Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification.

Five carbapenemase enzymes, coined the 'big five', have been identified as the biggest threat to worldwide antibiotic resistance based on their broad substrate affinity and global prevalence. Here we show the development of a molecular detection method for the gene sequences from the five carbapenemases utilising the isothermal amplification method of recombinase polymerase amplification (RPA). We demonstrate the successful detection of each of the big five carbapenemase genes with femtomolar detection limits using a spatially separated multiplex amplification strategy. The approach uses tailed oligonucleotides for hybridisation, reducing the complexity and cost of the assay compared to classical RPA detection strategies. The reporter probe, horseradish peroxidase, generates the measureable output on a benchtop microplate reader, but more notably, our study leverages the power of a portable Raman spectrometer, enabling up to a 19-fold enhancement in the limit of detection. Significantly, the development approach employed a solid-phase RPA format, wherein the forward primers targeting each of the five carbapenemase genes are immobilised to a streptavidin-coated microplate. The adoption of this solid-phase methodology is pivotal for achieving a successful developmental pathway when employing this streamlined approach. The assay takes 2 hours until result, including a 40 minutes RPA amplification step at 37 °C. This is the first example of using solid-phase RPA for the detection of the big five and represents a milestone towards the developments of an automated point-of-care diagnostic for the big five using RPA.

Effect of Ambient Environment on Laser Reduction of Graphene Oxide for Applications in Electrochemical Sensing.

Electrochemical sensors play an important role in a variety of applications. With the potential for enhanced performance, much of the focus has been on developing nanomaterials, in particular graphene, for such sensors. Recent work has looked towards laser scribing technology for the reduction of graphene oxide as an easy and cost-effective option for sensor fabrication. This work looks to develop this approach by assessing the quality of sensors produced with the effect of different ambient atmospheres during the laser scribing process. The graphene oxide was reduced using a laser writing system in a range of atmospheres and sensors characterised with Raman spectroscopy, XPS and cyclic voltammetry. Although providing a slightly higher defect density, sensors fabricated under argon and nitrogen atmospheres exhibited the highest average electron transfer rates of approximately 2 × 10-3 cms-1. Issues of sensor reproducibility using this approach are discussed.

Recent advances in antibiotic resistance diagnosis using SERS: focus on the "Big 5" challenges.

Antibiotic resistant bacteria constitute a global health threat. It is essential for healthcare professionals to prescribe the correct dose of an effective antibiotic to mitigate the bacterial infection in a timely manner to improve the therapeutic outcomes to the patient and prevent the dissemination of antibiotic resistance. To achieve this, there is a need to implement a rapid and ultra-sensitive clinical diagnosis to identify resistant bacterial strains and monitor the effect of antibiotics. In this review, we highlight the use of surface enhanced Raman scattering (SERS) as a powerful diagnostic technique for bacterial detection and evaluation. Initially, this is viewed through a lens covering why SERS can surpass other traditional techniques for bacterial diagnosis. This is followed by different SERS substrates design, detection strategies that have been used for various bacterial biomarkers, how SERS can be combined with other diagnostic platforms to improve its performance towards the bacterial detection and the application of SERS for antibiotic resistance diagnosis. Finally, the recent progress in SERS detection methods in the last decade for the "Big 5" antibiotic resistant challenges as demonstrators of public health major threats is reviewed, namely: Methicillin-resistant Staphylococcus aureus (MRSA), Carbapenem-resistant Enterobacteriaceae (CRE)/Extended-spectrum beta-lactamases (ESBLs), Mycobacterium tuberculosis (TB), Vancomycin-resistant Enterococcus (VRE) and Neisseria Gonorrhoea (NG). This review provides a comprehensive view of the current state of the art with regard to using SERS for assessing antibiotic resistance with a future outlook on where the field go head in the coming years.

Rapid ultra-sensitive diagnosis of clostridium difficile infection using a SERS-based lateral flow assay.

Clostridium difficile (C. diff) infection is one of the most contagious diseases associated with high morbidity and mortality rates in hospitalised patients. Accurate diagnosis can slow its spread by determining the most effective treatment. Herein, we report a novel testing platform as a proof-of-concept for the selective, sensitive, rapid and cost-effective diagnosis of C. diff infection (CDI) based on a duplex measurement. This was achieved by detecting two specific biomarkers, surface layer protein A (SlpA) and toxin B (ToxB), using a surface enhanced Raman scattering-based lateral flow assay (SERS-based LFA). The simultaneous duplex detection of SlpA with ToxB has not been described for the clinical diagnosis of CDI previously. The SlpA biomarker "AKDGSTKEDQLVDALA" was first reported by our group in 2018 as a species-specific identification tool. The second biomarker, ToxB, is the essential virulence biomarker of C. diff pathogenic strains and is required to confirm true infection pathogenicity. The proposed SERS-based LFA platform enabled rapid duplex detection of SlpA and ToxB on separate test lines using a duplex LF test strip within 20 minutes. The use of a handheld Raman spectrometer to scan test lines allowed for the highly sensitive quantitative detection of both biomarkers with a lowest observable concentration of 0.01 pg µL-1. The use of a handheld device in this SERS-based LFA instead of benchtop machine paves the way for rapid, selective, sensitive and cheap clinical evaluation of CDI at the point of care (POC) with minimal sample backlog.

Digital technologies in the public-health response to COVID-19.

Digital technologies are being harnessed to support the public-health response to COVID-19 worldwide, including population surveillance, case identification, contact tracing and evaluation of interventions on the basis of mobility data and communication with the public. These rapid responses leverage billions of mobile phones, large online datasets, connected devices, relatively low-cost computing resources and advances in machine learning and natural language processing. This Review aims to capture the breadth of digital innovations for the public-health response to COVID-19 worldwide and their limitations, and barriers to their implementation, including legal, ethical and privacy barriers, as well as organizational and workforce barriers. The future of public health is likely to become increasingly digital, and we review the need for the alignment of international strategies for the regulation, evaluation and use of digital technologies to strengthen pandemic management, and future preparedness for COVID-19 and other infectious diseases.

Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay.

Sensitive, specific, rapid, inexpensive and easy-to-use nucleic acid tests for use at the point-of-need are critical for the emerging field of personalised medicine for which companion diagnostics are essential, as well as for application in low resource settings. Here we report on the development of a point-of-care nucleic acid lateral flow test for the direct detection of isothermally amplified DNA. The recombinase polymerase amplification method is modified slightly to use tailed primers, resulting in an amplicon with a duplex flanked by two single stranded DNA tails. This tailed amplicon facilitates detection via hybridisation to a surface immobilised oligonucleotide capture probe and a gold nanoparticle labelled reporter probe. A detection limit of 1 × 10-11 M (190 amol), equivalent to 8.67 × 105 copies of DNA was achieved, with the entire assay, both amplification and detection, being completed in less than 15 minutes at a constant temperature of 37 °C. The use of the tailed primers obviates the need for hapten labelling and consequent use of capture and reporter antibodies, whilst also avoiding the need for any post-amplification processing for the generation of single stranded DNA, thus presenting an assay that can facilely find application at the point of need.

Aptamer Lateral Flow Assays for Ultrasensitive Detection of ß-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers.

In this work, different methodologies were evaluated in search of robust, simple, rapid, ultrasensitive, and user-friendly lateral flow aptamer assays. In one approach, we developed a competitive based lateral flow aptamer assay, in which ß-conglutin immobilized on the test line of a nitrocellulose membrane and ß-conglutin in the test sample compete for binding to AuNP labeled aptamer. The control line exploits an immobilized DNA probe complementary to the labeled aptamer, forcing displacement of the aptamer from the ß-conglutin-aptamer complex. In a second approach, the competition for aptamer binding takes place off-strip, and following competition, aptamer bound to the immobilized ß-conglutin is eluted and used as a template for isothermal recombinase polymerase amplification, exploiting tailed primers, resulting in an amplicon of a duplex flanked by single stranded DNA tails. The amplicon is rapidly and quantitatively detected using a nucleic acid lateral flow with an immobilized capture probe and a gold nanoparticle labeled reporter probe. The competitive lateral flow is completed in just 5 min, achieving a detection limit of 55 pM (1.1 fmol), and the combined competitive-amplification lateral flow requires just 30 min, with a detection limit of 9 fM (0.17 amol).

One-Port Electronic Detection Strategies for Improving Sensitivity in Piezoelectric Resonant Sensor Measurements.

This paper describes a one-port mechanical resonance detection scheme utilized on a piezoelectric thin film driven silicon circular diaphragm resonator and discusses the limitations to such an approach in degenerate mode mass detection sensors. The sensor utilizes degenerated vibration modes of a radial symmetrical microstructure thereby providing both a sense and reference mode allowing for minimization of environmental effects on performance. The circular diaphragm resonator was fabricated with thickness of 4.5 µm and diameter of 140 µm. A PZT thin film of 0.75 µm was patterned on the top surface for the purposes of excitation and vibration sensing. The device showed a resonant frequency of 5.8 MHz for the (1, 1) mode. An electronic interface circuit was designed to cancel out the large static and parasitic capacitance allowing for electrical detection of the mechanical vibration thereby enabling the frequency split between the sense and reference mode to be measured accurately. The extracted motional current, proportional to the vibration velocity, was fed back to the drive to effectively increase the Q factor, and therefore device sensitivity, by more than a factor of 8. A software phase-locked loop was implemented to automatically track the resonant frequencies to allow for faster and accurate resonance detection. Results showed that by utilizing the absolute mode frequencies as an indication of sensor temperature, the variation in sensor temperature due to the heating from the drive electronics was accounted for and led to an ultimate measurement sensitivity of 2.3 Hz.

Laser-scribed graphene presents an opportunity to print a new generation of disposable electrochemical sensors.

Graphene application within electrochemical sensing has been widely reported, but mainly as a composite, which adds summative effects to an underlying electrode. In this work we report the use of laser-scribed graphene as a distinct electrode patterned on a non-conducting flexible substrate. The laser-scribed graphene electrode compared favourably to established carbon macroelectrodes when evaluating both inner sphere and outer sphere redox probes, providing promise of extensive utility as an electrochemical sensor. The laser-scribed graphene electrode demonstrated the fastest heterogeneous electron transfer rate of all the electrodes evaluated with a k(0) of 0.02373 cm s(-1) for potassium ferricyanide, which exceeds commercially available edge plane pyrolytic graphite at 0.00260 cm s(-1), basal plane pyrolytic graphite at 0.00033 cm s(-1) and the very slow and effectively irreversible electrochemistry observed using single layer graphene. Finally and most significantly, a proof of principle system was fabricated using the laser-scribed graphene as working electrode, counter electrode and underlying base for the Ag/AgCl reference electrode, all in situ on the same planar flexible substrate, removing the requirement of macroscale external electrodes. The planar three electrode format operated with the same optimal electrode characteristics. Furthermore, the fabrication is inexpensive, scalable and compatible with a disposable biosensor format, considerably widening the potential applications in electrochemical bio-sensing for laser-scribed graphene.

Structure-function studies of an engineered scaffold protein derived from Stefin A. II: Development and applications of the SQT variant.

Constrained binding peptides (peptide aptamers) may serve as tools to explore protein conformations and disrupt protein-protein interactions. The quality of the protein scaffold, by which the binding peptide is constrained and presented, is of crucial importance. SQT (Stefin A Quadruple Mutant-Tracy) is our most recent development in the Stefin A-derived scaffold series. Stefin A naturally uses three surfaces to interact with its targets. SQT tolerates peptide insertions at all three positions. Peptide aptamers in the SQT scaffold can be expressed in bacterial, yeast and human cells, and displayed as a fusion to truncated pIII on phage. Peptides that bind to CDK2 can show improved binding in protein microarrays when presented by the SQT scaffold. Yeast two-hybrid libraries have been screened for binders to the POZ domain of BCL-6 and to a peptide derived from PBP2', specific to methicillin-resistant Staphylococcus aureus. Presentation of the Noxa BH3 helix by SQT allows specific interaction with Mcl-1 in human cells. Together, our results show that Stefin A-derived scaffolds, including SQT, can be used for a variety of applications in cellular and molecular biology. We will henceforth refer to Stefin A-derived engineered proteins as Scannins.

Discovery of biphasic thermal unfolding of ompc with implications for surface loop stability.

Escherichia coli outer membrane protein C (osmoporin) is a close homologue of OmpF or matrix porin, expressed under conditions of high osmolarity or ionic strength. Despite the fact that the proteins display very similar structures (rmsd = 0.78 Å), the channel activities (gating or selectivity) of the two proteins are markedly different, and compared to OmpF, there is much less published information about the stability and folding of OmpC. In this paper, we report a structural study of nine OmpC mutations that affect channel size and voltage gating. The secondary and tertiary structural analysis by circular dichroism (CD) indicated that the single-amino acid substitutions have little impact on the protein fold. However, a thermal denaturation study using CD and differential scanning calorimetry shows that different mutations lead to varied levels of destabilization, with the largest showing a 15 °C lower T(m) than the wild type and a 40% reduction in ΔH(cal). CD thermal denaturation measurements revealed that OmpC unfolds in a biphasic process, in which only the second phase is affected by the known mutations. The first stage of unfolding was shown to be reversible and separate from the main unfolding and loss of trimeric structure occurring in the second phase, leaving the flexible extracellular loops as the likely site of unfolding. The first phase is abolished as OmpC becomes more stable at lower pH.

Biomolecule patterning on analytical devices: a microfabrication-compatible approach.

The present work describes a methodology for patterning biomolecules on silicon-based analytical devices that reconciles 3-D biological functionalization with standard resist lift-off techniques. Unlike classic sol-gel approaches in which the biomolecule of interest is introduced within the sol mixture, a two-stage scenario has been developed. It consists first of patterning micrometer/submicrometer polycondensate scaffold structures, using classic microfabrication tools, that are then loaded with native biomolecules via a second simple incubation step under biologically friendly environmental conditions. The common compatibility issue between the biological and microfabrication worlds has been circumvented because native recognition biomolecules can be introduced into the host scaffold downstream from all compatibility issues. The scaffold can be generated on any silicon substrate via the polycondensation of aminosilane, namely, aminopropyltriethoxy silane (APTES), under conditions that are fully compatible with resist mask lithography. The scaffold porosity and high primary amine content allow proteins and nucleic acid sequences to penetrate the polycondensate and to interact strongly, thus giving rise to micrometer/submicrometer 3-D structures exhibiting high biological activity. The integration of such a biopatterning approach in the microfabrication process of silicon analytical devices has been demonstrated via the successful completion of immunoassays and nucleic acid assays.