Monitoring systems and quantitative measurement of biomolecules for the management of trauma.

Continued high morbidity and complications due to trauma related hemorrhage underscores the fact that our understanding of the detailed molecular events of trauma are inadequate to bring life-saving changes to practice. The current state of efficacy and advances in biomedical microdevice technology for trauma diagnostics concerning hemorrhage and hemorrhagic shock was considered with respect to vital signs and metabolic biomarkers. Tachycardia and hypotension are markers of hemorrhagic shock in decompensated trauma patients. Base deficit has been predicative of injury severity at hospital admission. Tissue oxygen saturation has been predicative of onset of multiple organ dysfunction syndrome. Blood potassium levels increase with onset of hemorrhagic shock. Lactate is a surrogate for tissue hypoxia and its clearance predicts mortality. Triage glucose measurements have been shown to be specific in predicting major injuries. No vital sign has yet to be proven effective as an independent predictor of trauma severity. Point of care (POC) devices allow for rapid results, easy sample preparation and processing, small sample volumes, small footprint, multifunctional analysis, and low cost. Advances in the field of in-vivo biosensors has provided a much needed platform by which trauma related metabolites can be monitored easily, rapidly and continuously. Multi-analyte monitoring biosensors have the potential to explore areas still undiscovered in the realm of trauma physiology.

Biomimetic hydrogels gate transport of calcium ions across cell culture inserts.

Control of the in vitro spatiotemporal availability of calcium ions is one means by which the microenvironments of hematopoietic stem cells grown in culture may be reproduced. The effects of cross-linking density on the diffusivity of calcium ions through cell culture compatible poly(2-hydroxyethyl methacrylate) [poly(HEMA)]-based bioactive hydrogels possessing 1.0 mol% 2-methacryloyloxyethyl phosphorylcholine (MPC), 5 mol% N,N-(dimethylamino)ethylmethacrylate (DMAEMA) and ca. 17 mol% n-butyl acrylate (n-BA) have been investigated to determine if varying cross-link density is a viable approach to controlling transport of calcium across hydrogel membranes. Cross-linking density was varied by changing the composition of cross-linker, tetraethyleneglycol diacrylate (TEGDA). The hydrogel membranes were formed by sandwich casting onto the external surface of track-etched polycarbonate membranes (T = 10 µm, φ = 0.4 µm pores) of cell culture inserts, polymerized in place by UV light irradiation and immersed in buffered (0.025 HEPES, pH 7.4) 0.10 M calcium chloride solution. The transport of calcium ions across the hydrogel membrane was monitored using a calcium ion selective electrode set within the insert. Degree of hydration (21.6 ± 1.0%) and void fraction were found to be constant across all cross-linking densities. Diffusion coefficients, determined using time-lag analysis, were shown to be strongly dependent on and to exponentially decrease with increasing cross-linking density. Compared to that found in buffer (2.0-2.5 × 10⁻6 cm²/s), diffusion coefficients ranged from 1.40 × 10⁻6 cm²/s to 1.80 × 10⁻7 cm²/s and tortuosity values ranged from 1.7 to 10.0 for the 1 and 12 mol% TEGDA cross-linked hydrogels respectively. Changes in tortuosity arising from variations in cross-link density were found to be the primary modality for controlling diffusivity through novel n-BA containing poly(HEMA)-based bioactive hydrogels.

On the intersection of molecular bioelectronics and biosensors: 20 Years of C3B.

Formed in 2000 at Virginia Commonwealth University, the Center for Bioelectronics, Biosensors and Biochips (C3B®) has subsequently been located at Clemson University and at Texas A&M University. Established as an industry-university collaborative center of excellence, the C3B has contributed new knowledge and technology in the areas of i) molecular bioelectronics, ii) responsive polymers, iii) multiplexed biosensor systems, and iv) bioelectronic biosensors. Noteworthy contributions in these areas include i) being the first to report direct electron transfer of oxidoreductase enzymes enabled by single walled carbon nanotubes and colloidal clays, ii) the molecular level integration of inherently conductive polymers with bioactive hydrogels using bi-functional monomers such as poly(pyrrole-co-3-pyrrolylbutyrate-conj-aminoethylmethacrylate) [PyBA-conj-AEMA] and 3-(1-ethyl methacryloylate)aniline to yield hetero-ladder electroconductive hydrogels, iii) the development of a multi-analyte physiological status monitoring biochip, and iv) the development of a bioanalytical Wien-bridge oscillator for the fused measurement to lactate and glucose. The present review takes a critical look of these contributions over the past 20 years and offers some perspective on the future of bioelectronics-based biosensors and systems. Particular attention is given to multiplexed biosensor systems and data fusion for rapid decision making.

Biofabrication Using Pyrrole Electropolymerization for the Immobilization of Glucose Oxidase and Lactate Oxidase on Implanted Microfabricated Biotransducers.

The dual responsive Electrochemical Cell-on-a-Chip Microdisc Electrode Array (ECC MDEA 5037) is a recently developed electrochemical transducer for use in a wireless, implantable biosensor system for the continuous measurement of interstitial glucose and lactate. Fabrication of the biorecognition membrane via pyrrole electropolymerization and both in vitro and in vivo characterization of the resulting biotransducer is described. The influence of EDC-NHS covalent conjugation of glucose oxidase with 4-(3-pyrrolyl) butyric acid (monomerization) and with 4-sulfobenzoic acid (sulfonization) on biosensor performance was examined. As the extent of enzyme conjugation was increased sensitivity decreased for monomerized enzymes but increased for sulfonized enzymes. Implanted biotransducers were examined in a Sprague-Dawley rat hemorrhage model. Resection after 4 h and subsequent in vitro re-characterization showed a decreased sensitivity from 0.68 (±0.40) to 0.22 (±0.17) µA·cm-2·mM-1, an increase in the limit of detection from 0.05 (±0.03) to 0.27 (±0.27) mM and a six-fold increase in the response time from 41 (±18) to 244 (±193) s. This evidence reconfirms the importance of biofouling at the bio-abio interface and the need for mitigation strategies to address the foreign body response.

Amperometric glucose biosensor based on electroconductive hydrogels.

Fabrication of an enzyme amperometric biosensor for glucose via electropolymerization of pyrrole in the presence of glucose oxidase onto a hydrogel coated platinum electrode is hereby established as a viable biotransducer fabrication method. Platinum micro- (φ=25 µm) and macro- (φ=100 µm) electrodes were electrochemically activated and chemically modified with 3-aminopropyl-trimethoxysilane (APTMS), functionalized with acryloyl(polyethyleneglycol)-N-hydroxysuccinamide (ACRL-PEG-NHS), dipped into a polyHEMA based hydrogel cocktail and UV cross-linked. Electropolymerization of Py in the presence of GOx produced glucose responsive biotransducers that showed; (i) a 4-fold reduction in sensitivity compared with directly electropolymerized PPy films, (ii) an electropolymerization charge density dependence of biotransducer sensitivity and enzyme activity that was maximal at 1.0 mC/cm(2) with an apparent K(M) of 33 mM, (iii) interference screening of ascorbic acid and (iv) a temporal increase in sensitivity with storage over a 17 days period. This method has the ability to precisely and quantitatively add enzyme catalytic bioactivity to metal or semiconductor biointerfaces for applications in biosensors, bioelectronics and bionics.

The effect of the physicochemical properties of bioactive electroconductive hydrogels on the growth and proliferation of attachment dependent cells.

The physicochemical properties of soft electrode materials for the abio-bio interface of advanced biosensors and next generation bionic devices in the form of electroconductive hydrogels (ECH) of interpenetrating networks of polypyrrole formed within poly(hydroxyethylmethacrylate)-based hydrogels were examined. The 1.5 mol% UV-crosslinked tetraethyleneglycol diacrylate (TEGDA) (step 1) poly(HEMA) and the electropolymerized (step 2) polypyrrole co-networks were covalently joined by the inclusion of a bifunctional monomer (1.5 mol%), 2-methacryloyloxyethyl-4(3-pyrrolyl)butanate (MPB) that served to covalently link the two networks. The optical absorbance, degree of hydration, the frequency dependent electrical impedance and the elastic modulus were examined as a function of electropolymerization charge density (step 2) (1-900 mC/cm(2)) used to prepare the linked, interpenetrating co-networks. The absorption at 430 nm showed a monotonic increase with electropolymerization charge density and correlated with the increase in elastic modulus [56 (± 32)-499 (± 293) kPa], the decrease in % hydration (68-0%) and the decrease in membrane electrical resistance. Polypyrrole (PPy) grows initially from the gel-electrode interface to fill voids within the hydrogel and ultimately onto the surface of the hydrogel. Growth of attachment dependent Rhabdomyosarcoma (RMS13) and pheochromocytoma (PC 12) cells reflects this evolution, showing an increase to a maximal value and then to decrease again at high electropolymerization charge density.

Bioactive electroconductive hydrogels: the effects of electropolymerization charge density on the storage stability of an enzyme-based biosensor.

Electrode-supported hydrogels were conferred with the biospecificity of enzymes during the process of electropolymerization to give rise to a class of bioactive, stimuli-responsive co-joined interpenetrating networks of inherently conductive polymers and highly hydrated hydrogels. Glucose responsive biotransducers were prepared by potentiostatic electropolymerization [750 mV vs. Ag/AgCl (3 M KCl)] of pyrrole at Poly(hydoxyethyl methacrylate)-based hydrogel-coated Pt micro-electrodes (Φ = 100 µm) from aqueous solutions of pyrrole and glucose oxidase (GOx; 0.4 M pyrrole, 1.0 mg/ml GOx) to 1.0 and 10.0 mC/cm². Polypyrrole was them over-oxidized by cyclic voltammetry (0-1.2 V vs. Ag/AgCl, 40 cycles in PBKCl, pH = 7.0). Biotransducers were stored at 4 °C in PBKCl for up to 18 days. Amperometric dose-response at 0.4 V vs. Ag/AgCl followed by Lineweaver-Burk analysis produced enzyme kinetic parameters as a function of electropolymerization charge density and storage time. Apparent Michaelis constant (K (Mapp)) increased from 18.6-152.0 mM (1.0 mC/cm²) and from 2.7-6.1 mM (10.0 mC/cm²). Biotransducer sensitivity increased to 21.2 nA/mM after 18 days and to 12.8 pA/mM after 10 days for the 1.0 and 10.0 mC/cm² membranes, respectively. Maximum current, I (max), also increased over time to 2.7 nA (1.0 mC/cm²) and to 170 pA (10.0 mC/cm²). Electropolymerization of polypyrrole is shown to be an effective means for imparting bioactivity to a hydrogel-coated microelectrode. GOx was shown to be stabilized and to increase activity over time within the electroconductive hydrogel.

Implantable enzyme amperometric biosensors.

The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems.