentation , Biosensing Techniques/methods , Biosensing Techniques/instrumentation , Electrodes , Enzyme-Linked Immunosorbent Assay/methods , Interleukin-6/blood , Interleukin-6/analysis , Benzidines/chemistryABSTRACT
A major societal challenge is the development of the necessary tools for early diagnosis of diseases such as cancer and sepsis. Consequently, there is a concerted push to develop low-cost and non-invasive methods of analysis with high sensitivity and selectivity. A notable trend is the development of highly sensitive methods that are not only amenable for point-of-care (POC) testing, but also for wearable devices allowing continuous monitoring of biomarkers. In this context, a non-invasive test for the detection of a promising biomarker, the protein Interleukin-6 (IL-6), could represent a significant advance in the clinical management of cancer, in monitoring the chemotherapy response, or for prompt diagnosis of sepsis. This work reports a capacitive electrochemical impedance spectroscopy sensing platform tailored towards POC detection and treatment monitoring in human serum. The specific recognition of IL-6 was achieved employing gold surfaces modified with an anti-IL6 nanobody (anti-IL-6 VHH) or a specific IL-6 aptamer. In the first system, the anti-IL-6 VHH was covalently attached to the gold surface using a binary self-assembled-monolayer (SAM) of 6-mercapto-1-hexanol (MCH) and 11-mercaptoundecanoic acid. In the second system, the aptamer was chemisorbed onto the surface in a mixed SAM layer with MCH. The analytical performance for each label-free sensor was evaluated in buffer and 10% human serum samples and then compared. The results of this work were generated using a low-cost, thin film eight-channel gold sensor array produced on a flexible substrate providing useful information on the future design of POC and wearable impedance biomarker detection platforms.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Neoplasms , Sepsis , Humans , Biosensing Techniques/methods , Interleukin-6 , Aptamers, Nucleotide/chemistry , Gold/chemistry , Biomarkers , Electrodes , Electrochemical Techniques/methodsABSTRACT
Tetracycline antibiotics are used extensively in veterinary medicine, but the majority of the administrated dose is eliminated unmodified from the animal through various excretion routes including urine, faeces and milk. In dairy animals, limits on residues secreted in milk are strictly controlled by legislation. Tetracyclines (TCs) have metal chelation properties and form strong complexes with iron ions under acidic conditions. In this study, we exploit this property as a strategy for low cost, rapid electrochemical detection of TC residues. TC-Fe(III) complexes in a ratio of 2:1 were created in acidic conditions (pH 2.0) and electrochemically measured on plasma-treated gold electrodes modified with electrodeposited gold nanostructures. DPV measurements showed a reduction peak for the TC-Fe(III) complex that was observed at 50 mV (vs. Ag/AgCl QRE). The limit of detection in buffer media was calculated to be 345 nM and was responsive to increasing TC concentrations up to 2 mM, added to 1 mM FeCl3. Whole milk samples were processed to remove proteins and then spiked with tetracycline and Fe(III) to explore the specificity and sensitivity in a complex matrix with minimal sample preparation, under these conditions the LoD was 931 nM. These results demonstrate a route towards an easy-to-use sensor system for identification of TC in milk samples taking advantage of the metal chelating properties of this antibiotic class.
Subject(s)
Gold , Tetracycline , Animals , Tetracycline/analysis , Milk/chemistry , Ferric Compounds/chemistry , Anti-Bacterial Agents/chemistry , Tetracyclines/analysis , Chelating Agents , ElectrodesABSTRACT
Electrochemical DNA (e-DNA) biosensors are feasible tools for disease monitoring, with their ability to translate hybridization events between a desired nucleic acid target and a functionalized transducer, into recordable electrical signals. Such an approach provides a powerful method of sample analysis, with a strong potential to generate a rapid time to result in response to low analyte concentrations. Here, we report a strategy for the amplification of electrochemical signals associated with DNA hybridization, by harnessing the programmability of the DNA origami method to construct a sandwich assay to boost charge transfer resistance (RCT) associated with target detection. This allowed for an improvement in the sensor limit of detection by two orders of magnitude compared to a conventional label-free e-DNA biosensor design and linearity for target concentrations between 10 pM and 1 nM without the requirement for probe labeling or enzymatic support. Additionally, this sensor design proved capable of achieving a high degree of strand selectivity in a challenging DNA-rich environment. This approach serves as a practical method for addressing strict sensitivity requirements necessary for a low-cost point-of-care device.
Subject(s)
Biosensing Techniques , Electrochemical Techniques , Electrochemical Techniques/methods , DNA/genetics , Nucleic Acid Hybridization/methods , Biosensing Techniques/methodsABSTRACT
One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that M. tuberculosis (TB) is a disease most often associated with low- and middle-income countries, it is critical that low-cost and easy-to-use technological solutions are developed, which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). This work presents a low-cost, simple-to-use AST sensor that can detect drug susceptibility on the basis of changing RNA abundance for the typically slow-growing M. tuberculosis (TB) pathogen in 96 h using screen-printed electrodes and standard molecular biology laboratory reactionware. In order to find out the sensitivity of applied sensor platform, a different concentration (108 -103 CFU/mL) of M. tuberculosis was performed, and limit of detection and limit of quantitation were calculated as 103.82 and 1011.59 CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. These findings pave the way for the development of a comprehensive, low-cost, and simple-to-use AST system for prescribing in TB and multidrug-resistant tuberculosis.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Humans , Mycobacterium tuberculosis/genetics , Antitubercular Agents , Tuberculosis, Multidrug-Resistant/diagnosis , Tuberculosis, Multidrug-Resistant/drug therapy , Microbial Sensitivity TestsABSTRACT
In this study, an implantable stereo-electroencephalography (sEEG) depth electrode was functionalised with an enzyme coating for enzyme-based biosensing of glucose and L-glutamate. This was done because personalised medicine could benefit from active real-time neurochemical monitoring on small spatial and temporal scales to further understand and treat neurological disorders. To achieve this, the sEEG depth electrode was characterised using cyclic voltammetry (CV), differential pulse voltammetry (DPV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) using several electrochemical redox mediators (potassium ferri/ferrocyanide, ruthenium hexamine chloride, and dopamine). To improve performance, the Pt sensors on the sEEG depth electrode were coated with platinum black and a crosslinked gelatin-enzyme film to enable enzymatic biosensing. This characterisation work showed that producing a useable electrode with a good electrochemical response showing the expected behaviour for a platinum electrode was possible. Coating with Pt black improved the sensitivity to H2O2 over unmodified electrodes and approached that of well-defined Pt macro disc electrodes. Measured current showed good dependence on concentration, and the calibration curves report good sensitivity of 29.65 nA/cm2/µM for glucose and 8.05 nA/cm2/µM for L-glutamate with a stable, repeatable, and linear response. These findings demonstrate that existing clinical electrode devices can be adapted for combined electrochemical and electrophysiological measurement in patients and obviate the need to develop new electrodes when existing clinically approved devices and the associated knowledge can be reused. This accelerates the time to use and application of in vivo and wearable biosensing for diagnosis, treatment, and personalised medicine.
Subject(s)
Biosensing Techniques , Glutamic Acid , Humans , Hydrogen Peroxide , Electrodes , Glucose/chemistry , Biosensing Techniques/methods , Electrodes, Implanted , Electrochemical Techniques/methods , Platinum/chemistryABSTRACT
3D-printing has become a fundamental part of research in many areas of investigation since it provides rapid and personalized production of parts that meet very specific user needs. Biosensing is not an exception, and production of electrochemical sensors that can detect a variety of redox mediators and biologically relevant molecules has been widely reported. However, most 3D-printed electrochemical sensors detailed in the literature rely on big, individual, single-material electrodes that require large sample volumes to perform effectively. Our work exploits multi-material fused filament fabrication 3D-printing to produce a compact electrochemical sensor able to operate with only 100 µL of sample. We report cyclic voltammetry, differential pulse voltammetry, and chronoamperometry results to assess sensor performance and sensitivity. We investigated the influence of layer print orientation and layer thickness on the electrochemical performance of the sensor, and used the optimal parameters to produce the final device. The integrated 3D-printed platform successfully detects electrochemical activity for hexaammineruthenium(III) chloride and potassium ferricyanide (0.1 mM to 2 mM in 100 mM KCl), dopamine (50 µM to 1 mM in 1×PBS), and glucose via mediated amperometric glucose oxidase enzyme-based sensing (1 mM to 12 mM in 1×PBS), indicating good acceptance of biological modification. These results reveal the exciting potential of multi-material 3D-printing and how it can be used for the rapid development of efficient, small, integrated, personalized electrochemical biosensors.
Subject(s)
Biosensing Techniques , Dopamine , Chlorides , Electrochemical Techniques , Electrodes , Glucose , Glucose Oxidase , Printing, Three-DimensionalABSTRACT
SARS-CoV-2 diagnostic practices broadly involve either quantitative polymerase chain reaction (qPCR)-based nucleic amplification of viral sequences or antigen-based tests such as lateral flow assays (LFAs). Reverse transcriptase-qPCR can detect viral RNA and is the gold standard for sensitivity. However, the technique is time-consuming and requires expensive laboratory infrastructure and trained staff. LFAs are lower in cost and near real time, and because they are antigen-based, they have the potential to provide a more accurate indication of a disease state. However, LFAs are reported to have low real-world sensitivity and in most cases are only qualitative. Here, an antigen-based electrochemical aptamer sensor is presented, which has the potential to address some of these shortfalls. An aptamer, raised to the SARS-CoV-2 spike protein, was immobilized on a low-cost gold-coated polyester substrate adapted from the blood glucose testing industry. Clinically relevant detection levels for SARS-CoV-2 are achieved in a simple, label-free measurement format using sample incubation times as short as 15 min on nasopharyngeal swab samples. This assay can readily be optimized for mass manufacture and is compatible with a low-cost meter.
Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnosis , Dielectric Spectroscopy , Electrodes , Humans , RNA, Viral , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Spike Glycoprotein, CoronavirusABSTRACT
Isothermal amplification reactions represent an important and exciting approach to achieve widespread, low cost, and easily implemented molecular diagnostics. This work presents a modified recombinase polymerase amplification (RPA) reaction, which can be directly coupled to a simple electrochemical measurement to ultimately allow development of a nucleic acid-based assay for antibiotic resistance genes. It is shown that use of reagents from a standard RPA reaction kit allows incorporation of horse radish peroxidase-labeled thymine nucleotides into amplified DNA strands, which can be detected via an amperometric signal readout for detection of important gene sequences. The assay is exemplified through detection of fragments of the oxacillin resistance gene in Escherichia coli cells bearing a drug resistance plasmid, achieving a potential limit of detection of 319 cfus/mL and an unoptimized time to result of 60 min. This work serves as a suitable demonstration of the potential for a system to deliver detection of key drug resistance genes at clinically relevant levels.
Subject(s)
Nucleic Acid Amplification Techniques , Oxacillin , Escherichia coli/genetics , Horseradish Peroxidase , RecombinasesABSTRACT
A point-of-care blood test for the detection of an emerging biomarker, CCL17/TARC, could prove transformative for the clinical management of classic Hodgkin lymphoma (cHL). Primary care diagnosis is challenging due to nonspecific clinical presentation and lack of a diagnostic test, leading to significant diagnostic delays. Treatment monitoring encounters false-positive and negative results, leading to avoidable chemotherapy toxicity, or undertreatment, impacting patient morbidity and mortality. Here, we present an amperometric CCL17/TARC immunosensor, based on the utilization of a thiolated heterobifunctional cross-linker and sandwich antibody assay, to facilitate novel primary care triage and chemotherapy monitoring strategies for cHL. The immunosensor shows excellent analytical performance for clinical testing; linearity (R2 = 0.986), detection limit (194 pg/mL), and lower and upper limits of quantitation (387-50â¯000 pg/mL). The biosensor differentiated all 42 newly diagnosed cHL patients from healthy volunteers, based on serum CCL17/TARC concentration, using blood samples collected prior to treatment intervention. The immunosensor also discriminated between paired blood samples of all seven cHL patients, respectively, collected prior to treatment and during chemotherapy, attributed to the decrease in serum CCL17/TARC concentration following chemotherapy response. Overall, we have shown, for the first time, the potential of an electrochemical CCL17/TARC biosensor for primary care triage and chemotherapy monitoring for cHL, which would have positive clinical and psychosocial implications for patients, while streamlining current healthcare pathways.
Subject(s)
Biosensing Techniques , Chemokine CCL17/blood , Hodgkin Disease , Electrochemical Techniques , Hodgkin Disease/diagnosis , Hodgkin Disease/drug therapy , Humans , Immunoassay , TriageABSTRACT
Antimicrobial drug residues in food are strictly controlled and monitored by national laws in most territories. Tetracyclines are a major broad-spectrum antibiotic class, active against a wide range of Gram-positive and Gram-negative bacteria, and they are the leading choice for the treatment of many conditions in veterinary medicine in recent years. In dairy farms, milk from cows being treated with antibiotic drugs, such as tetracyclines, is considered unfit for human consumption. Contamination of the farm bulk tank with milk containing these residues presents a threat to confidence of supply and results in financial losses to farmers and dairy. Real-time monitoring of milk production for antimicrobial residues could reduce this risk and help to minimise the release of residues into the environment where they can cause reservoirs of antimicrobial resistance. In this article, we review the existing literature for the detection of tetracyclines in cow's milk. Firstly, the complex nature of the milk matrix is described, and the test strategies in commercial use are outlined. Following this, emerging biosensors in the low-cost biosensors field are contrasted against each other, focusing upon electrochemical biosensors. Existing commercial tests that identify antimicrobial residues within milk are largely limited to beta-lactam detection, or non-specific detection of microbial inhibition, with tests specific to tetracycline residues less prevalent. Herein, we review a number of emerging electrochemical biosensor detection strategies for tetracyclines, which have the potential to close this gap and address the industry challenges associated with existing tests.
Subject(s)
Milk/chemistry , Tetracyclines/analysis , Animals , Anti-Bacterial Agents , Biosensing Techniques , Cattle , Dairying , Drug Residues , Female , Gram-Negative Bacteria , Gram-Positive Bacteria , Humans , TetracyclineABSTRACT
The goal of achieving enhanced diagnosis and continuous monitoring of human health has led to a vibrant, dynamic and well-funded field of research in medical sensing and biosensor technologies. The field has many sub-disciplines which focus on different aspects of sensor science; engaging engineers, chemists, biochemists and clinicians, often in interdisciplinary teams. The trends which dominate include the efforts to develop effective point of care tests and implantable/wearable technologies for early diagnosis and continuous monitoring. This review will outline the current state of the art in a number of relevant fields, including device engineering, chemistry, nanoscience and biomolecular detection, and suggest how these advances might be employed to develop effective systems for measuring physiology, detecting infection and monitoring biomarker status in wild animals. Special consideration is also given to the emerging threat of antimicrobial resistance and in the light of the current SARS-CoV-2 outbreak, zoonotic infections. Both of these areas involve significant crossover between animal and human health and are therefore well placed to seed technological developments with applicability to both human and animal health and, more generally, the reviewed technologies have significant potential to find use in the measurement of physiology in wild animals. This article is part of the theme issue 'Measuring physiology in free-living animals (Part II)'.
Subject(s)
Biosensing Techniques/instrumentation , COVID-19/diagnosis , Synthetic Biology/methods , Wearable Electronic Devices , Zika Virus Infection/veterinary , Zoonoses/diagnosis , Animals , Animals, Wild/microbiology , Animals, Wild/parasitology , Animals, Wild/virology , Biomarkers/analysis , Cell Engineering/methods , Humans , Monitoring, Physiologic/instrumentation , Monitoring, Physiologic/methods , Nanotechnology/instrumentation , Nanotechnology/methods , Point-of-Care Testing , Zika Virus Infection/diagnosisABSTRACT
DNA origami structures represent an exciting class of materials for use in a wide range of biotechnological applications. This study reports the design, production, and characterization of a DNA origami "zipper" structure, which contains nine pH-responsive DNA locks. Each lock consists of two parts that are attached to the zipper's opposite arms: a DNA hairpin and a single-stranded DNA that are able to form a DNA triplex through Hoogsteen base pairing. The sequences of the locks were selected in a way that the zipper adopted a closed configuration at pH 6.5 and an open state at pH 8.0 (transition pKa 7.6). By adding thiol groups, it was possible to immobilize the zipper structure onto gold surfaces. The immobilization process was characterized electrochemically to confirm successful adsorption of the zipper. The open and closed states were then probed using differential pulse voltammetry and electrochemical impedance spectroscopy with solution-based redox agents. It was found that after immobilization, the open or closed state of the zipper in different pH regimes could be determined by electrochemical interrogation. These findings pave the way for development of DNA origami-based pH monitoring and other pH-responsive sensing and release strategies for zipper-functionalized gold surfaces.
Subject(s)
Biosensing Techniques , DNA , DNA, Single-Stranded , Electrochemical Techniques , Gold , Hydrogen-Ion ConcentrationABSTRACT
Accurate and rapid diagnostic tests are critical to reducing the impact of SARS-CoV-2. This study presents early, but promising measurements of SARS-CoV-2 using the ACE2 enzyme as the recognition element to achieve clinically relevant detection. The test provides a scalable route to sensitive, specific, rapid and low cost mass testing.
Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Biosensing Techniques/methods , Electrochemical Techniques/methods , Enzymes, Immobilized/chemistry , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2/metabolism , Biosensing Techniques/instrumentation , COVID-19/diagnosis , COVID-19 Testing/instrumentation , COVID-19 Testing/methods , Electrochemical Techniques/instrumentation , Electrodes , Enzymes, Immobilized/metabolism , Fluorocarbons/chemistry , Gold/chemistry , Humans , Limit of Detection , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolismABSTRACT
Circulating tumour DNA (ctDNA) is widely used in liquid biopsies due to having a presence in the blood that is typically in proportion to the stage of the cancer and because it may present a quick and practical method of capturing tumour heterogeneity. This paper outlines a simple electrochemical technique adapted towards point-of-care cancer detection and treatment monitoring from biofluids using a label-free detection strategy. The mutations used for analysis were the KRAS G12D and G13D mutations, which are both important in the initiation, progression and drug resistance of many human cancers, leading to a high mortality rate. A low-cost DNA sensor was developed to specifically investigate these common circulating tumour markers. Initially, we report on some developments made in carbon surface pre-treatment and the electrochemical detection scheme which ensure the most sensitive measurement technique is employed. Following pre-treatment of the sensor to ensure homogeneity, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low-cost screen printed carbon electrodes using diazonium chemistry and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide coupling. Prior to electrochemical detection, the sensor was functionalised with target DNA amplified by standard and specialist PCR methodologies (6.3% increase). Assay development steps and DNA detection experiments were performed using standard voltammetry techniques. Sensitivity (as low as 0.58 ng/µL) and specificity (>300%) was achieved by detecting mutant KRAS G13D PCR amplicons against a background of wild-type KRAS DNA from the representative cancer sample and our findings give rise to the basis of a simple and very low-cost system for measuring ctDNA biomarkers in patient samples. The current time to receive results from the system was 3.5 h with appreciable scope for optimisation, thus far comparing favourably to the UK National Health Service biopsy service where patients can wait for weeks for biopsy results.
Subject(s)
Biosensing Techniques , Point Mutation , DNA , Electrochemical Techniques , GTP-Binding Protein alpha Subunits, G12-G13 , Mutation , Proto-Oncogene Proteins p21(ras)ABSTRACT
Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6- [Formula: see text]/min) either at room temperature or 50° C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis.
Subject(s)
Microfluidic Analytical Techniques , Microfluidics , BacteriaABSTRACT
Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long-term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore, in vitro antibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using streptomycin as antibiotics. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for M. tuberculosis. A commercially available and low-cost screen-printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitor M. smegmatis nucleic acid sequences using the techniques of square-wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24-H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods.
Subject(s)
Antitubercular Agents/analysis , Gold/chemistry , Streptomycin/analysis , Antitubercular Agents/pharmacology , Electrodes , Gold/economics , Microbial Sensitivity Tests , Mycobacterium smegmatis/drug effects , Streptomycin/pharmacologyABSTRACT
Antibiotic resistance has been cited by the World Health Organisation (WHO) as one of the greatest threats to public health. Mitigating the spread of antibiotic resistance requires a multipronged approach with possible interventions including faster diagnostic testing and enhanced antibiotic stewardship. This study employs a low-cost diagnostic sensor test to rapidly pinpoint the correct antibiotic for treatment of infection. The sensor comprises a screen-printed gold electrode, modified with an antibiotic-seeded hydrogel to monitor bacterial growth. Electrochemical growth profiles of the common microorganism, Escherichia coli (E. coli) (ATCC 25922) were measured in the presence and absence of the antibiotic streptomycin. Results show a clear distinction between the E. coli growth profiles depending on whether streptomycin is present, in a timeframe of ≈2.5 h (p < 0.05), significantly quicker than the current gold standard of culture-based antimicrobial susceptibility testing. These results demonstrate a clear pathway to a low cost, phenotypic and reproducible antibiotic susceptibility testing technology for the rapid detection of E. coli within clinically relevant concentration ranges for conditions such as urinary tract infections.
Subject(s)
Biosensing Techniques , Electrodes , Escherichia coli , Microbial Sensitivity Tests/methods , Anti-Bacterial Agents , Drug Resistance, Microbial , Escherichia coli Infections , HumansABSTRACT
It is well-known that two major issues, preventing improved outcomes from cancer are late diagnosis and the evolution of drug resistance during chemotherapy, therefore technologies that address these issues can have a transformative effect on healthcare workflows. In this work we present a simple, low-cost DNA biosensor that was developed specifically to detect mutations in a key oncogene (KRAS). The sensor employed was a screen-printed array of carbon electrodes, used to perform parallel measurements of DNA hybridisation. A DNA amplification reaction was developed with primers for mutant and wild type KRAS sequences which amplified target sequences from representative clinical samples to detectable levels in as few as twenty cycles. High levels of sensitivity were demonstrated alongside a clear exemplar of assay specificity by showing the mutant KRAS sequence was detectable against a significant background of wild type DNA following amplification and hybridisation on the sensor surface. The time to result was found to be 3.5 h with considerable potential for optimisation through assay integration. This quick and versatile biosensor has the potential to be deployed in a low-cost, point-of-care test where patients can be screened either for early diagnosis purposes or monitoring of response to therapy.