More Than One Enzyme: Exploring Alternative FMN-Dependent L-Lactate Oxidases for Biosensor Development.

The α-hydroxy acid oxidoreductase (HAOx) family contains a diverse group of enzymes that can be applied in biosensors for L-lactate detection, most prominently lactate oxidase (LOx). The limited availability and a lack of diversity of L-lactate-oxidizing enzymes have currently hindered advancements in L-lactate biosensor development. Until now, the field has mostly relied on a single, commercially available enzyme, namely Aerococcus viridans L-lactate oxidase (AvLOx). In this study, we present newly discovered alternative L-lactate oxidases that exhibit a narrow substrate specificity and varied kinetic efficiencies toward L-lactate, making them suitable for integration into existing biosensor configurations. Some of these FMN-dependent L-lactate oxidases could be obtained in substantial amounts from routine E. coli expression, potentially facilitating commercial production. Using electrochemical characterization with a mediated biosensor setup, we present 7 enzymes that perform comparable or even better than commercial AvLOx. Finally, we show that their electrochemical performance is not directly correlating with their biochemical performance, making predictions of the suitability of enzymes for biosensor applications extremely difficult. Our research emphasizes the significance of expanding the enzyme toolbox of L-lactate oxidases for the development of improved L-lactate biosensors.

Voltammetric methods for electrochemical characterization and quantification of artemether-based antimalarials.

Every year substandard and falsified (SF) artemisinin derivative-based antimalarials are responsible for the loss of 450 000 deaths and billions of GBP. The lack of infrastructure and funds to support pharmaceutical quality control in many low-and-middle-income countries contributes to this problem. This work assesses fitness for purpose of voltammetric methods for identification and quantification of artemether in the presence of excipients. Electrochemical characterization of artemether using cyclic voltammetry shows that the reduction of artemether is chemically irreversible within the potential range of -0.4 V to -1.4 V. A chronocoulometric quantification algorithm for artemether is created and tested with pure artemether, as well as filtered and unfiltered Riamet® tablets. Filtration of Riamet® tablets provides no additional benefit for the quantification of artemether in Riamet®. In addition, artemether's response to pH indicates possible protonation and coupled homogeneous chemistry. Finally, sodium sulfite is an effective means of removing dissolved oxygen and improving artemether signal resolution in air-equilibrated PBS. This concludes that electrochemical analysis is a promising method for artemether identification and quantification.

Surface immobilization strategies for the development of electrochemical nucleic acid sensors.

Following the recent pandemic and with the emergence of cell-free nucleic acids in liquid biopsies as promising biomarkers for a broad range of pathologies, there is an increasing demand for a new generation of nucleic acid tests, with a particular focus on cost-effective, highly sensitive and specific biosensors. Easily miniaturized electrochemical sensors show the greatest promise and most typically rely on the chemical functionalization of conductive materials or electrodes with sequence-specific hybridization probes made of standard oligonucleotides (DNA or RNA) or synthetic analogues (e.g. Peptide Nucleic Acids or PNAs). The robustness of such sensors is mostly influenced by the ability to control the density and orientation of the probe at the surface of the electrode, making the chemistry used for this immobilization a key parameter. This exhaustive review will cover the various strategies to immobilize nucleic acid probes onto different solid electrode materials. Both physical and chemical immobilization techniques will be presented. Their applicability to specific electrode materials and surfaces will also be discussed as well as strategies for passivation of the electrode surface as a way of preventing electrode fouling and reducing nonspecific binding.

Continuous Measurement of Lactate Concentration in Human Subjects through Direct Electron Transfer from Enzymes to Microneedle Electrodes.

Microneedle lactate sensors may be used to continuously measure lactate concentration in the interstitial fluid in a minimally invasive and pain-free manner. First- and second-generation enzymatic sensors produce a redox-active product that is electrochemically sensed at the electrode surface. Direct electron transfer enzymes produce electrons directly as the product of enzymatic action; in this study, a direct electron transfer enzyme specific to lactate has been immobilized onto a microneedle surface to create lactate-sensing devices that function at low applied voltages (0.2 V). These devices have been validated in a small study of human volunteers; lactate concentrations were raised and lowered through physical exercise and subsequent rest. Lactazyme microneedle devices show good agreement with concurrently obtained and analyzed serum lactate levels.

Neomycin, but Not Neamine, Blocks Angiogenic Factor Induced Nitric Oxide Release through Inhibition of Akt Phosphorylation.

Angiogenesis, the growth of new blood vessels, is a critical factor of carcinogenesis. Neomycin and neamine, two drugs blocking the nuclear translocation of angiogenin (ANG), have been proven to inhibit tumour growth in vivo. However, the high toxicity of neomycin prevents its therapeutic use, thus indicating that the less toxic neamine may be a better candidate. Endothelial cells were cultured on a biocompatible multiple microelectrode array (MMA). The release of NO evoked by ANG or vascular endothelial growth factor (VEGF) was detected electrochemically. The effects of neomycin and neamine on ANG- and VEGF-induced NO releases have been investigated. Neomycin totally blocks NO release for concentrations down to the pM range, probably through the inhibition of the Akt kinase phosphorylation, as revealed by confocal microscopy. On the other hand, both ANG- and VEGF-induced NO releases were not significantly hindered by the presence of high concentrations of neamine. The inhibition of the Akt pathway and NO release are expected to lead to a severe decrease in tissue growth and repair, thus indicating a possible cause for the toxicity of neomycin. Furthermore, the data presented here show that ANG- and VEGF-induced NO releases are not dependent on the nuclear translocation of angiogenin, as these events were not abolished by the presence of neamine.

Rapid Measurement of Lactate in the Exhaled Breath Condensate: Biosensor Optimization and In-Human Proof of Concept.

Lactate concentration is of increasing interest as a diagnostic for sepsis, septic shock, and trauma. Compared with the traditional blood sample media, the exhaled breath condensate (EBC) has the advantages of non-invasiveness and higher user acceptance. An amperometric biosensor was developed and its application in EBC lactate detection was investigated in this paper. The sensor was modified with PEDOT:PSS-PB, and two different lactate oxidases (LODs). A rotating disk electrode and Koutecky-Levich analysis were applied for the kinetics analysis and gel optimization. The optimized gel formulation was then tested on disposable screen-printed sensors. The disposable sensors exhibited good performance and presented a high stability for both LOD modifications. Finally, human EBC analysis was conducted from a healthy subject at rest and after 30 min of intense aerobic cycling exercise. The sensor coulometric measurements showed good agreement with fluorometric and triple quadrupole liquid chromatography mass spectrometry reference methods. The EBC lactate concentration increased from 22.5 µM (at rest) to 28.0 µM (after 30 min of cycling) and dropped back to 5.3 µM after 60 min of rest.

Synthetic biology and bioelectrochemical tools for electrogenetic system engineering.

Synthetic biology research and its industrial applications rely on deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and electrodes), allowing for the direct integration of electronics with biological processes. However, the use of electrogenetic systems is limited by poor activity, tunability, and standardization. In this work, we developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Last, we demonstrated electrochemical activation of gene expression under aerobic conditions using a novel, modular bioelectrochemical device. These genetic and electrochemical tools facilitate the design and improve the performance of electrogenetic systems. Furthermore, the genetic design strategies used can be applied to other redox-responsive promoters to further expand the available tools for electrogenetics.

Optimizing antimicrobial use: challenges, advances and opportunities.

An optimal antimicrobial dose provides enough drug to achieve a clinical response while minimizing toxicity and development of drug resistance. There can be considerable variability in pharmacokinetics, for example, owing to comorbidities or other medications, which affects antimicrobial pharmacodynamics and, thus, treatment success. Although current approaches to antimicrobial dose optimization address fixed variability, better methods to monitor and rapidly adjust antimicrobial dosing are required to understand and react to residual variability that occurs within and between individuals. We review current challenges to the wider implementation of antimicrobial dose optimization and highlight novel solutions, including biosensor-based, real-time therapeutic drug monitoring and computer-controlled, closed-loop control systems. Precision antimicrobial dosing promises to improve patient outcome and is important for antimicrobial stewardship and the prevention of antimicrobial resistance.

Aerosolised fluorescein can quantify FFP mask faceseal leakage: a cost-effective adaptation to the existing point of care fit test.

BACKGROUND: A qualitative fit test using bitter-tasting aerosols is the commonest way to determine filtering face-piece (FFP) mask leakage. This taste test is subjective and biased by placebo. We propose a cheap, quantitative modification of the taste test by measuring the amount of fluorescein stained filter paper behind the mask using image analysis. METHODS: A bitter-tasting fluorescein solution was aerosolised during mask fit tests, with filter paper placed on masks' inner surfaces. Participants reported whether they could taste bitterness to determine taste test 'pass' or 'fail' results. Filter paper photographs were digitally analysed to quantify total fluorescence (TF). RESULTS: Fifty-six healthcare professionals were fit tested; 32 (57%) 'passed' the taste test. TF between the taste test 'pass' and 'fail' groups was significantly different (p<0.001). A cut-off (TF = 5.0 × 106 units) was determined at precision (78%) and recall (84%), resulting in 5/56 participants (9%) reclassified from 'pass' to 'fail' by the fluorescein test. Seven out of 56 (12%) reclassified from 'fail' to 'pass'. CONCLUSION: Fluorescein is detectable and sensitive at identifying FFP mask leaks. These low-cost adaptations can enhance exiting fit testing to determine 'pass' and 'fail' groups, protecting those who 'passed' the taste test but have high fluorescein leak, and reassuring those who 'failed' the taste test despite having little fluorescein leak.

Lifting the lid on the potentiostat: a beginner's guide to understanding electrochemical circuitry and practical operation.

Students who undertake practical electrochemistry experiments for the first time will come face to face with the potentiostat. To many this is simply a box containing electronics which enables a potential to be applied between a working and reference electrode, and a current to flow between the working and counter electrode, both of which are outputted to the experimentalist. Given the broad generality of electrochemistry across many disciplines it is these days very common for students entering the field to have a minimal background in electronics. This article serves as an introductory tutorial to those with no formalized training in this area. The reader is introduced to the operational amplifier, which is at the heart of the different potentiostatic electronic circuits and its role in enabling a potential to be applied and a current to be measured is explained. Voltage follower op-amp circuits are also highlighted, given their importance in measuring voltages accurately. We also discuss digital to analogue and analogue to digital conversion, the processes by which the electrochemical cell receives input signals and outputs data and data filtering. By reading the article, it is intended the reader will also gain a greater confidence in problem solving issues that arise with electrochemical cells, for example electrical noise, uncompensated resistance, reaching compliance voltage, signal digitisation and data interpretation. We also include trouble shooting tables that build on the information presented and can be used when undertaking practical electrochemistry.

Nucleic acid sensing via electrochemical oligonucleotide-templated reactions.

Short single-stranded nucleic acids as found in a variety of bodily fluids have recently emerged as minimally invasive biomarkers for a broad range of pathologies, most notably cancer. Because of their small size, low natural abundance and high sequence homology between family members they are challenging to detect using standard technologies suitable for use at the point-of-care. Herein we report the design, engineering and testing of a novel sensing strategy: electrochemically active molecular probes based on peptide nucleic acid (PNA) scaffolds for the detection of single-stranded oligonucleotides, in particular microRNAs (or miRs). As a proof-of-principle, a wide range of probes were designed and tested to detect miR-141, a known diagnostic biomarker for prostate cancer. Optimal quantitative sensing of miR-141 was achieved via the first example of an electrochemical oligonucleotide-templated reaction (EOTR), whereby two PNA probes - functionalized with an aniline and a 1,4-catechol respectively - preferentially react with each other upon simultaneous hybridization to the same RNA target strand, serving here as a template. Quantitative, electrochemical detection of the product of this bio-orthogonal reaction showed direct correlation between adduct formation and miR-141 concentration. Coupling the specificity of OTR with the speed and sensitivity of electrochemical sensing delivers EOTRs as a promising new technique for fast, low-cost, quantitative and sequence-specific detection of short nucleic acids from liquid biopsies.

Decreased 14-3-3 expression correlates with age-related regional reductions in CNS dopamine and motor function in the pond snail, Lymnaea.

Ageing is associated in many organisms with a reduction in motor movements. We have previously shown that the rate of feeding movements of the pond snail, Lymnaea, decreased with age but the underlying cause is not fully understood. Here, we show that dopamine in the cerebro-buccal complex is an important signalling molecule regulating feeding frequency in Lymnaea and that ageing is associated with a decrease in CNS dopamine. A proteomic screen of young and old CNSs highlighted a group of proteins that regulate stress responses. One of the proteins identified was 14-3-3, which can enhance the synthesis of dopamine. We show that the Lymnaea 14-3-3 family exists as three distinct isoforms. The expression of the 29 kDa isoform (14-3-3Lym3) in the cerebro-buccal complex decreased with age and correlated with feeding rate. Using a 14-3-3 antagonist (R18) we were able to reduce the synthesis of L-DOPA and dopamine in ex vivo cerebro-buccal complexes. Together these data suggest that an age-related reduction in 14-3-3 can decrease CNS dopamine leading to a consequential reduction in feeding rate.

Exhaled breath condensate based breath analyser - a disposable hydrogen peroxide sensor and smart analyser.

Exhaled breath condensate (EBC) based breath analysis has gained significant interest in pulmonary disease diagnostics over the past few years due to the non-invasiveness and simplicity of the technique. Most approaches to date have separated EBC collection from the subsequent lab-based analysis. Here we report a low-cost disposable hydrogen peroxide (H2O2)sensor modified with a new composite consisting of Prussian Blue (PB), PEDOT:PSS, and crosslinker ethylene glycol (EG) and divinyl sulfone (DVS) combined with a thermoelectric EBC collection device. Theoretical modelling suggests that previously reported large variations between analyte concentrations arise from poor control of the condensation process. Experimental data confirm the model predictions and proof-of-principle human samples were analysed showing good agreement with fluorometric methods.

Self-assembly Synthesis of Molecularly Imprinted Polymers for the Ultrasensitive Electrochemical Determination of Testosterone.

Molecularly imprinted polymers (MIPs) can often bind target molecules with high selectivity and specificity. When used as MIPs, conductive polymers may have unique binding capabilities; they often contain aromatic rings and functional groups, which can undergo π－π and hydrogen bonding interactions with similarly structured target (or template) molecules. In this work, an electrochemical method was used to optimize the synthetic self-assembly of poly(aniline-co-metanilic acid) and testosterone, forming testosterone-imprinted electronically conductive polymers (TIECPs) on sensing electrodes. The linear sensing range for testosterone was from 0.1 to 100 pg/mL, and the limit of detection was as low as ~pM. Random urine samples were collected and diluted 1000-fold to measure testosterone concentration using the above TIECP sensors; results were compared with a commercial ARCHITECT ci 8200 system. The testosterone concentrations in the tested samples were in the range of 0.33 ± 0.09 to 9.13 ± 1.33 ng/mL. The mean accuracy of the TIECP-coated sensors was 90.3 ± 7.0%.

Development of a Minimally Invasive Microneedle-Based Sensor for Continuous Monitoring of ß-Lactam Antibiotic Concentrations in Vivo.

Antimicrobial resistance poses a global threat to patient health. Improving the use and effectiveness of antimicrobials is critical in addressing this issue. This includes optimizing the dose of antibiotic delivered to each individual. New sensing approaches that track antimicrobial concentration for each patient in real time could allow individualized drug dosing. This work presents a potentiometric microneedle-based biosensor to detect levels of ß-lactam antibiotics in vivo in a healthy human volunteer. The biosensor is coated with a pH-sensitive iridium oxide layer, which detects changes in local pH as a result of ß-lactam hydrolysis by ß-lactamase immobilized on the electrode surface. Development and optimization of the biosensor coatings are presented, giving a limit of detection of 6.8 µM in 10 mM PBS solution. Biosensors were found to be stable for up to 2 weeks at -20 °C and to withstand sterilization. Sensitivity was retained after application for 6 h in vivo. Proof-of-concept results are presented showing that penicillin concentrations measured using the microneedle-based biosensor track those measured using both discrete blood and microdialysis sampling in vivo. These preliminary results show the potential of this microneedle-based biosensor to provide a minimally invasive means to measure real-time ß-lactam concentrations in vivo, representing an important first step toward a closed-loop therapeutic drug monitoring system.

Microneedle biosensors for real-time, minimally invasive drug monitoring of phenoxymethylpenicillin: a first-in-human evaluation in healthy volunteers.

BACKGROUND: Enhanced methods of drug monitoring are required to support the individualisation of antibiotic dosing. We report the first-in-human evaluation of real-time phenoxymethylpenicillin monitoring using a minimally invasive microneedle-based ß-lactam biosensor in healthy volunteers. METHODS: This first-in-human, proof-of-concept study was done at the National Institute of Health Research/Wellcome Trust Imperial Clinical Research Facility (Imperial College London, London, UK). The study was approved by London-Harrow Regional Ethics Committee. Volunteers were identified through emails sent to a healthy volunteer database from the Imperial College Clinical Research Facility. Volunteers, who had to be older than 18 years, were excluded if they had evidence of active infection, allergies to penicillin, were at high risk of skin infection, or presented with anaemia during screening. Participants wore a solid microneedle ß-lactam biosensor for up to 6 h while being dosed at steady state with oral phenoxymethylpenicillin (five 500 mg doses every 6 h). On arrival at the study centre, two microneedle sensors were applied to the participant's forearm. Blood samples (via cannula, at -30, 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240 min) and extracellular fluid (ECF; via microdialysis, every 15 min) pharmacokinetic (PK) samples were taken during one dosing interval. Phenoxymethylpenicillin concentration data obtained from the microneedles were calibrated using locally estimated scatter plot smoothing and compared with free-blood and microdialysis (gold standard) data. Phenoxymethylpenicillin PK for each method was evaluated using non-compartmental analysis. Area under the concentration-time curve (AUC), maximum concentration, and time to maximum concentration were compared. Bias and limits of agreement were investigated with Bland-Altman plots. Microneedle biosensor limits of detection were estimated. The study was registered with ClinicalTrials.gov, number NCT03847610. FINDINGS: Ten healthy volunteers participated in the study. Mean age was 42 years (SD 14). Seven (70%) were men. Microdialysis and microneedle results were similar for phenoxymethylpenicillin ECF maximum concentration (0·74 mg/L vs 0·64 mg/L; 95% CI -0·24 to 0·44; p=0·53), time to maximum concentration (1·18 h vs 1·10 h; -0·52 to 0·67; p=0·79), and AUC (1·54 mg × h/L vs 1·67 mg × h/L; -1·10 to 0·85; p=0·79). In total, 440 time points were compared with mean difference between measurements -0·16 mg/L (95% CI -1·30 to 0·82). Mean phenoxymethylpenicillin AUCs for free serum and microneedle PK were similar (1·77 mg × h/L [SD 0·59] vs 1·67 mg × h/L [1·00]; -0·77 to 0·97; p=0·81). Median coefficient of variation between sensors within individuals was 7% (IQR 4-17). Limit of detection for the microneedles was estimated at 0·17 mg/L. INTERPRETATION: This study is proof-of-concept of real-time, microneedle sensing of penicillin in vivo. Future work will explore microneedle use in patient populations, their role in data generation to inform dosing recommendations, and their incorporation into closed-loop control systems for automated drug delivery. FUNDING: National Institute for Health Research Imperial Biomedical Research Centre, Mérieux Foundation.

Public acceptability of computer-controlled antibiotic management: An exploration of automated dosing and opportunities for implementation.

A paucity of data describing citizen perceptions of novel technologies, including those containing unsupervised computer-controlled systems is currently available. We explored citizen perceptions of using a microneedle biosensor and automated dose control system at a university public festival. Groups of citizens (from 2-6 people per group) attended a short demonstration of a microneedle biosensor and automated dosing system versus a traditional phlebotomy approach over a two-day public festival. Individual groups discussed and reached consensus on a number of short questions regarding their perceptions on the acceptability of such technology. Over the two days, 100 groups participated (56/100 day 1 and 44/100 day 2). The majority of individuals reported high acceptability of microneedle technology (median Likert score 9/10), but the majority believed that doctors should decide what dose of antibiotic is delivered (75/100; 75%). Groups concurred with the acceptability of microneedles to reduce blood tests and pain associated with them. However, concerns were reported over unsupervised computer-controlled programmes making decision about antibiotic dosing. This was driven by concerns over computer error and the inability of systems to contextualise decision making to the human and social context. Future work must consider the greater role of citizen engagement in the development of such technologies, to ensure their acceptability upon implementation in clinical practice.

Molecular methods in electrochemical microRNA detection.

High-throughput profiling/sensing of nucleic acids has recently emerged as a highly promising strategy for the early diagnosis and improved prognosis of a broad range of pathologies, most notably cancer. Among the potential biomarker candidates, microRNAs (miRNAs), a class of non-coding RNAs of 19-25 nucleotides in length, are of particular interest due to their role in the post-transcriptional regulation of gene expression. Developing miRNA sensing technologies that are quantitative, ultrasensitive and highly specific has proven very challenging because of their small size, low natural abundance and the high degree of sequence similarity among family members. When compared to optical based methods, electrochemical sensors offer many advantages in terms of sensitivity and scalability. This non-comprehensive review aims to break-down and highlight some of the most promising strategies for electrochemical sensing of microRNA biomarkers.

Monolithic nano-porous polymer in microfluidic channels for lab-chip liquid chromatography.

In this paper, a nano-porous polymer has been integrated into the microfluidics device as on-chip monolithic liquid chromatography column for separation of chemical and biological samples. Monolithic nano-porous polymer (MNP) was formed and firmly grafted on the surface of the microfluidic channel. Neurotransmitters, 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptamine (serotonin, 5-HT), were successfully separated with the developed on-chip MNP column.

Microelectrode generator-collector systems for electrolytic titration: theoretical and practical considerations.

Electochemical generator-collector systems, where one electrode is used to generate a reagent, have a potentially large field of application in sensing and measurement. We present a new theoretical description for coplanar microelectrode disc-disc systems where the collector is passive (such as a potentiometric sensor) and the generator is operating at constant flux. This solution is then used to develop a leading order solution for such a system where the reagent reacts reversibly in solution, such as in acid-base titration, where a hydrogen ion flux is generated by electrolysis of water. The principal novel result of the theory is that such devices are constrained by a maximum reagent flux. The hydrogen ion concentration at the collector will only reflect the buffer capacity of the bulk solution if this constraint is met. Both mathematical solutions are evaluated with several microfabricated devices and reasonable agreement with theory is demonstrated.