This work presents the design, fabrication, and characterization of a robust 3D printed microfluidic analysis system that integrates with FDA-approved clinical microdialysis probes for continuous monitoring of human tissue metabolite levels. The microfluidic device incorporates removable needle type integrated biosensors for glucose and lactate, which are optimized for high tissue concentrations, housed in novel 3D printed electrode holders. A soft compressible 3D printed elastomer at the base of the holder ensures a good seal with the microfluidic chip. Optimization of the channel size significantly improves the response time of the sensor. As a proof-of-concept study, our microfluidic device was coupled to lab-built wireless potentiostats and used to monitor real-time subcutaneous glucose and lactate levels in cyclists undergoing a training regime.

Spreading depolarizations occur spontaneously and frequently in injured human brain. They propagate slowly through injured tissue often cycling around a local area of damage. Tissue recovery after an spreading depolarization requires greatly augmented energy utilisation to normalise ionic gradients from a virtually complete loss of membrane potential. In the injured brain, this is difficult because local blood flow is often low and unreactive. In this study, we use a new variant of microdialysis, continuous on-line microdialysis, to observe the effects of spreading depolarizations on brain metabolism. The neurochemical changes are dynamic and take place on the timescale of the passage of an spreading depolarization past the microdialysis probe. Dialysate potassium levels provide an ionic correlate of cellular depolarization and show a clear transient increase. Dialysate glucose levels reflect a balance between local tissue glucose supply and utilisation. These show a clear transient decrease of variable magnitude and duration. Dialysate lactate levels indicate non-oxidative metabolism of glucose and show a transient increase. Preliminary data suggest that the transient changes recover more slowly after the passage of a sequence of multiple spreading depolarizations giving rise to a decrease in basal dialysate glucose and an increase in basal dialysate potassium and lactate levels.

Antimicrobial resistance poses a global threat to patient health. Improving the use and effectiveness of antimicrobials is critical in addressing this issue. This includes optimising the dose of antibiotic delivered to each individual. New sensing approaches that track antimicrobial concentration for each patient in real time could allow individualised drug dosing. This work presents a potentiometric microneedlebased biosensor to detect levels of β-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 β-lactam hydrolysis by β-lactamase immobilised on the electrode surface. Development and optimisation 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 two weeks at-20°C and to withstand sterilisation. Sensitivity was retained after application for 6 hours in vivo. Proofof-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 β-lactam concentrations in vivo, representing an important first step towards a closed-loop therapeutic drug monitoring system.

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 β-lactam biosensor in healthy volunteers.

The (306)VQIVYK(311) sequence in the tau peptide is essential for the formation of intracellular amyloid fibrils related to Alzheimer's disease, where it forms interdigitating cross-beta-structures. The inherent conformational preferences of the capped hexapeptide Ac-VQIVYK-NHMe were characterized in the gas phase. IR/UV double-resonance spectroscopy of the peptide isolated in a cold molecular beam was used to probe the conformation of the neutral peptide. The influence of protonation at the lysine side chain was investigated at 298 K by characterizing the protonated peptide ion, Ac-VQIVYK(H(+))-NHMe, with IRMPD spectroscopy in the fingerprint and amide I/II band region in an FTICR mass spectrometer. The conformations for both neutral and protonated peptides were predicted by an extensive conformational search procedure followed by cluster analysis and then DFT calculations. Comparison of the experimental and computed IR spectra, with consideration of the relative energies, was used to assign the dominant conformations observed. The neutral peptide adopts a beta-hairpin-like conformation with two loosely extended peptide chains, demonstrating the preference of the sequence for extended beta-strand-like structures. In the protonated peptide, the lysine NH3(+) disrupts this extended conformation by binding to the backbone and induces a transition to a random-coil-like structure.

In Alzheimer's disease, the tau protein forms intracellular amyloid fibrils in which the (306)VQIVYK(311) sequence adopts parallel beta-sheets, enabling fibril formation via cross-beta "steric zippers". We investigated aggregation of the protected segment (Ac-VQIVYK-NHMe) using IR/UV hole-burning spectroscopy in the NH stretch region in a cold molecular beam combined with DFT calculations in order to characterize its structure and identify the noncovalent interactions generally responsible for aggregation and stabilization in amyloid peptides. The computed and experimental IR spectra suggest that the tau-protein fragments form extended beta-strands that are combined in a beta-sheet through characteristic backbone hydrogen bonds, indicating that this secondary structure is energetically most attractive and readily forms in the gas phase, without any "guiding" interactions from a solvent or protein environment.

Application of machine perfusion in preclinical porcine and human pancreas models is feasible and successful; the development of these translational models could be beneficial in improving pancreas preservation before transplantation and allowing organ viability assessment and optimization.

We describe an enzyme-based electroanalysis system for real-time analysis of a clinical microdialysis sampling stream during surgery. Free flap tissue transfer is used widely in reconstructive surgery after resection of tumours or in other situations such as following major trauma. However, there is a risk of flap failure, due to thrombosis in the flap pedicle, leading to tissue ischaemia. Conventional clinical assessment is particularly difficult in such ‘buried’ flaps where access to the tissue is limited. Rapid sampling microdialysis (rsMD) is an enzyme-based electrochemical detection method, which is particularly suited to monitoring metabolism. This online flow injection system analyses a dialysate flow stream from an implanted microdialysis probe every 30 s for levels of glucose and lactate. Here, we report its first use in the monitoring of free flap reconstructive surgery, from flap detachment to re-vascularisation and overnight in the intensive care unit. The on-set of ischaemia by both arterial clamping and failure of venous drainage was seen as an increase in lactate and decrease in glucose levels. Glucose levels returned to normal within 10 min of successful arterial anastomosis, whilst lactate took longer to clear. The use of the lactate/glucose ratio provides a clear predictor of ischaemia on-set and subsequent recovery, as it is insensitive to changes in blood flow such as those caused by topical vasodilators, like papaverine. The use of storage tubing to preserve the time course of dialysate, when technical difficulties arise, until offline analysis can occur, is also shown. The potential use of rsMD in free flap surgery and tissue monitoring is highly promising.FigureFree flap surgery timeline: The flap is raised and MD probe inserted. Glucose and lactate levels were monitored at 1 minute intervals throughout flap removal and the reconstruction of the tongue. Grey lines indicate key events as communicated by the surgeons in real time.