A Conductive Microcavity Created by Assembly of Carbon Nanotube Buckypapers for Developing Electrochemically Wired Enzyme Cascades.

We describe the creation of a conductive microcavity based on the assembly of two pieces of carbon nanotube buckypaper for the entrapment of two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), as well as a redox mediator: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). The hollow electrode, employing GOx, HRP, and the mediator, as an electrochemical enzyme cascade model, is utilized for glucose sensing at a potential of 50 mV vs. Ag/AgCl. This bienzyme electrode demonstrates the ability to oxidize glucose by GOx and subsequently convert H2O2 to water via the electrical wiring of HRP by ABTS. Different redox mediators (ABTS, potassium hexacyanoferrate (III), and hydroquinone) are tested for HRP wiring, with ABTS being the best candidate for the electroenzymatic reduction of H2O2. To demonstrate the possibility to optimize the enzyme cascade configuration, the enzyme ratio is studied with 1 mg HRP combined with variable amounts of GOx (1-4 mg) and 2 mg GOx combined with variable amounts of HRP (0.5-2 mg). The bienzyme electrode shows continuous operational stability for over a week and an excellent storage stability in phosphate buffer, with a decay of catalytic current by only 29% for 1 mM glucose after 100 days.

Exploring the role of Mn2+ in the structure, magnetic properties, and radar absorption performance of MnxFe3-xO4-DEA/MWCNT nanocomposites.

Iron oxide/carbon-based nanocomposites are known as an ideal combination of magnetic-conductive materials that were recently developed in radar absorption application; one example is the Fe3O4/multiwalled carbon nanotubes (MWCNTs). In this study, we try to boost their radar absorption ability by Mn-ion doping. Mn is an appropriate Fe substitute that is predicted to alter the magnetic properties and enhance the conductivity, which are crucial to developing their radar absorption properties. Diethylamine (DEA) is also used as a capping agent to improve the size and shape of the nanocomposite. In this study, a MnxFe3-xO4-DEA/MWCNT nanocomposite is successfully prepared by the coprecipitation method using a variation of x = 0, 0.25, 0.5, 0.75, and 1. We found that the sample's magnetic saturation (Ms) decreases, while the reflection loss (RL) increases with increasing the molar fraction of Mn. The enhancement of the radar wave absorption in the sample is dominated by dielectric losses due to the increase of electrical conductivity and interfacial polarization with the addition of Mn in the nanocomposites. We believe that our finding could shed light on the role of doping elements to develop the radar absorption properties, and further pave the way for the real implementation of iron oxides/graphene-based nanocomposite as radar-absorbing materials (RAMs).

Glucose Oxidase/Egg White Protein Microparticles with a Redox Mediator for Glucose Biosensors on a Screen-Printed Electrode and a Decomposable Electrode.

Glucose oxidase (GOx) is a typical model enzyme used to create biosensors. Exploring a strategy to prepare ready-to-use functional enzymatic microparticles combining GOx and food-based proteins offers compelling advantages. However, no reports exist on the integration of egg white materials to synthesize functional biorecognition particles with glucose oxidation catalytic functions for electrochemical biosensors. Here, we demonstrate functional microparticles combining egg white proteins, GOx, and 9,10-phenanthrenequinone (PQ). The egg white proteins crosslink to form three-dimensional scaffolds to accommodate GOx and redox molecules. The PQ mediator enhances electron transfer between the electrode surface and the GOx enzyme's flavin adenine dinucleotides. The functional microparticles are directly applied to the printed electrode. The performance of these microparticles is evaluated using a screen-printed carbon nanotube (CNT)-modified electrode coated with GOx/PQ/egg white protein microparticles. The analytical performance of the system exhibits a linear range of 0.125-40 mM, with a maximum current (Imax) and a Michaelis-Menten constant (Km) being 0.2 µA and 4.6 mM, respectively. Additionally, a decomposable electrode composed of CNTs and edible oil conjugated with functional enzyme microparticles is shown to undergo degradation under gastric conditions. Utilizing food-based proteins to accommodate enzymes and to create redox-active microparticles for catalyzing glucose oxidation offers advantages in developing affordable and degradable bioelectrodes. This concept holds promise for advancing biocompatible electrodes in biosensor and bioelectronics applications.

Dual-Role Peptide with Capping and Cleavage Site Motifs in Nanoparticle-Based One-Pot Colorimetric and Electrochemical Protease Assay.

A new method for enzyme substrate assembly and its use in proteolytic enzyme assays with colorimetric and electrochemical detection is presented. The novelty of the method is the use of dual-function synthetic peptide containing both gold clustering and protease-sensitive moieties, which not only induces the simple formation of the peptide-decorated gold nanoparticle test substrates but also allows for the detection of proteolysis in the same batch. Protease-treated nanoparticles with a destabilized peptide shell became more prone to electroactivity, and thus, the model enzyme plasmin activity could be quantified with stripping square wave voltammetry analysis as well, giving an alternative method to conduct aggregation-based assays. Spectrophotometric and electrochemical calibration data proved to be linear within the 40-100 nM active enzyme concentration range, with possible extensions of the dynamic range by varying substrate concentration. The simple initial components and the ease of synthesis make the assay substrate preparation economic and easy to implement. The possibility of cross-check analytical results with two independent measurement techniques in the same batch greatly increases the applicability of the proposed system.

A wearable electrode based on copper nanoparticles for rapid determination of paraquat.

The application of copper-based nanoparticles synthesized via green synthesis and their integration with a wearable electrode is reported for designing a flexible catalytic electrode on a glove for onsite electroanalysis of paraquat. A copper precursor and an orange extract from Citrus reticulata are used to synthesize an economical electrocatalytic material for supporting the selective and sensitive detection of paraquat. The electrode yields multidimensional fingerprints due to two redox couples in a square wave voltammogram, corresponding to the presence of paraquat. The developed lab-on-a-finger sensor provides the fast electroanalysis of paraquat within 10 s, covering a wide range from 0.50 to 1000 µM, with a low detection limit down to 0.31 µM and high selectivity. It is also possible to use this sensor at a fast scan rate as high as 6 V s-1 (< 0.5 s for a scan). This wearable glove sensor allows the user to directly touch and analyze samples, such as surfaces of vegetables and fruits, to screen the contamination. It is envisioned that these glove-embedded sensors can be applied to the on-site analysis of food contamination and environments.

Redox-Mediated Gold Nanoparticles with Glucose Oxidase and Egg White Proteins for Printed Biosensors and Biofuel Cells.

Glucose oxidase (GOx)-based electrodes are important for bioelectronics, such as glucose sensors. It is challenging to effectively link GOx with nanomaterial-modified electrodes while preserving enzyme activity in a biocompatible environment. To date, no reports have used biocompatible food-based materials, such as egg white proteins, combined with GOx, redox molecules, and nanoparticles to create the biorecognition layer for biosensors and biofuel cells. This article demonstrates the interface of GOx integrated with egg white proteins on a 5 nm gold nanoparticle (AuNP) functionalized with a 1,4-naphthoquinone (NQ) and conjugated with a screen-printed flexible conductive carbon nanotube (CNT)-modified electrode. Egg white proteins containing ovalbumin can form three-dimensional scaffolds to accommodate immobilized enzymes and adjust the analytical performance. The structure of this biointerface prevents the escape of enzymes and provides a suitable microenvironment for the effective reaction. The bioelectrode's performance and kinetics were evaluated. Using redox-mediated molecules with the AuNPs and the three-dimensional matrix made of egg white proteins improves the transfer of electrons between the electrode and the redox center. By engineering the layer of egg white proteins on the GOx-NQ-AuNPs-mediated CNT-functionalized electrodes, we can modulate analytical performances such as sensitivity and linear range. The bioelectrodes demonstrate high sensitivity and can prolong the stability by more than 85% after 6 h of continuous operation. The use of food-based proteins with redox molecule-modified AuNPs and printed electrodes demonstrates advantages for biosensors and energy devices due to their small size, large surface area, and ease of modification. This concept holds a promise for creating biocompatible electrodes for biosensors and self-sustaining energy devices.

Screen-Printable Functional Nanomaterials for Flexible and Wearable Single-Enzyme-Based Energy-Harvesting and Self-Powered Biosensing Devices.

Developing flexible bioelectronics is essential to the realization of artificial intelligence devices and biomedical applications, such as wearables, but their potential is limited by sustainable energy supply. An enzymatic biofuel cell (BFC) is promising for power supply, but its use is limited by the challenges of incorporating multiple enzymes and rigid platforms. This paper shows the first example of screen-printable nanocomposite inks engineered for a single-enzyme-based energy-harvesting device and a self-powered biosensor driven by glucose on bioanode and biocathode. The anode ink is modified with naphthoquinone and multiwalled carbon nanotubes (MWCNTs), whereas the cathode ink is modified with Prussian blue/MWCNT hybrid before immobilizing with glucose oxidase. The flexible bioanode and the biocathode consume glucose. This BFC yields an open circuit voltage of 0.45 V and a maximum power density of 266 µW cm-2. The wearable device coupled with a wireless portable system can convert chemical energy into electric energy and detect glucose in artificial sweat. The self-powered sensor can detect glucose concentrations up to 10 mM. Common interfering substances, including lactate, uric acid, ascorbic acid, and creatinine, have no effect on this self-powered biosensor. Additionally, the device can endure multiple mechanical deformations. New advances in ink development and flexible platforms enable a wide range of applications, including on-body electronics, self-sustainable applications, and smart fabrics.

Advances in wearable electrochemical antibody-based sensors for cortisol sensing.

Cortisol is a crucial hormone involving many physiological processes. Hence, cortisol detection is essential. This review highlights the key progress made on wearable electrochemical sensors using antibodies. It covers the design, principle, and electroanalytical methodology for detecting cortisol noninvasively. This article also analyzes and collects the analytical performances of electrochemical cortisol sensors. The development of these sensors continues to face challenges such as biofouling, sample management, sensitivity, flexibility, stability, and recognition layer performance. It is also necessary to develop a sensitive electrode and material. This article also presents potential strategies for designing antibody electrodes and provides examples of sensing systems. Additionally, it discusses the challenges in translating research into practical applications.

Single-step salt-template-based scalable production of 2D carbon sheets heterostructured with nickel nanocatalysts for lowering overpotential of hydrogen evolution reaction.

Non-precious metals have been considered as suitable alternatives for high-performance hydrogen evolution reactions (HER). Although the incorporation of carbon substances is shown to improve the number of active sites, electron transfer pathways, and long-term stability, there have been rare reports on their single-step scalable production. Herein, we realize free-standing two-dimensional (2D) carbon sheets heterostructured with nickel (Ni) nanocatalysts by pyrolyzing ultrathin layers of acetate tetrahydrate (i.e. the single precursor for both Ni and C sources) over water-soluble salt crystals. Such a salt-templated methodology is environmentally friendly and readily scalable without the implementation of sophisticated equipment. The resulting 2D carbon sheets exhibit an average small thickness of âˆ¼ 3 nm and lateral dimensions with tens of micrometers, where a large number of nano-sized Ni particles with an average diameter of 14 nm are uniformly dispersed. Such 2D Ni-C sheets demonstrate a small overpotential of 111 mV at 10 mA/cm2 and a low Tafel slope of 86 mV/dec for HER in 1 M KOH, which is significantly improved over those of reported non-precious metals composited with carbon substances. This work offers new insight into the design and practical production of non-precious metal matrixes for economical HER.

A self-powered ingestible wireless biosensing system for real-time in situ monitoring of gastrointestinal tract metabolites.

Information related to the diverse and dynamic metabolite composition of the small intestine is crucial for the diagnosis and treatment of various diseases. However, our current understanding of the physiochemical dynamics of metabolic processes within the small intestine is limited due to the lack of in situ access to the intestinal environment. Here, we report a demonstration of a battery-free ingestible biosensing system for monitoring metabolites in the small intestine. As a proof of concept, we monitor the intestinal glucose dynamics on a porcine model. Battery-free operation is achieved through a self-powered glucose biofuel cell/biosensor integrated into a circuit that performs energy harvesting, biosensing, and wireless telemetry via a power-to-frequency conversion scheme using magnetic human body communication. Such long-term biochemical analysis could potentially provide critical information regarding the complex and dynamic small intestine metabolic profiles.

Tackling the challenges of developing microneedle-based electrochemical sensors.

Microneedles provide unique advantages in biomedical areas, particularly biosensing, because of their opportunities for minimally invasive and convenient detection. This review introduces concepts of electrochemical microneedle-based sensors for minimally invasive analysis together with insights into skin compartments for designing successful microneedles. We discuss requirements for developing microneedle-based biosensors, including electrical and electrochemical behaviors of materials (such as metals, nanomaterials, and conducting polymers) which are the key factors for sensitive biosensors. We further emphasize immobilization strategies to attach biorecognition elements to electrode materials. Moreover, we detail advanced techniques that require state-of-the-art fabrications and materials to realize porous, biodegradable, and antifouling materials to enhance biosensing performances. We also emphasize studies of cytotoxicity and damage to the skin to elucidate biocompatible potentials. Furthermore, we highlight the latest advances in microneedles published from 2019 to 2022. This review also includes an overview of key remaining obstacles and opportunities where developments will push the advances continuously. The conclusion and outlook on the status of laboratory and commercial availability are added. We prospect that microneedle sensors will be integrated into a closed-loop system biodevice for diverse applications, e.g., detection and therapy. This review discusses concepts of electrochemical microneedle-based sensors for minimally invasive monitoring. We discuss requirements including geometry, mechanical, electrical, electrochemical properties, and immobilization strategy. We illustrate advanced techniques and materials to enrich biosensor features, e.g., porosity, dissolvable ability, and anti-fouling capability. Concern of toxicity is highlighted. We also illustrate recent applications and outlooks of recently published study that exploits microneedle-based biosensors.

Lab on a body for biomedical electrochemical sensing applications: The next generation of microfluidic devices.

This chapter highlights applications of microfluidic devices toward on-body biosensors. The emerging application of microfluidics to on-body bioanalysis is a new strategy to establish systems for the continuous, real-time, and on-site determination of informative markers present in biofluids, such as sweat, interstitial fluid, blood, saliva, and tear. Electrochemical sensors are attractive to integrate with such microfluidics due to the possibility to be miniaturized. Moreover, on-body microfluidics coupled with bioelectronics enable smart integration with modern information and communication technology. This chapter discusses requirements and several challenges when developing on-body microfluidics such as difficulties in manipulating small sample volumes while maintaining mechanical flexibility, power-consumption efficiency, and simplicity of total automated systems. We describe key components, e.g., microchannels, microvalves, and electrochemical detectors, used in microfluidics. We also introduce representatives of advanced lab-on-a-body microfluidics combined with electrochemical sensors for biomedical applications. The chapter ends with a discussion of the potential trends of research in this field and opportunities. On-body microfluidics as modern total analysis devices will continue to bring several fascinating opportunities to the field of biomedical and translational research applications.

In Silico Analysis of Glucose Oxidase from Aspergillus niger: Potential Cysteine Mutation Sites for Enhancing Protein Stability.

Glucose oxidase (GOx) holds considerable advantages for various applications. Nevertheless, the thermal instability of the enzyme remains a grand challenge, impeding the success in applications outside the well-controlled laboratories, particularly in practical bioelectronics. Many strategies to modify GOx to achieve better thermal stability have been proposed. However, modification of this enzyme by adding extra disulfide bonds is yet to be explored. This work describes the in silico bioengineering of GOx from Aspergillus niger by judiciously analyzing characteristics of disulfide bonds found in the Top8000 protein database, then scanning for amino acid residue pairs that are suitable to be replaced with cysteines in order to establish disulfide bonds. Next, we predicted and assessed the mutant GOx models in terms of disulfide bond quality (bond length and α angles), functional impact by means of residue conservation, and structural impact as indicated by Gibbs free energy. We found eight putative residue pairs that can be engineered to form disulfide bonds. Five of these are located in less conserved regions and, therefore, are unlikely to have a deleterious impact on functionality. Finally, two mutations, Pro149Cys and His158Cys, showed potential for stabilizing the protein structure as confirmed by a structure-based stability analysis tool. The findings in this study highlight the opportunity of using disulfide bond modification as a new alternative technique to enhance the thermal stability of GOx.

Cavitas electrochemical sensors for the direct determination of salivary thiocyanate levels.

Noninvasive diagnosis using salivary samples to detect thiocyanate provides vital information on individual health. This article demonstrates the first example of a wearable sensing device to noninvasively assess thiocyanate levels. The customized screen-printed electrode system is integrated into a form of a mouthguard squarewave-voltammetric sensor toward the convenient and fast detection of the salivary biomarker within 15 s. The sensor with a protective film to mitigate the effect of biofouling offers high sensitivity and selectivity toward the detection of thiocyanate ions. Partial least square regression is applied to analyze the high-order squarewave-voltammetric data over the applied potential range of 0-1.75 V vs Ag/AgCl and quantify the thiocyanate concentration in a complex matrix. The mouthguard sensor operating under physiological conditions can monitor a wide range of thiocyanate (up to 11 mM) with a low detection limit of 30 µM. The demonstration introduces a unique approach, that obviates the requirement for blood sampling, to study thiocyanate levels of healthy people, cigarette smokers, or people with other health conditions. It is envisioned that the new cavitas device possesses a substantial promise for diverse biomedical diagnosis applications.

Advances in emergent biological recognition elements and bioelectronics for diagnosing COVID-19.

Coronaviruses pose a serious threat to public health. Tremendous efforts are dedicated to advance reliable and effective detection of coronaviruses. Currently, the coronavirus disease 2019 (COVID-19) diagnosis mainly relies on the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic materials by using reverse transcription-polymerase chain reaction (RT-PCR) assay. However, simpler and more rapid and reliable alternatives are needed to meet high demand during the pandemic. Biosensor-based diagnosis approaches become alternatives for selectively and rapidly detecting virus particles because of their biorecognition elements consisting of biomaterials that are specific to virus biomarkers. Here, we summarize biorecognition materials, including antibodies and antibody-like molecules, that are designed to recognize SARS-CoV-2 biomarkers and the advances of recently developed biosensors for COVID-19 diagnosis. The design of biorecognition materials or layers is crucial to maximize biosensing performances, such as high selectivity and sensitivity of virus detection. Additionally, the recent representative achievements in developing bioelectronics for sensing coronavirus are included. This review includes scholarly articles, mainly published in 2020 and early 2021. In addition to capturing the fast development in the fields of applied materials and biodiagnosis, the outlook of this rapidly evolving technology is summarized. Early diagnosis of COVID-19 could help prevent the spread of this contagious disease and provide significant information to medical teams to treat patients.

Label-free potentiometric aptasensing platform for the detection of Pb2+ based on guanine quadruplex structure.

Potentiometric aptasensors enhanced by integrating advanced nanomaterials are of particular interest for the detection of multiplex species (e.g., proteins, bacteria, micro-organisms) due to their low cost, ease of operation, and low detection limits. However, potentiometric detection of small ionic species aptasensors is still challenging. This article describes the first example of a label-free G-quadruplex-based potentiometric aptasensing platform for the detection of Pb2+. Polyion oligonucleotide-labeled gold nanoparticles (AuNPs-DNA) as probes are modified on Au electrode, providing high-density negative charge on the electrode surface. These signal-amplifying probes can selectively form G-quadruplexes with the presence of Pb2+ ions and reduce the negative charges on the electrode surface, hence achieving potentiometric detection of Pb2+ ions with high selectivity. The AuNPs-DNA-based aptasensor shows an acceptable sensitivity over a wide range from 10-11 to 10-6 M with a detection limit of 8.5 pM. Furthermore, confirmed by coupled plasma mass spectrometry, the sensing platform is capable of performing effective and accurate detection of Pb2+ level in real water samples. The presented aptasensor offers a fast, convenient, low-maintenance, and highly sensitive alternative for on-site water pollution detections.

Wearable, stable, highly sensitive hydrogel-graphene strain sensors.

A stable and highly sensitive graphene/hydrogel strain sensor is designed by introducing glycerol as a co-solvent in the formation of a hydrogel substrate and then casting a graphene solution onto the hydrogel in a simple, two-step method. This hydrogel-based strain sensor can effectively retain water in the polymer network due to the formation of strong hydrogen bonding between glycerol and water. The addition of glycerol not only enhances the stability of the hydrogel over a wider temperature range, but also increases the stretchability of the hydrogel from 800% to 2000%. The enhanced sensitivity can be attributed to the graphene film, whereby the graphene flakes redistribute to optimize the contact area under different strains. The careful design enables this sensor to be used in both stretching and bending modes. As a demonstration, the as-prepared strain sensor was applied to sense the movement of finger knuckles. Given the outstanding performance of this wearable sensor, together with the proposed scalable fabrication method, this stable and sensitive hydrogel strain sensor is considered to have great potential in the field of wearable sensors.

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.

Enzymatic glucose/oxygen biofuel cells: Use of oxygen-rich cathodes for operation under severe oxygen-deficit conditions.

A glucose/oxygen biofuel cell (BFC) that can operate continuously under oxygen-free conditions is described. The oxygen-deficit limitations of metabolite/oxygen enzymatic BFCs have been addressed by using an oxygen-rich cathode binder material, polychlorotrifluoroethylene (PCTFE), which provides an internal oxygen supply for the BFC reduction reaction. This oxygen-rich cathode component mitigates the potential power loss in oxygen-free medium or during external oxygen fluctuations through internal supply of oxygen, while the bioanode employs glucose oxidase-mediated reactions. The internal oxygen supply leads to a prolonged energy-harvesting in oxygen-free solutions, e.g., maintaining over 90% and 70% of its initial power during 10- and 24-h operations, respectively, in the absence of oxygen. The new strategy holds considerable promise for energy-harvesting and self-powered biosensing applications in oxygen-deficient conditions.