Eyeglasses-based tear biosensing system: Non-invasive detection of alcohol, vitamins and glucose.

We report on a wearable tear bioelectronic platform, integrating a microfluidic electrochemical detector into an eyeglasses nose-bridge pad, for non-invasive monitoring of key tear biomarkers. The alcohol-oxidase (AOx) biosensing fluidic system allowed real-time tear collection and direct alcohol measurements in stimulated tears, leading to the first wearable platform for tear alcohol monitoring. Placed outside the eye region this fully wearable tear-sensing platform addresses drawbacks of sensor systems involving direct contact with the eye as the contact lenses platform. Integrating the wireless electronic circuitry into the eyeglasses frame thus yielded a fully portable, convenient-to-use fashionable sensing device. The tear alcohol sensing concept was demonstrated for monitoring of alcohol intake in human subjects over multiple drinking courses, displaying good correlation to parallel BAC measurements. We also demonstrate for the first time the ability to monitor tear glucose outside the eye and the utility of wearable devices for monitoring vitamin nutrients in connection to enzymatic flow detector and rapid voltammetric scanning, respectively. These developments pave the way to build an effective eyeglasses system capable of chemical tear analysis.

Wearable biosensors for healthcare monitoring.

Wearable biosensors are garnering substantial interest due to their potential to provide continuous, real-time physiological information via dynamic, noninvasive measurements of biochemical markers in biofluids, such as sweat, tears, saliva and interstitial fluid. Recent developments have focused on electrochemical and optical biosensors, together with advances in the noninvasive monitoring of biomarkers including metabolites, bacteria and hormones. A combination of multiplexed biosensing, microfluidic sampling and transport systems have been integrated, miniaturized and combined with flexible materials for improved wearability and ease of operation. Although wearable biosensors hold promise, a better understanding of the correlations between analyte concentrations in the blood and noninvasive biofluids is needed to improve reliability. An expanded set of on-body bioaffinity assays and more sensing strategies are needed to make more biomarkers accessible to monitoring. Large-cohort validation studies of wearable biosensor performance will be needed to underpin clinical acceptance. Accurate and reliable real-time sensing of physiological information using wearable biosensor technologies would have a broad impact on our daily lives.

Wearable Bioelectronics: Enzyme-Based Body-Worn Electronic Devices.

In this Account, we detail recent progress in wearable bioelectronic devices and discuss the future challenges and prospects of on-body noninvasive bioelectronic systems. Bioelectronics is a fast-growing interdisciplinary research field that involves interfacing biomaterials with electronics, covering an array of biodevices, encompassing biofuel cells, biosensors, ingestibles, and implantables. In particular, enzyme-based bioelectronics, built on diverse biocatalytic reactions, offers distinct advantages and represents a centerpiece of wearable biodevices. Such wearable bioelectronic devices predominately rely on oxidoreductase enzymes and have already demonstrated considerable promise for on-body applications ranging from highly selective noninvasive biomarker monitoring to epidermal energy harvesting. These systems can thus greatly increase the analytical capability of wearable devices from the ubiquitous monitoring of mobility and vital signs, toward the noninvasive analysis of important chemical biomarkers. Wearable enzyme electrodes offer exciting opportunities to a variety of areas, spanning from healthcare, sport, to the environment or defense. These include real-time noninvasive detection of biomarkers in biofluids (such as sweat, saliva, interstitial fluid and tears), and the monitoring of environmental pollutants and security threats in the immediate surrounding of the wearer. Furthermore, the interface of enzymes with conducting flexible electrode materials can be exploited for developing biofuel cells, which rely on the bioelectrocatalytic oxidation of biological fuels, such as lactate or glucose, for energy harvesting applications. Crucial for such successful application of enzymatic bioelectronics is deep knowledge of enzyme electron-transfer kinetics, enzyme stability, and enzyme immobilization strategies. Such understanding is critical for establishing efficient electrical contacting between the redox enzymes and the conducting electrode supports, which is of fundamental interest for the development of robust and efficient bioelectronic platforms. Furthermore, stretchable and flexible bioelectronic platforms, with mechanical properties similar to those of biological tissues, are essential for handling the rigors of on-body operation. As such, special attention must be given to changes in the behavior of enzymes due to the uncontrolled conditions of on-body operation (including diverse outdoor activities and different biofluids), for maintaining the attractive performance that these bioelectronics devices display in controlled laboratory settings. Therefore, a focus of this Account is on interfacing biocatalytic layers onto wearable electronic devices for creating efficient and stable on-body electrochemical biosensors and biofuel cells. With proper attention to key challenges and by leveraging the advantages of biocatalysis, electrochemistry, and flexible electronics, wearable bioelectronic devices could have a tremendous impact on diverse biomedical, fitness, and defense fields.

Simultaneous Monitoring of Sweat and Interstitial Fluid Using a Single Wearable Biosensor Platform.

The development of wearable biosensors for continuous noninvasive monitoring of target biomarkers is limited to assays of a single sampled biofluid. An example of simultaneous noninvasive sampling and analysis of two different biofluids using a single wearable epidermal platform is demonstrated here. The concept is successfully realized through sweat stimulation (via transdermal pilocarpine delivery) at an anode, alongside extraction of interstitial fluid (ISF) at a cathode. The system thus allows on-demand, controlled sampling of the two epidermal biofluids at the same time, at two physically separate locations (on the same flexible platform) containing different electrochemical biosensors for monitoring the corresponding biomarkers. Such a dual biofluid sampling and analysis concept is implemented using a cost-effective screen-printing technique with body-compliant temporary tattoo materials and conformal wireless readout circuits to enable real-time measurement of biomarkers in the sampled epidermal biofluids. The performance of the developed wearable device is demonstrated by measuring sweat-alcohol and ISF-glucose in human subjects consuming food and alcoholic drinks. The different compositions of sweat and ISF with good correlations of their chemical constituents to their blood levels make the developed platform extremely attractive for enhancing the power and scope of next-generation noninvasive epidermal biosensing systems.

Wearable Electrochemical Alcohol Biosensors.

The rapid development of wearable sensing platforms in recent years has led to an array of viable monitoring applications for various target analytes. As a significant biomarker with high impact in diverse areas, the reliable on-body detection and continuous monitoring of alcohol has become a focus of many such systems. Currently, several commercial sensing platforms are available that are capable of transdermal monitoring of alcohol consumption using insensible sweat. Drawbacks of existing alcohol sensing platforms that apply this sensing strategy have led to efforts in developing wearable biosensors capable of real-time alcohol detection in sampled biofluids such as sensible sweat and skin interstitial fluid. This review discusses the current trends in wearable electrochemical alcohol biosensing and highlights recent advances in such systems toward continuous, real-time monitoring of alcohol consumption. Our perspective on this important field is given with an outlook on the future of wearable electrochemical alcohol biosensors.

Wearable non-invasive epidermal glucose sensors: A review.

The growing recent interest in wearable and mobile technologies has led to increased research efforts toward development of non-invasive glucose monitoring platforms. Continuous glucose monitoring addresses the limitations of finger-stick blood testing and provides the opportunity for optimal therapeutic interventions. This article reviews recent advances and challenges toward the development of non-invasive epidermal electrochemical glucose sensing systems. Recent reports claim success in glucose monitoring in human subjects using skin-worn electrochemical sensors. Such epidermal electrochemical biosensors obviate the disadvantages of minimally-invasive subcutaneous glucose biosensors and offer promise for improved glycemic control. The ability of such systems to monitor glucose non-invasively offers an attractive route toward advancing the management of diabetes and achieving improved glycemic control. However, realizing the potential diagnostic impact of these new epidermal sensing strategies would require extensive efforts toward addressing key technological challenges and establishing a reliable correlation to gold standard blood glucose meters.

Epidermal Microfluidic Electrochemical Detection System: Enhanced Sweat Sampling and Metabolite Detection.

Despite tremendous recent efforts, noninvasive sweat monitoring is still far from delivering its early analytical promise. Here, we describe a flexible epidermal microfluidic detection platform fabricated through hybridization of lithographic and screen-printed technologies, for efficient and fast sweat sampling and continuous, real-time electrochemical monitoring of glucose and lactate levels. This soft, skin-mounted device judiciously merges lab-on-a-chip and electrochemical detection technologies, integrated with a miniaturized flexible electronic board for real-time wireless data transmission to a mobile device. Modeling of the device design and sweat flow conditions allowed optimization of the sampling process and the microchannel layout for achieving attractive fluid dynamics and rapid filling of the detection reservoir (within 8 min from starting exercise). The wearable microdevice thus enabled efficient natural sweat pumping to the electrochemical detection chamber containing the enzyme-modified electrode transducers. The fabricated device can be easily mounted on the epidermis without hindrance to the wearer and displays resiliency against continuous mechanical deformation expected from such epidermal wear. Amperometric biosensing of lactate and glucose from the rapidly generated sweat, using the corresponding immobilized oxidase enzymes, was wirelessly monitored during cycling activity of different healthy subjects. This ability to monitor sweat glucose levels introduces new possibilities for effective diabetes management, while similar lactate monitoring paves the way for new wearable fitness applications. The new epidermal microfluidic electrochemical detection strategy represents an attractive alternative to recently reported colorimetric sweat-monitoring methods, and hence holds considerable promise for practical fitness or health monitoring applications.

Wearable Ring-Based Sensing Platform for Detecting Chemical Threats.

This work describes a wireless wearable ring-based multiplexed chemical sensor platform for rapid electrochemical monitoring of explosive and nerve-agent threats in vapor and liquid phases. The ring-based sensor system consists of two parts: a set of printed electrochemical sensors and a miniaturized electronic interface, based on a battery-powered stamp-size potentiostat, for signal processing and wireless transmission of data. A wide range of electrochemical capabilities have thus been fully integrated into a 3D printed compact ring structure, toward performing fast square-wave voltammetry and chronoamperometric analyses, along with interchangeable screen-printed sensing electrodes for the rapid detection of different chemical threats. High analytical performance is demonstrated despite the remarkable miniaturization and integration of the ring system. The attractive capabilities of the wearable sensor ring system have been demonstrated for sensitive and rapid voltammetric and amperometric monitoring of nitroaromatic and peroxide explosives, respectively, along with amperometric biosensing of organophosphate (OP) nerve agents. Such ability of the miniaturized wearable sensor ring platform to simultaneously detect multiple chemical threats in both liquid and vapor phases and alert the wearer of such hazards offers considerable promise for meeting the demands of diverse defense and security scenarios.

Bactericidal Specificity and Resistance Profile of Poly(Quaternary Ammonium) Polymers and Protein-Poly(Quaternary Ammonium) Conjugates.

Antibacterial polymers are potentially powerful biocides that can destroy bacteria on contact. Debate in the literature has surrounded the mechanism of action of polymeric biocides and the propensity for bacteria to develop resistance to them. There has been particular interest in whether surfaces with covalently coupled polymeric biocides have the same mechanism of action and resistance profile as similar soluble polymeric biocides. We designed and synthesized a series of poly(quaternary ammonium) polymers, with tailorable molecular structures and architectures, to engineer their antibacterial specificity and their ability to delay the development of bacterial resistance. These linear poly(quaternary ammonium) homopolymers and block copolymers, generated using atom transfer radical polymerization, had structure-dependent antibacterial specificity toward Gram positive and negative bacterial species. When single block copolymers contained two polymer segments of differing antibacterial specificity, the polymer combined the specificities of its two components. Nanoparticulate human serum albumin-poly(quaternary ammonium) conjugates of these same polymers, synthesized via "grafting from" atom transfer radical polymerization, were strongly biocidal and also exhibited a marked decrease in the rate of bacterial resistance development relative to linear polymers. These protein-biocide conjugates mimicked the behavior of surface-presented polycationic biocides rather than their nonproteinaceous counterparts.

Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates.

The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.

Polymer-based protein engineering grown ferrocene-containing redox polymers improve current generation in an enzymatic biofuel cell.

Enzymatic biofuel cells (EBFCs) are capable of generating electricity from physiologically present fuels making them promising power sources for the future of implantable devices. The potential application of such systems is limited, however, by inefficient current generation. Polymer-based protein engineering (PBPE) offers a unique method to tailor enzyme function through tunable modification of the enzyme surface with functional polymers. In this study, we report on the modification of glucose oxidase (GOX) with ferrocene-containing redox polymers to increase current generation efficiency in an enzyme-modified anode. Poly(N-(3-dimethyl(ferrocenyl)methylammonium bromide)propyl acrylamide) (pFcAc) was grown from covalently attached, water-soluble initiator molecules on the surface of GOX in a "grafting-from" approach using atom transfer radical polymerization (ATRP). The covalently-coupled ferrocene-containing polymers on the enzyme surface promoted the effective "wiring" of the GOX active site to an external electrode. The resulting GOX-pFcAc conjugates generated over an order of magnitude increase in current generation efficiency and a 4-fold increase in maximum EBFC power density (≈1.7µWcm(-2)) with similar open circuit voltage (0.27V) compared to native GOX when physically adsorbed onto paddle-shaped electrodes made up of electrospun polyacrylonitrile fibers coated with gold nanoparticles and multi-wall carbon nanotubes. The formation of electroactive enzyme-redox polymer conjugates using PBPE represents a powerful new tool for the improvement of mediated enzyme-based bioelectronics without the need for free redox mediators or anode/cathode compartmentalization.

Membrane/mediator-free rechargeable enzymatic biofuel cell utilizing graphene/single-wall carbon nanotube cogel electrodes.

Enzymatic biofuel cells (EBFCs) utilize enzymes to convert chemical energy present in renewable biofuels into electrical energy and have shown much promise in the continuous powering of implantable devices. Currently, however, EBFCs are greatly limited in terms of power and operational stability with a majority of reported improvements requiring the inclusion of potentially toxic and unstable electron transfer mediators or multicompartment systems separated by a semipermeable membrane resulting in complicated setups. We report on the development of a simple, membrane/mediator-free EBFC utilizing novel electrodes of graphene and single-wall carbon nanotube cogel. These cogel electrodes had large surface area (∼ 800 m(2) g(-1)) that enabled high enzyme loading, large porosity for unhindered glucose transport and moderate electrical conductivity (∼ 0.2 S cm(-1)) for efficient charge collection. Glucose oxidase and bilirubin oxidase were physically adsorbed onto these electrodes to form anodes and cathodes, respectively, and the EBFC produced power densities up to 0.19 mW cm(-2) that correlated to 0.65 mW mL(-1) or 140 mW g(-1) of GOX with an open circuit voltage of 0.61 V. Further, the electrodes were rejuvenated by a simple wash and reloading procedure. We postulate these porous and ultrahigh surface area electrodes will be useful for biosensing applications, and will allow reuse of EBFCs.

Enzyme catalytic efficiency: a function of bio-nano interface reactions.

Biocatalyst immobilization onto carbon-based nanosupports has been implemented in a variety of applications ranging from biosensing to biotransformation and from decontamination to energy storage. However, retaining enzyme functionality at carbon-based nanosupports was challenged by the non-specific attachment of the enzyme as well as by the enzyme-enzyme interactions at this interface shown to lead to loss of enzyme activity. Herein, we present a systematic study of the interplay reactions that take place upon immobilization of three pure enzymes namely soybean peroxidase, chloroperoxidase, and glucose oxidase at carbon-based nanosupport interfaces. The immobilization conditions involved both single and multipoint single-type enzyme attachment onto single and multi-walled carbon nanotubes and graphene oxide nanomaterials with properties determined by Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray analysis (EDX), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Our analysis showed that the different surface properties of the enzymes as determined by their molecular mapping and size work synergistically with the carbon-based nanosupports physico-chemical properties (i.e., surface chemistry, charge and aspect ratios) to influence enzyme catalytic behavior and activity at nanointerfaces. Knowledge gained from these studies can be used to optimize enzyme-nanosupport symbiotic reactions to provide robust enzyme-based systems with optimum functionality to be used for fermentation, biosensors, or biofuel applications.