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.

Vincent J Vezzaa, Adrian Butterwortha, Perrine Lasserrea, Ewen O Blaira, Alexander MacDonalda, Stuart Hannaha, Christopher Rinaldib, Paul A Hoskissonc, Andrew C Wardd, Alistair Longmuire, Steven Setforde, Eoghan CW Farmerf, Michael...

No abstract

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.

Testing outside the laboratory environment, such as point of care testing, is a rapidly evolving area with advances in the integration of sample handling, measurement and sensing elements widely reported.

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.

Si3N4 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 (Si3N4) 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 Si3N4 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. Si3N4 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.