Microfluidic biosensor systems for real-time in vivo clinical bioanalysis

The aim of this thesis was to develop online biosensing systems for dialysate tissue metabo- lite detection in real time, to provide an insight into the health of tissue in various in vivo applications. An autocalibration system was developed using LabSmith programmable components to improve the accuracy of results obtained over long monitoring times. A method of col- lecting dialysate into storage tubes for online analysis while retaining temporal resolution was developed and validated. Microfluidic biosensor systems were developed for online measurement of glucose and lactate. One approach employed the use of biosensors, using a combined needle electrode with enzyme encapsulated in a hydrogel layer. The dynamic range of the biosensors was extended by adding an outer polyurethane layer. An alternative approach used automated syringe pumps and valves to develop a microfluidic system for in-flow enzyme addition to the dialysate stream. The existing rsMD system was applied for detection of tissue ischaemia during and after free flap surgery, by measuring dialysate glucose and lactate levels in real time. The system was able to detect flap failure, both during surgery and afterwards in the intensive therapy unit (ITU), much earlier than traditional methods. The rsMD system was adapted to enable monitoring of lactate levels in two dialysate streams and was applied for monitoring isolated porcine kidneys during two methods of cold preservation and subsequent re-warming. Significant differences in the lactate concentrations were observed between the two techniques. The system was extended for use with human transplant kidneys and with both porcine and human pancreases. A novel 3D printed wearable biosensor system was developed for direct integration with a clinical microdialysis probe. The system considerably improved the lag time and dispersional smearing compared with the existing rsMD system. The device was used in a proof-of-concept study with wireless potentiostats to monitor cyclists during exercise.Open Acces

Real-time detection of carboplatin using a microfluidic system

A microfluidic sensor system based on a carbon nanotube-epoxy composite electrode was fabricated to allow detection of the presence of the anti-cancer drug carboplatin in healthy tissue in real time during chemotherapy. Detection of carboplatin was carried out by observing the effects of the drug on the differential pulse voltammetry of free purine bases using a novel carbon nanotube-epoxy composite electrode. In free solution these electrodes performed better than glassy carbon electrodes for oxidation of the free purine bases AMP and GMP, and than DNA-modified carbon nanotube-epoxy composite sensors for detection of carboplatin. On-line carboplatin detection was performed using a computer-controlled microfluidic platform. The methodology for on-line carboplatin detection was optimised in terms of the analysis time and of to allow repeated carboplatin measurement using the same electrode. Microdialysis sampling and our microfluidic platform were combined to give a proof of concept system for real-time carboplatin detection with a limit of detection of 0.014 mM carboplatin in the sampled media. This paper is dedicated to Craig Lunte’s pioneering work in analysis and microdialysis

Clinical value of bioelectrical properties of cancerous tissue in advanced epithelial ovarian cancer patients

Currently, there are no valid pre-operatively established biomarkers or algorithms that can accurately predict surgical and clinical outcome for patients with advanced epithelial ovarian cancer (EOC). In this study, we suggest that profiling of tumour parameters such as bioelectrical-potential and metabolites, detectable by electronic sensors, could facilitate the future development of devices to better monitor disease and predict surgical and treatment outcomes. Biopotential was recorded, using a potentiometric measurement system, in ex vivo paired non-cancerous and cancerous omental tissues from advanced stage EOC (n = 36), and lysates collected for metabolite measurement by microdialysis. Consistently different biopotential values were detected in cancerous tissue versus non-cancerous tissue across all cases (p < 0.001). High tumour biopotential levels correlated with advanced tumour stage (p = 0.048) and tumour load, and negatively correlated with stroma. Within our EOC cohort and specifically the high-grade serous subtype, low biopotential levels associated with poorer progression-free survival (p = 0.0179, p = 0.0143 respectively). Changes in biopotential levels significantly correlated with common apoptosis related pathways. Lactate and glucose levels measured in paired tissues showed significantly higher lactate/glucose ratio in tissues with low biopotential (p < 0.01, n = 12). Our study proposes the feasibility of biopotential and metabolite monitoring as a biomarker modality profiling EOC to predict surgical and clinical outcomes

Cold ischaemia time: is too long really too bad?. Studies using a porcine kidney ex-vivo reperfusion model

Introduction: Post-ischaemic hypothermic machine perfusion (HMP) may be beneficial in recovery of marginal kidney grafts. The full capacity of conventional HMP (with passive oxygenation) to recondition an organ has not been realised. We investigated whether HMP can ameliorate ischemic damage caused by extremely prolonged static cold storage (SCS). Methods: Porcine kidneys underwent 4-h (SCS4,n ¼ 4) or 52-h (SCS52,n ¼ 4) SCS, followed by 10 h of HMP and were then subjected to 2 h of isolated normothermic reperfusion (NRP). Results: There was a post-SCS graft weight loss in SCS52 vs SCS4 kidneys. SCS52 kidneys showed viable perfusion dynamics during HMP, with significantly shorter times to reach viable parameters vs SCS4 kidneys (p < 0.027). During NRP SCS52 kidneys demonstrated similar trends in perfusion dynamics, renal function, oxygen consumptions, lactate production, and tubular injury to SCS4 kidneys. Conclusion: Graft weight loss after SCS, reducing resistance to perfusion, may facilitate better HMP dynamics and graft reconditioning. Clinicians utilising HMP should be aware of this phenomenon when using HMP in kidneys exposed to extreme periods of SCS. HMP after an extended period of SCS can resuscitate kidneys to a level equitable of viability as those after a short period of SCS. Utilising passive oxygenation however may be limiting such recovery and interventions utilising active oxygenation may provide benefit in such organs

An ex vivo comparison of adenosine and lidocaine solution and University of Wisconsin solution for hypothermic machine perfusion of porcine kidneys: potential for development

Background: The optimal hypothermic machine perfusion (HMP) solution has not yet been developed. An adenosine and lidocaine (AL) solution has been shown to be protective in cardiac preservation. The aim of the present study was to examine a modified AL solution with low Ca2+, 16 mM Mg2+, and 4% albumin on kidney preservation compared with University Wisconsin solution (UW). Methods: Twenty donation of organs after cardiac death porcine kidneys underwent HMP for 10 h (AL, n = 10; UW, n = 10) and then 2 h of normothermic reperfusion. Perfusion dynamics, functional parameters, histology, and real-time microdialysis were used to assess kidney responses and viability. Results: During HMP, modified AL-perfused kidneys maintained higher flow rates (21.5 versus 17.9 mL/min/100 g, P = 0.01), with perfusion flow index during the first 3 h 25% greater than with UW (AL = 0.50 + 0.2, UW = 0.40 + 0.17 mL/min/100 g/mmHg; P = 0.03), followed by an increase in UW kidneys which was not significantly different to AL over the remaining 7 h (0.54 versus 0.55 mL/min/100 g/mmHg, respectively). During warm reperfusion, there were no significant differences between the two HMP groups in creatinine clearance, oxygen, and glucose consumption between groups. Modified AL kidneys had significantly lower perfusate lactates (3.1 versus 4.1 mmol/L, P = 0.04) during reperfusion and lower cortical lactate levels (AL = 0.66 +/- 0.31, UW = 0.89 +/- 0.53 mM, P = 0.33). Histology showed similar degrees of reperfusion injury. Conclusions: We conclude that HMP with modified AL solution showed improved perfusion compared with UW and lower perfusate lactate levels during warm reperfusion. Further modification of the AL composition is warranted and may lead to more rapid kidney stabilization and improved graft viability assessment, potentially expanding donor pools

Fiber-Based Electrochemical Biosensors for Monitoring pH and Transient Neurometabolic Lactate

Developing tools that are able to monitor transient neurochemical dynamics is important to decipher brain chemistry and function. Multifunctional polymer-based fibers have been recently applied to monitor and modulate neural activity. Here, we explore the potential of polymer fibers comprising six graphite-doped electrodes and two microfluidic channels within a flexible polycarbonate body as a platform for sensing pH and neurometabolic lactate. Electrodes were made into potentiometric sensors (responsive to pH) or amperometric sensors (lactate biosensors). The growth of an iridium oxide layer made the fiber electrodes responsive to pH in a physiologically relevant range. Lactate biosensors were fabricated via platinum black growth on the fiber electrode, followed by an enzyme layer, making them responsive to lactate concentration. Lactate fiber biosensors detected transient neurometabolic lactate changes in an in vivo mouse model. Lactate concentration changes were associated with spreading depolarizations, known to be detrimental to the injured brain. Induced waves were identified by a signature lactate concentration change profile and measured as having a speed of ∼2.7 mm/min (n = 4 waves). Our work highlights the potential applications of fiber-based biosensors for direct monitoring of brain metabolites in the context of injury

Real-time continuous measurement of lactate through a minimally invasive microneedle patch: a phase I clinical study

IntroductionDetermination of blood lactate levels supports decision-making in a range of medical conditions. Invasive blood-sampling and laboratory access are often required, and measurements provide a static profile at each instance. We conducted a phase I clinical study validating performance of a microneedle patch for minimally invasive, continuous lactate measurement in healthy volunteers.MethodsFive healthy adult participants wore a solid microneedle biosensor patch on their forearms and undertook aerobic exercise for 30 min. The microneedle biosensor quantifies lactate concentrations in interstitial fluid within the dermis continuously and in real-time. Outputs were captured as sensor current and compared with lactate concentrations from venous blood and microdialysis.ResultsThe biosensor was well-tolerated. Participants generated a median peak venous lactate of 9.25 mmol/L (IQR 6.73–10.71). Microdialysate concentrations of lactate closely correlated with blood. Microneedle biosensor current followed venous lactate concentrations and dynamics, with good agreement seen in all participants. There was an estimated lag-time of 5 min (IQR −4 to 11 min) between microneedle and blood lactate measurements.ConclusionThis study provides first-in-human data on use of a minimally invasive microneedle patch for continuous lactate measurement, providing dynamic monitoring. This low-cost platform offers distinct advantages to frequent blood sampling in a wide range of clinical settings, especially where access to laboratory services is limited or blood sampling is infeasible. Implementation of this technology in healthcare settings could support personalised decision-making in a variety of hospital and community settings.Trial registration numberNCT04238611.</jats:sec