Antibiotic resistance is a growing concern in the treatment of infectious disease worldwide. Point-of-care (PoC) assays which rapidly identify antibiotic resistance in a sample will allow for ...immediate targeted therapy which improves patient outcomes and helps maintain the effectiveness of current antibiotic stockpiles. Electrochemical assays offer many benefits, but translation from a benchtop measurement system to low-cost portable electrodes can be challenging. Using electrochemical and physical techniques, this study examines how different electrode surfaces and bio-recognition elements, i.e. the self-assembled monolayer (SAM), affect the performance of a biosensor measuring the hybridisation of a probe for antibiotic resistance to a target gene sequence in solution. We evaluate several commercially available electrodes which could be suitable for PoC testing with different SAM layers and show that electrode selection also plays an important role in overall biosensor performance.
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ceramics show excellent characteristics of mechanical and chemical resistance in combination with good biocompatibility, antibacterial property and radiolucency. Therefore, they are ...intensively studied as structural materials in skeletal implant applications. Despite their attractive properties, there are limited data in the field about in vitro studies of cellular growth on ceramic implant materials. In this study, the growth of bone cells was investigated on porous silicon nitride (Si
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) ceramic implant by using electrochemical impedance spectroscopy (EIS). Partial sintering was performed at 1700 °C with limited amount of sintering additive for the production of porous Si
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scaffolds. All samples were then sterilized by using ethylene oxide followed by culturing MG-63 osteosarcoma cells on the substrates for in vitro assays. At 20 and 36 h, EIS was performed and results demonstrated that magnitude of the impedance as a result of the changes in the culture medium increased after incubation with osteosarcoma cells. The changes are attributed to the cellular uptake of charged molecules from the medium. Si
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samples appear to show large impedance magnitude changes, especially between 100 and 1 Hz. Impedance changes were also correlated with WST-1 measurements (36 h) and DAPI results.
•Adsorption of PCR amplicons electrochemically detected on low-cost PCB electrodes with methlyene blue (MB).•Amplicon length and MB concentration are both critical to achieving linear sensor ...performance.•Detection of SARS-CoV-2 nucleocapsid gene amplicons from 10 pg/μl (1.7 fM).•N1 fragment successfully amplified and detected in wastewater spiked with SARS-CoV-2 control RNA.
We present a low-cost electrochemical DNA biosensor based on printed circuit board (PCB) electrodes for wastewater monitoring using portable PCR instruments, such as miniPCR®, without the requirement for qPCR reagents. PCB electrodes are attractive candidates for low-cost and sensitive DNA biosensors of relevance in a pandemic such as COVID-19, and facilitate the opportunity to map disease spread in Low-Middle Income Countries (LMICs) through monitoring of environmental samples such as wastewater. The biosensor reported in this work is capable of detecting PCR amplicons through the intercalation of methylene blue (MB) with DNA, which increases the voltammogram peak current at the redox potential of MB. We describe how these changes are likely to result from the adsorption of MB-DNA complex on the electrode surface. The electrodes are reusable, easy to clean, do not undergo any surface modification and represent a cost-effective solution with long shelf-life. We also explore the impact that MB concentration and DNA length have upon our biosensor performance and provide insights useful to other investigators in the field. The sensor reported here is capable of detecting SARS-CoV-2 nucleocapsid gene amplicons at concentrations as low as 10 pg/μl (approximately 1.7 fM) and can detect nucleotides amplified after 10 PCR cycles. Furthermore, using the PCB electrode and approaches described here, SARS-CoV-2 amplicons were detected in simulated wastewater sample, by spiking wastewater collected from a sewage treatment plant in Mumbai, India with SARS-CoV-2 RNA.
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.
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.
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.
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 (R CT) 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.
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.
Recent demonstrations of normal tissue sparing by high-dose, high-dose-rate FLASH radiation therapy have driven considerable interest in its application to improve clinical outcomes. However, ...significant uncertainty remains about the underlying mechanisms of FLASH sparing and how deliveries can be optimized to maximize benefit from this effect. Rapid oxygen depletion has been suggested as a potential mechanism by which these effects occur, but this has yet to be quantitatively tested against experimental data.
Models of oxygen kinetics during irradiation were used to develop a time-dependent model of the oxygen enhancement ratio in mammalian cells that incorporates oxygen depletion. The characteristics of this model were then explored in terms of the dose and dose-rate dependence of the oxygen enhancement ratio. This model was also fit to experimental data from both in vitro and in vivo data sets.
In cases of FLASH radiation therapy, this model suggests that oxygen levels can be depleted by amounts that are sufficient to affect radiosensitivity only in conditions of intermediate oxygen tension, with no effect seen at high or very low initial oxygen levels. The model also effectively reproduced the dose, dose rate, and oxygen tension dependence of responses to FLASH radiation therapy in a range of systems, with model parameters compatible with published data.
Oxygen depletion provides a credible quantitative model to understand the biological effects of FLASH radiation therapy and is compatible with a range of experimental observations of FLASH sparing. These results highlight the need for more detailed quantification of oxygen depletion under high-dose-rate radiation exposures in relevant systems and the importance of oxygen tension in target tissues for FLASH sparing to be observed.
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