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.

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.

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.

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.

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.

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.

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.

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.

Both sides nanopatterned tubular collagen scaffolds as tissue-engineered vascular grafts.

Two major requirements for a tissue-engineered vessel are the establishment of a continuous endothelium and adequate mechanical properties. In this study, a novel tubular collagen scaffold possessing nanopatterns in the form of channels (with a 650 nm periodicity) on both sides was designed and examined after seeding and co-culturing with vascular cells. Initially, the exterior of the tube was seeded with human vascular smooth muscle cells (VSMCs), cultured for 14 days, and then human internal thoracic artery endothelial cells (HITAECs) were seeded on the inside of the tube and cultured for a further week. Microscopy revealed that nano-scale patterns could be reproduced on collagen with high fidelity and preserved during incubation in vitro. The VSMCs were circumferentially orientated with the help of these nanopatterns and formed multilayers on the exterior, while HITAECs formed a continuous layer on the interior, as is the case in natural vessels. Both cell types were observed to proliferate and retain their phenotypes in the co-culture.

Surface plasmon resonance imaging for biosensing.

Surface plasmon resonance imaging (SPRI) is a useful tool for the study of surface biomolecular interactions allowing for label-free detection and elegant instrumentation. SPRI imaging system is described in this review with an emphasis on recent applications with examples of different biological interactions and high throughput analysis. Signal amplification in SPRI using nanoparticle and waveguide-based optical coupling is introduced. Finally the detection sensitivity of the SPRI system is examined in terms of other competitive methods.

Influence of nanopatterns on endothelial cell adhesion: Enhanced cell retention under shear stress.

In this study, nanopatterned crosslinked films of collagen Type I were seeded with human microvascular endothelial cells and tested for their suitability for vascular tissue engineering. Since the films will be rolled into tubes with concentric layers of collagen, nutrient transfer through the collagen films is quite crucial. Molecular diffusivity through the collagen films, cell viability, cell proliferation and cell retention following shear stress were studied. Cells were seeded onto linearly nanogrooved films (groove widths of 332.5, 500 and 650nm), with the grooves aligned in the direction of flow. The nanopatterns did not affect cell proliferation or initial cell alignment; however, they significantly affected cell retention under fluid flow. While cell retention on unpatterned films was 35+/-10%, it was 75+/-4% on 332.5nm patterned films and even higher, 91+/-5%, on 650nm patterned films. The films were found to have diffusion coefficients of ca. 10(-6)cm(2)s(-1) for O(2) and 4-acetaminophenol, which is comparable to that observed in natural tissues. This constitutes another positive asset of these films for consideration as a scaffold material for vascular tissue engineering.

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.

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.

Technology developments to initiate a next generation of cochlear implants.

In the framework of the EU-supported research project Healthy Aims, we developed a range of novel electrode arrays and related technologies for use in hearing prosthesis. This paper summarizes our ongoing research activities on alternative electrode manufacturing routes, functional electrode interfaces and smart intra-cochlear and intra-modiolus electrode arrays.

Sensor biocompatibility: final frontier in bioanalytical measurement.

Chemical and biosensors operate as direct sample contact systems; as such, the response of the biomatrix to their presence is as important as their response to the targeted analyte. There needs to be greater research emphasis on the response of biofluids to sensors because of the impact on sensor performance. Mainly, this is the result of deposited biolayers through the adsorption of colloids and proteins, followed by a more complex surface-active system: whole cells. Sensor surface engineering and controlled sample presentation through fluidics are likely to be able to mitigate some of these effects. Overall there needs to be a convergence with rapidly evolving research strategies in biomaterials if sensors are to make the full transition from the laboratory to the real world.

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.

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.

Magnetic counter-gravity flow separation of electrically prepolarised lymphoid cells.

A novel principle is proposed for a differential separation of live cells (such as leucocytes) from a main flow. A microfluidic device with planar insulated electrodes as the side walls of the channel was manufactured and tested. An array of insulated vertical conductor wires was inserted along the axis of the channel and used to impose Lorentz forces upon polarisable particles that moved with the flow. Polystyrene microspheres and lymphoid cell lines (DOHH2 and K562) were used to test the ability of the setting to impose a force field that induced consistent vertical motion. The direction of electric current was found to directly influence the number of cells or microspheres that were sampled at the surface of the flow. Lorentz force was considered to be active upon cells due to an overall polarisation of the membrane surface. The consequence of the magnetic force was that the polarised cells were moved vertically upwards (opposing gravity). The setting was effective for increasing the number of extracted cells from a main flow or for increasing the concentration of DOHH2 cells in a mixed population with K562 in culture medium. The limitations of the work parameters (potential-current) were found to be dependent upon the cell type.

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.