High temporal resolution delayed analysis of clinical microdialysate streams.

This paper presents the use of tubing to store clinical microdialysis samples for delayed analysis with high temporal resolution, offering an alternative to traditional discrete offline microdialysis sampling. Samples stored in this way were found to be stable for up to 72 days at -80 °C. Examples of how this methodology can be applied to glucose and lactate measurement in a wide range of in vivo monitoring experiments are presented. This paper presents a general model, which allows for an informed choice of tubing parameters for a given storage time and flow rate avoiding high back pressure, which would otherwise cause the microdialysis probe to leak, while maximising temporal resolution.

In-vivo quantification of lactate using Near Infrared reflectance spectroscopy.

Elevated lactate levels in blood (hyperlactatemia) are indications of hypoperfusion or sepsis in critical care conditions. Quantification and monitoring of this important marker is performed using intermittent blood sampling, which fails to provide a complete scenario to aid clinicians in diagnosis. The feasibility of Near Infrared (NIR) Spectroscopy as an alternative to state-of-the-art techniques in critical care environments for non-invasive and continuous monitoring of lactate has previously been established. Nevertheless, the challenge lies in translating this research from bench to bedside monitoring. For this reason, a pilot investigation was carried out with a portable NIR spectrometer, where spectra in the range of 900-1300 nm were collected from 8 healthy human volunteers undertaking a high intensity incremental exercise protocol for lactate monitoring. This paper reports on the measurement set-up, spectra acquisition and analysis of diffuse NIR reflectance spectra of varying concentrations of lactate. The results obtained by 2D correlation analysis and linear regression are promising and show that the wavelengths 923 nm, 1047 nm, 1142 nm, 1233 nm, 1280 nm and 1330 nm are significant for lactate concentration determination in the NIR region. This provides the necessary confidence for using NIR sensor technology for lactate detection in critical care.

Bio-sensing using recessed gold-filled capillary amperometric electrodes.

A novel recessed electrode is reported for amperometric detection of hydrogen peroxide and via glucose oxidase for the detection of glucose. The electrode utilised electrodeposited platinum over a gold wire surface, which proved to be an effective peroxide-detecting surface. Compared with a traditional exposed electrode surface, the recessed tip facilitated an extended linear range for glucose from 4 to over 14 mM. Bio-fouling, as assessed by exposure to bovine serum albumin, was also significantly reduced. Though response time at the recess was increased, it was within an acceptable range for physiological monitoring. Moreover, the recess enabled precise measurement of the hydrogen peroxide diffusion coefficient; this was based on a bipartite expression for the transient amperometric current at the recessed structure following a step change in ambient hydrogen peroxide concentration. An important aspect of the diffusion measurement was the curve fitting routine used to map on to the theoretical response curve.

Near Infrared and Aquaphotomic analysis of water absorption in lactate containing media.

Increased concentrations of lactate levels in blood are often seen in patients with life-threatening cellular hypoperfusion or infections. State-of-the-art techniques used in clinical practice for measuring serum lactate concentrations rely on intermittent blood sampling and do not permit continuous monitoring of this all important parameter in critical care environments.In recent years, Near Infrared (NIR) Spectroscopy has been established as a possible alternative to existing methods that can mitigate these constraints and be used for non-invasive continuous monitoring of lactate. Nevertheless, the dominant absorption of -OH overtone bands of water in the NIR presents a challenge and complicates the accurate detection of other absorbers such as lactate. For this reason, comprehensive analysis of the -OH overtone bands with systematic lactate concentration changes is essential. This paper reports on the analysis of NIR spectra of two aqueous systems of varying concentrations of lactate in saline and whole blood using the principles of Aquaphotomics.The results show distinctive conformational and structural differences in lactate-water binding, which arise due to the molecular interactions of bonds present in respective solvents.

Comparison of a Genetic Algorithm Variable Selection and Interval Partial Least Squares for quantitative analysis of lactate in PBS.

Blood lactate is an important biomarker that has been linked to morbidity and mortality of critically ill patients, acute ischemic stroke, septic shock, lung injuries, insulin resistance in diabetic patients, and cancer. Currently, the clinical measurement of blood lactate is done by collecting intermittent blood samples. Therefore, noninvasive, optical measurement of this significant biomarker would lead to a big leap in healthcare. This study, presents a quantitative analysis of the optical properties of lactate. The benefits of wavelength selection for the development of accurate, robust, and interpretable predictive models have been highlighted in the literature. Additionally, there is an obvious, time- and cost-saving benefit to focusing on narrower segments of the electromagnetic spectrum in practical applications. To this end, a dataset consisting of 47 spectra of Na-lactate and Phosphate Buffer Solution (PBS) was produced using a Fourier transform infrared spectrometer, and subsequently, a comparative study of the application of a genetic algorithm-based wavelength selection and two interval selection methods was carried out. The high accuracy of predictions using the developed models underlines the potential for optical measurement of lactate. Moreover, an interesting finding is the emergence of local features in the proposed genetic algorithm, while, unlike the investigated interval selection methods, no explicit constraints on the locality of features was imposed. Finally, the proposed genetic algorithm suggests the formation of α-hydroxy-esters methyl lactate in the solutions while the other investigated methods fail to indicate this.

Near Infrared Spectrometric Investigations on the behaviour of Lactate.

In patients with life-threatening illnesses, the metabolic production and disposal of lactate are impaired, which leads to a build-up of blood lactate. In critical care units, the changes in lactate levels are measured through intermittent, invasive, blood sampling and in vitro assay. Continuous monitoring is lacking, yet such monitoring could allow early assessment of severity and prognosis to guide therapy. Currently, there is no routine means to measure lactate levels continuously, particularly non-invasively. The motivation of this study was to understand the interaction of lactate with light in the Near Infra Red (NIR) region of the electromagnetic spectrum. This was to create an opportunity to explore the possibility of a non-invasive sensing technology to monitor lactate continuously.In vitro studies were performed using solution samples with varying concentration levels of sodium lactate in isotonic Phosphate Buffer Solution (PBS) at constant pH (7.4). These samples were prepared using stoichiometric solution compositions and spectra for each sample were taken using a state-of-the-art spectrometer in the NIR region. The spectra were then analysed qualitatively by 2D correlation analysis, which identified the regions of interest. Further analysis of these regions using linear regression at four randomly selected wavelengths showed bathochromic shifts, which, moreover, showed systematic variation correlating with lactate concentration.

In vitro quantification of lactate in Phosphate Buffer Saline (PBS) samples.

Continuous measurement of lactate levels in the blood is a prerequisite in intensive care patients who are susceptible to sepsis due to their suppressed immune system and increased metabolic demand. Currently, there exists no noninvasive tool for continuous measurement of lactate in clinical practice. The current mode of measurement is based on arterial blood gas analyzers which require sampling of arterial blood. In this work, we propose the use of Near Infra-Red (NIR) spectroscopy together with multivariate models as a means to non-invasively predict the concentration of lactate in the blood. As the first step towards this objective, we examined the possibility of accurately predicting concentrations of sodium lactate (NaLac) from the NIR spectra of 37 isotonic phosphate buffer saline (PBS) samples containing NaLac ranging from 0 to 20 mmol/L. NIR spectra of PBS samples were collected using the Lambda 1050 dual beam spectrometer over a spectral range of 800 - 2600 nm with a quartz cell of 1 mm optical path. Estimates and calibration of the lactate concentration with the NIR spectra were made using Partial Least-Squares (PLS) regression analysis and leave-one-out cross-validation on filtered spectra. The regression analysis showed a correlation coefficient of 0.977 and a standard error of 0.89 mmol/L between the predicted and prepared samples. The results suggest that NIR spectroscopy together with multivariate models can be a valuable tool for non-invasive assessment of blood lactate concentrations.

An electrochemical study of acrylate bone adhesive permeability and selectivity change during in vitro ageing: A model approach to the study of biomaterials and membrane barriers.

This study assessed the solute permeability of a family of UV and moisture cured acrylates-based adhesives during in vitro ageing in pH 7.4 buffer. Acrylates have a potential role in bone fracture fixation, but their inability to allow microsolute exchange between the fractured bone surfaces may contribute to ineffective healing. Cyclic voltammetry and chronoamperometry were used to determine the diffusion coefficients for various electrochemically active probe molecules (O2, H2O2, acetaminophen, catechol, uric acid and ascorbic acid) at proprietary acrylic, urethane - acrylate and cyanoacrylate adhesives. All adhesives proved to be impermeable for up to 9 days ageing, following which a near-exponential increase in permeability resulted for all solutes. At 18 days, the diffusion coefficients were in the range of 10-5 cm2s-1 for O2 and H2O2 and 10-6 cm2s-1 for the organic solutes; no transport selectivity was seen between the latter. Adhesive joint strength showed a direct, inverse, correlation with permeability, with the more hydrophilic cyanoacrylates showing the greatest loss of strength. Adhesive permeabilisation does not appear to be compatible with the retention of bonding strength, but it serves as a new non-destructive predictor of adhesion strength change during ageing and practical use.

A test method to monitor in vitro storage and degradation effects on a skin substitute.

We have investigated a potential test method to monitor changes through possible degradation of a collagen/glycosaminoglycan tissue engineering scaffold in vitro. The method used cyclic voltammetry where the degradation process was measured by determining changes in the apparent diffusion coefficients of thermodynamically reversible couples, ferrocyanide and 1,4-benzoquinone, through the scaffold before and after degradation at low pH and at different temperatures. Scaffold samples were degraded in vitro by exposure to pH 3 for 44 days and also stored in pH 7.4 phosphate saline buffer for one week. Sample temperatures used were 21 degrees C, 37 degrees C and 40 degrees C. The greatest apparent degradation was observed for scaffolds stored at 40 degrees C. Prior to storage, effective diffusion coefficients were 4.4x10(-6) cm2 s(-1) and 2.6x10(-10) cm2 s(-1) for ferrocyanide and 1,4-benzoquinone, respectively. For these respective compounds values changed to 1.2x10(-6) cm2 s(-1) and 1.0x10(-6) cm2 s(-1) after 37 degrees C degradation and 2.6x10(-6) cm2 s(-1) and 5.5x10(-8) cm2 s(-1) after pH 3 degradation.

Impedimetric sensing of cells on polypyrrole-based conducting polymers.

Polypyrrole (PPy) is a conducting polymer that may be electrochemically generated with the incorporation of any anionic species, including net-negatively charged biological molecules such as proteins and polysaccharides. In this article, dermatan and chloride-loaded PPy films were prepared on gold sputter-coated coverslips and various skin derived cells were studied on them by electrochemical impedance spectroscopy. Impedance spectra in the frequency range 1-100 kHz were either determined at specific times or impedance was monitored continuously at specific frequencies. An equivalent impedance circuit was fitted to the recorded impedance spectra to obtain parameters whose contributions could be mapped to intracellular and intercellular current pathways, and the membrane properties of cells. Results show cell-induced impedance changes were detected over PPy modified electrodes and were dependent on cell density and type, monitoring frequency, material composition, and treatment. Lower cell densities were detected on PPy when compared with bare gold. Keratinocyte confluence, as determined by impedimetric analysis, was reached more rapidly on PPy than on gold. This was consistent with previous, more cumbersome, biochemical assays. Electrical equivalent circuit analysis provided evidence that the technique may be extended to discriminate cell type because of the intracellular and intercellular resistance, and cell membrane capacitance being related to cell morphology.

Modifying surfaces and interfaces for improved biomaterial performance.

Greater understanding of the fundamental processes that drive a body's response to an implant material is leading to new approaches in biomaterial surface engineering. Some recent work is reported. The discussion looks, respectively, at the important roles of the surface water layer and microscopic molecular domains, the new emphasis on conducting polymers and future developments.

Biocompatible materials developments for new medical implants.

Recent work on modifying silicone rubber to improve water permeability and biocompatibility is described. In addition, modifications to the interface between an active implanted device and the body are reported, which have led to reduced power consumption and improved device performance.

An electrochemical study of microporous track-etched membrane permeability and the effect of surface protein layers.

Microporous track-etched membranes serve as important permeable growth surfaces for cell culture where diffusive solute transport is needed across two growth compartments. This study has established effective solute diffusion coefficients for four probe micro-solutes: hydrogen peroxide, pyrocatechol, acetaminophen and ascorbic acid across three track-etched membranes formulated, respectively, from polycarbonate and polyethylene terephthalate. Chronoamperometry and cyclic voltammetry were used for the diffusion measurements. These showed substantially reduced intra-pore diffusion in relation to available pore area. Diffusion coefficients ranging from 1.43×10-10 to 3.17×10-7cm2s-1 were demonstrated. This strongly suggests that water organisation in micro-pores is not equivalent to that of bulk water. Superimposed protein layers of Type I and IV collagen, Type I collagen-fibronectin, Type I collagen-heparin, and Type I collagen-chondroitin sulphate increased diffusional resistance, but with disproportional retardation of ascorbate diffusion due to charge repulsion at collagen-heparin and collagen-chondroitin sulphate combinations. Diffusive resistance at natural tendon and cartilage was considerably smaller; diffusion coefficients ranged from 8.33×10-6 to 1.09×10-8cm2s-1.

Polypyrrole-based conducting polymers and interactions with biological tissues.

Polypyrrole (PPy) is a conjugated polymer that displays particular electronic properties including conductivity. In biomedical applications, it is usually electrochemically generated with the incorporation of any anionic species including also negatively charged biological macromolecules such as proteins and polysaccharides to give composite materials. In biomedical research, it has mainly been assessed for its role as a reporting interface in biosensors. However, there is an increasing literature on the application of PPy as a potentially electrically addressable tissue/cell support substrate. Here, we review studies that have considered such PPy based conducting polymers in direct contact with biological tissues and conclude that due to its versatile functional properties, it could contribute to a new generation of biomaterials.

Characterization of a laminar flow cell for the prevention of biosensor fouling.

A flow cell using dual parallel laminar flows was designed and fabricated with the aim to prevent biosensor fouling. Y-shaped entrances allowed the introduction of sample and electrolyte streams, with the electrolyte serving as a mobile protective layer near the biosensor. Potassium permanganate was used to quantify the diffusion in the flow cell. Optical intensity analysis of potassium permanganate along a series of transverse lines across the flow cell was carried out under different flow conditions. It was found that the error function, erf(y/[square root](Dt)), where y was the position along the transverse line, D the diffusion coefficient of the solute and t was the time, gave reasonable approximation to the diffusion of potassium permanganate in the cell. The diffusion coefficient of potassium permanganate was determined in stop-flow measurements and the value, D=4.0 x 10(-5)cm(2)/s, agreed to previously reported values. Velocity distribution in the flow cell was simulated numerically to reveal the development of two inflows into one single laminar flow. Results from the study provided preliminary data on solute diffusion characteristics in the flow cell and supported the working principle of laminar flow cells in preventing biosensor fouling.

Bicarbonate and other buffer systems can enhance the rate of H+ diffusion through mucus in vitro.

The effect of various diffusible buffers on mucus H+ permeability, and in particular the potency of the HCO3-/CO2 buffer system relative to other selected buffers is reported here. The diffusional resistance of mucus and water was demonstrated to be dependent on buffer concentration, and the contrast between the two types of layer was most pronounced for low DH+ values near neutrality. This concentration dependence was most marked with mucus layers in the buffer systems investigated. Furthermore, the nature and pKa values of the diffusible buffer systems used in this study had a profound effect on measured DH+. The effect was particularly striking in the case of HCO3- buffer with mucus. Possible implications of these in vitro findings in mucosal protection from acid are discussed.

A liposomal enzyme electrode for measuring glucose.

Enzyme electrodes have been described for measuring glucose but have been limited by the saturation kinetics of the glucose oxidase not allowing clinically relevant glucose concentrations to be measured (0-25 mM). One way of alleviating this problem is to use diffusion-controlled membranes which result in the enzyme experiencing a smaller substrate concentration than that of the bulk solution. As an extension of this concept we have encapsulated glucose oxidase in liposomes whereby the lipid bilayer wall provides the diffusion-limiting membrane as well as providing a biocompatible layer which is of particular relevance when blood glucose is to be measured. Linear ranges were found to embrace the required glucose concentrations and moreover by using liposomes prepared from different lipids, e.g., dimyristoyl (14:0) phosphatidylcholine (DMPC), dipalmitoyl (16:0) phosphatidylcholine (DPPC) and distearoyl (18:0) phosphatidylcholine (DSPC), the electrode response was shown to depend on the bilayer permeabilities in relation to the lipid phase transition temperatures and as a consequence the linear ranges were duly altered.

Surface plasmon resonance: a study of the effect of biotinylation on the selection of antibodies for use in immunoassays.

Surface plasmon resonance is a valuable optical phenomenon for monitoring biomolecular interactions in real time. In this project anti-mouse-Fc was coupled to the carboxymethyl dextran coating on the surface of a CM5 sensor chip (BIAcore) using amine coupling. Monoclonal antibodies (MAbs) to Luteinizing Hormone (LH) were then captured on this surface in the correct orientation for binding. LH (500 IU/l) was injected over the surface and the subsequent binding and dissociation events were monitored. The resulting optical response curves allowed fast analysis of the binding interactions of eight selected MAbs. It was possible to develop a two-site immunometric assay for LH using a pair of these MAbs. The effect of biotinylating the MAbs, using various biotin:antibody coupling ratios, on their subsequent binding to both LH and avidin conjugated alkaline phosphatase was also investigated. This approach has allowed rapid evaluation of the effect of changes in both reagent and reaction conditions on immunoassay performance and appears to be a valuable adjunct to immunosensor and immunoassay development.

Changes in plasma and whole blood lactate in response to arterial occlusion.

The development of lactic acidaemia in the adult forearm in response to partial and total arterial occlusion is described in both plasma and whole blood. Under conditions of total occlusion of the brachial artery lactate levels in the plasma compartment rise at a significantly faster rate; mean difference in gradients -11.6 95% confidence limits -15.6 and -7.6. Thus in these conditions they provide a more sensitive index of tissue hypoxia. Under conditions of partial occlusion there is no significant difference; mean difference in gradients 1.0 95% confidence limits 9.4 and -7.3. Though plasma lactate levels are more sensitive to the ischaemic state in certain circumstances, whole blood measurements are equally applicable to the monitoring of trends in blood lactic acid.

Ion exchanger modified PVC membranes--selectivity studies and response amplification of oxalate and lactate enzyme electrodes.

We report on a novel method of oxidase enzyme electrode response amplification, using unplasticized PVC and plasticized PVC, respectively. The anion exchanger tricaprylylmethylammonium chloride (Aliquat 336s) and hydrophobic isopropylmyristate (IPM) plasticizer have been used together to modify PVC. Resulting structures are anionic substrate selective and hydrogen peroxide impermeable and can be used as outer membranes of a classical dual membrane amperometric enzyme electrode where an oxidase is used to generate H2O2 for electrochemical detection. Their effect on sensor sensitivity and linearity is considered.