Electrochemical Biosensing of Glucose Based on the Enzymatic Reduction of Glucose.

In this work, the enzyme aldehyde reductase, also known as aldose reductase, was synthesized and cloned from a human gene. Spectrophotometric measurements show that in presence of the nicotinamide adenine dinucleotide phosphate cofactor (NADPH), the aldehyde reductase catalyzed the reduction of glucose to sorbitol. Electrochemical measurements performed on an electrodeposited poly(methylene green)-modified gold electrode showed that in the presence of the enzyme aldehyde reductase, the electrocatalytic oxidation current of NADPH decreased drastically after the addition of glucose. These results demonstrate that aldehyde reductase is an enzyme that allows the construction of an efficient electrochemical glucose biosensor based on glucose reduction.

Comparison of Dual Beam Dispersive and FTNIR Spectroscopy for Lactate Detection.

Near Infrared (800-2500 nm) spectroscopy has been extensively used in biomedical applications, as it offers rapid, in vivo, bed-side monitoring of important haemodynamic parameters, which is especially important in critical care settings. However, the choice of NIR spectrometer needs to be investigated for biomedical applications, as both the dual beam dispersive spectrophotomer and the FTNIR spectrometer have their own advantages and disadvantages. In this study, predictive analysis of lactate concentrations in whole blood were undertaken using multivariate techniques on spectra obtained from the two spectrometer types simultaneously and results were compared. Results showed significant improvement in predicting analyte concentration when analysis was performed on full range spectral data. This is in comparison to analysis of limited spectral regions or lactate signature peaks, which yielded poorer prediction models. Furthermore, for the same region, FTNIR showed 10% better predictive capability than the dual beam dispersive NIR spectrometer.

Monitoring with In Vivo Electrochemical Sensors: Navigating the Complexities of Blood and Tissue Reactivity.

The disruptive action of an acute or critical illness is frequently manifest through rapid biochemical changes that may require continuous monitoring. Within these changes, resides trend information of predictive value, including responsiveness to therapy. In contrast to physical variables, biochemical parameters monitored on a continuous basis are a largely untapped resource because of the lack of clinically usable monitoring systems. This is despite the huge testing repertoire opening up in recent years in relation to discrete biochemical measurements. Electrochemical sensors offer one of the few routes to obtaining continuous readout and, moreover, as implantable devices information referable to specific tissue locations. This review focuses on new biological insights that have been secured through in vivo electrochemical sensors. In addition, the challenges of operating in a reactive, biological, sample matrix are highlighted. Specific attention is given to the choreographed host rejection response, as evidenced in blood and tissue, and how this limits both sensor life time and reliability of operation. Examples will be based around ion, O2, glucose, and lactate sensors, because of the fundamental importance of this group to acute health care.

Identification and Quantitative Determination of Lactate Using Optical Spectroscopy-Towards a Noninvasive Tool for Early Recognition of Sepsis.

Uninterrupted monitoring of serum lactate levels is a prerequisite in the critical care of patients prone to sepsis, cardiogenic shock, cardiac arrest, or severe lung disease. Yet there exists no device to continuously measure blood lactate in clinical practice. Optical spectroscopy together with multivariate analysis is proposed as a viable noninvasive tool for estimation of lactate in blood. As an initial step towards this goal, we inspected the plausibility of predicting the concentration of sodium lactate (NaLac) from the UV/visible, near-infrared (NIR), and mid-infrared (MIR) spectra of 37 isotonic phosphate-buffered saline (PBS) samples containing NaLac ranging from 0 to 20 mmol/L. UV/visible (300-800 nm) and NIR (800-2600 nm) spectra of PBS samples were collected using the PerkinElmer Lambda 1050 dual-beam spectrophotometer, while MIR (4000-500 cm-1) spectra were collected using the Spectrum two FTIR spectrometer. Absorption bands in the spectra of all three regions were identified and functional groups were assigned. The concentration of lactate in samples was predicted using the Partial Least-Squares (PLS) regression analysis and leave-one-out cross-validation. The regression analysis showed a correlation coefficient (R2) of 0.926, 0.977, and 0.992 for UV/visible, NIR, and MIR spectra, respectively, between the predicted and reference samples. The RMSECV of UV/visible, NIR, and MIR spectra was 1.59, 0.89, and 0.49 mmol/L, respectively. The results indicate that optical spectroscopy together with multivariate models can achieve a superior technique in assessing lactate concentrations.

Investigations into the Effects of pH on Quantitative Measurements of Lactate in Biological Media Using ATR-FTIR Spectroscopy.

Quantification of lactate/lactic acid in critical care environments is essential as lactate serves as an important biochemical marker for the adequacy of the haemodynamic circulation in shock and of cell respiration at the onset of sepsis/septic shock. Hence, in this study, ATR-FTIR was explored as a potential tool for lactate measurement, as the current techniques depend on sample preparation and fails to provide rapid response. Moreover, the effects of pH on PBS samples (7.4, 7, 6.5 and 6) and change in solution conditions (PBS to whole blood) on spectral features were also investigated. A total 189 spectra from five sets of lactate containing media were obtained. Results suggests that lactate could be measured with more than 90% accuracy in the wavenumber range of 1500-600 cm-1. The findings of this study further suggest that there exist no effects of change in pH or media, when estimating lactate concentration changes in this range of the Mid-IR spectral region.

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

3D Printed Microfluidic Device with Integrated Biosensors for Online Analysis of Subcutaneous Human Microdialysate.

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.

Chemosensors and biosensors based on polyelectrolyte microcapsules containing fluorescent dyes and enzymes.

The concept of enzyme-assisted substrate sensing based on use of fluorescent markers to detect the products of enzymatic reaction has been investigated by fabrication of micron-scale polyelectrolyte capsules containing enzymes and dyes in one entity. Microcapsules approximately 5 µm in size entrap glucose oxidase or lactate oxidase, with peroxidase, together with the corresponding markers Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride (Ru(dpp)) complex and dihydrorhodamine 123 (DHR123), which are sensitive to oxygen and hydrogen peroxide, respectively. These capsules are produced by co-precipitation of calcium carbonate particles with the enzyme followed by layer-by-layer assembly of polyelectrolytes over the surface of the particles and incorporation of the dye in the capsule interior or in the multilayer shell. After dissolution of the calcium carbonate the enzymes and dyes remain in the multilayer capsules. In this study we produced enzyme-containing microcapsules sensitive to glucose and lactate. Calibration curves based on fluorescence intensity of Ru(dpp) and DHR123 were linearly dependent on substrate concentration, enabling reliable sensing in the millimolar range. The main advantages of using these capsules with optical recording is the possibility of building single capsule-based sensors. The response from individual capsules was observed by confocal microscopy as increasing fluorescence intensity of the capsule on addition of lactate at millimolar concentrations. Because internalization of the micron-sized multi-component capsules was feasible, they could be further optimized for in-situ intracellular sensing and metabolite monitoring on the basis of fluorescence reporting.

A naringin-derived bioink enhances the shape fidelity of 3D bioprinting and efficiency of cartilage defect repair.

3D bioprinting is a major area of interest in health sciences for customized manufacturing, but lacks specific bioinks to enhance the shape fidelity of 3D bioprinting and efficiency of tissue repair for particular clinical purposes. A naringin derived bioink, which contains 1.5 mM methylacryloyl naringin and 0.15 mM methylacryloyl gelatin, improves the fidelity of 3D bioprinting due to 405 nm light absorption of methylacryloyl naringin. The naringin derived bioink promotes the growth of chondrocytes due to preserving bioactivities of naringin and functions as a medical ingredient from which it has been described as a medical bioink in this study. It facilitates cartilage regeneration by upregulating the transcription of chondrogenesis-related genes like SOX9 and genes against oxidative stress like SOD1 and SOD2 and maintains chondrocytes active resulting from the significantly enhanced COL II/COL I ratio. According to a rabbit cartilage defect model, the proposed naringin derived medical bioink significantly improves the efficiency and quality of cartilage defect repair, suggesting that the bioink is suitable for cartilage defect repair applications and a feasible strategy is provided for the formulation of medical bioinks for specific clinical purposes.

Fabrication of biomaterials via controlled protein bubble generation and manipulation.

In this work, we utilize a recently developed microbubbling process to generate controlled protein (bovine serum albumin, BSA) coated bubbles and then manipulate these to fabricate a variety of structures suitable for several generic biomedical applications, tissue engineering, and biosensor coatings. Using BSA solutions with varying concentrations (20, 25, and 30 wt %) and cross-linking (terephthaloyl chloride) mechanisms, structures were fabricated including porous thin films with variable pore sizes and thickness (partially cross-linked coupled to bubble breakdown), scaffolds with variable pore morphologies (fully cross-linked), and coated bubbles (no cross-linking), which can be used as stand-alone delivery devices and contrast agents. The movement of typical biosensor chemicals (catechol and hydrogen peroxide) across appropriate film structures was studied. The potential of formed scaffold structures for tissue engineering applications was demonstrated using mouse cell lines (L929). In addition to low cost, providing uniform structure generation and high output, the size of the bubbles can easily be controlled by adjusting simplistic processing parameters. The combination of robust processing and chemical modification to uniform macromolecule bubbles can be utilized as a competing, yet novel, tool with current technologies and processes in advancing the biomaterials and biomedical engineering remits.

Synthesis and characterisation of enhanced barrier polyurethane for encapsulation of implantable medical devices.

Polymeric membranes have been used as interfaces between implantable devices and biological tissues to operate as a protective barrier from water exchanging and to enhance biocompatibility. Polyurethanes have been used as biocompatible membranes for decades. In this study, copolymers of polyether urethane (PEU) with polydimethylsiloxane (PDMS) were synthesised with the goal of creating materials with low water permeability and high elasticity. PDMS was incorporated into polymer backbone as a part of the soft segment during polyurethane synthesis and physical properties as well as water permeability of resulting copolymer were studied in regard to PDMS content. Increase in PDMS content led to increase of microphase separation of the copolymer and corresponding increase in elastic modulus. Surface energy of the polymer was decreased by incorporating PDMS compared to unmodified PEU. PDMS in copolymer formed a hydrophobic surface which caused reduction in water permeability and water uptake of the membranes. Thus, PDMS containing polyurethane with its potent water resistant properties demonstrated a great promise for use as an implantable encapsulation material.

Development of electrochemical biosensors based on sol-gel enzyme encapsulation and protective polymer membranes.

Protective polymer coatings have been used to enhance the retention of enzymes in sol-gel films as immobilisation phases in electrochemical biosensors. Carbon film electrodes were electrochemically modified with poly(neutral red) (PNR). These electrodes were coated with oxysilane sol-gels incorporating glucose oxidase and an outer coating of carboxylated PVC (CPVC) or polyurethane (PU), with and without Aliquat-336 or isopropyl myristate (IPM) plasticizer, was applied. The biosensors were characterised electrochemically using cyclic voltammetry and amperometry, electrochemical impedance spectroscopy and scanning electron microscopy. Impedance spectra showed that the electrode surface is most active when the sol-gel-GOx layer is not covered with a membrane. However, membranes without plasticizer extend the lifetime of the biosensor to more than 2 months when PU is used as an outer membrane. The linear range of the biosensors was found to be 0.05-0.50 mM of glucose and the biosensor with PU outer membrane exhibited higher sensitivity (ca.117 nA mM(-1)) in the region of linear response than that with CPVC. The biosensors were applied to glucose measurement in natural samples of commercial orange juice.

Assessment of tissue scaffold degradation using electrochemical techniques.

Degradation of a commercially available collagen-glycosaminoglycan dermal equivalent matrix was studied using electrochemical techniques. Degradation was accelerated by exposure to gamma radiation followed by storage at elevated temperatures or exposure to enzymes. The time-dependent diffusion of a small, electrochemically active, molecular probe, potassium ferrocyanide, through the matrix was monitored via changes in the oxidation peak currents of cyclic voltammograms. These measurements were made using a two-compartment diffusion chamber with the sample positioned well away from the working electrodes and a single-compartment electrode cell where the matrix was in direct contact with the working electrode. The relative merits of these two approaches are considered. Regardless of the approach chosen, amperometry appears well suited to monitoring progressive diffusivity changes through mechanically weak porous structures subject to different solution environments.

Challenges for successful implantation of biofuel cells.

There is a growing interest in the design and engineering of operational biofuel cells that can be implanted. This review highlights the recent progress in the electrochemistry of biofuel cell technologies, but with a particular emphasis on the medical and physiological aspects that impact the biocompatibility of biofuel cells operating inside a living body. We discuss the challenge of supplying power to implantable medical devices, with regard to the limitations of lithium battery technology and why implantable biofuel cells can be a promising alternative to provide the levels of power required for medical devices. In addition to the challenge of designing a biofuel cell that provides a stable level of sufficient power, the review highlights the biocompatibility and biofouling problems of implanting a biofuel cell that have a major impact on the availability of the substrates inside body that provide fuel for the biofuel cell. These physiological challenges and associated ethical considerations are essential to consider for biofuel cells that are designed to be implanted for long-term operation inside a living animal and eventually to human clinical applications.

Bifunctional aptamer-mediated catalytic hairpin assembly for the sensitive and homogenous detection of rare cancer cells.

The presence of cancer cells in body fluids confirms the occurrence of metastasis and guides treatment. A simple, fast, and homogeneous fluorescent method was developed to detect cancer cells based on catalytic hairpin assembly (CHA) and bifunctional aptamers. The bifunctional aptamer had a recognition domain for binding to target cancer cells and an initiator domain for triggering the CHA reaction. In the presence of target cells, the bifunctional aptamer was released from the inhibitor and initiated a cascade reaction of assembly and disassembly of the hairpins. Separation of the fluorophores from the quenchers produced fluorescence signals. The proposed strategy showed high specificity for discriminating normal cells and leukocytes, and the detection limit was 10 cells/mL, which was lower than that of previous aptasensors. This assay was further tested using four kinds of clinical samples spiked with target cells to confirm its applicability. We developed a simple, rapid, and cost-effective method for the detection of cancer cells that did not require purification, and the approach holds great potential for bioanalysis and early diagnosis.

Preparation, characterization and applications of ultrathin cellulose acetate Langmuir-Blodgett films.

The preparation and characterization of mono- and multilayers of cellulose acetate (CA) Langmuir-Blodgett films on indium tin oxide and gold surfaces were studied in detail for the first time. These layers were characterized by their thickness, wettability, morphology and structure using various surface techniques. The thickness of a monolayer of CA based on XPS measurement was one nanometre. Multilayers of CA Langmuir films were homogeneously transferred onto solid surfaces. The permeation of different molecules across these films was studied using electrochemistry in various redox solutions. Our findings suggest that a membrane like structure is formed, which is less permeable as the number of layers increases. Finally, potential applications of these ultrathin films as supports for accommodating biomolecules or metal nanoparticles are presented.

Effective diffusion coefficient determination within cylindrical granules of adsorbents using a direct simulation method.

Analytical expressions for solute adsorption kinetics within porous carbon cylindrical granules of adsorbents with a one point formula for effective diffusion coefficient determination are available based on the assumption that solute transport is the rate limiting step and that it follows Fick's Second Law. Here the first practical application of this theory is provided with an initial, estimated diffusion coefficient refined by fitting calculated kinetic adsorption curves to experimental data determined for activated carbons. In an ideal experiment, experimental error (noise) is negligible, and no data refinement is needed. However, real experimental data are always more or less noise contaminated. Where such noise is significant, a simulation method offers the best value for effective diffusion coefficient. For this specific system, surface modification, pH and temperature effects on adsorption kinetics were analysed quantitatively as a basis of determining effective diffusion coefficients through the porous structure.

Characterisation of the nanoporous structure of collagen-glycosaminoglycan hydrogels by freezing-out of bulk and bound water.

The nanoporous structure of collagen-glycosaminoglycan (CG) hydrogels was studied using 1H NMR spectroscopy and thermally stimulated depolarisation (TSD) current with layer-by-layer freezing-out of bulk and interfacial water. The depression of the freezing point of water is related to the size of the nanopore, to which it is confined. Changes in the Gibbs free energy of the unfrozen interfacial water are related to the amount of bound water in the hydrogel matrix and to the re-arrangement of the 3D network structure of the biopolymer. Analysis of the thermodynamic properties of bulk and interfacial water using the layer-by-layer freezing-out technique combined with NMR and TSDC provides valuable information about the structural features of CG hydrogels that can be used for characterisation of different types of hydrogels and soft tissue scaffolds, artificial skin substitutes and other biomaterials.

Culture of human keratinocytes on polypyrrole-based conducting polymers.

Variously loaded polypyrrole films, including those containing proteins and polysaccharides, were prepared on gold-coated polycarbonate coverslips. The characteristics of human keratinocytes were studied on these films by microscopy, biochemical assays, and immunocytochemistry. We found keratinocyte viability to be load dependent. For chloride, polyvinyl sulphate, dermatan sulphate, and collagen-loaded polypyrrole films, keratinocyte viability as assessed by the AlamarBlue assay was respectively 47.22, 60.43, 87.71, and 22.65% of tissue culture polystyrene controls after 5 days. This was found to require a previously unreported polymer washing step prior to cell seeding due to the observed toxicity of untreated films. In the case of bare polycarbonate and gold substrates, viability was respectively 75.44 and 61.04% of tissue culture polystyrene controls after 5 days. Keratinocytes stained positive for PCNA (proliferation), K10 (suprabasal differentiation), and K16 (hyperproliferation) markers although cell morphology was poor for organotypical cultures on dermatan- loaded polypyrrole compared with de-epidermalized dermis. From our studies, we concluded that optimized polypyrrole films adequately support keratinocyte growth in submerged cultures with some improvements needed for organotypical cultures. Polypyrrole composites are attractive candidates for tissue-engineering applications since they may incorporate biomolecules and are electrically addressable with the potential to both direct and report on cell activity.