Amperometric Biosensor Research Articles

An Electroactive and Self-Assembling Bio-Ink, based on Protein-Stabilized Nanoclusters and Graphene, for the Manufacture of Fully Inkjet-Printed Paper-Based Analytical Devices.

Hundreds of new electrochemical sensors are reported in literature every year. However, only a few of them makes it to the market. Manufacturability, or rather the lack of it, is the parameter that dictates if new sensing technologies will remain forever in the laboratory in which they are conceived. Inkjet printing is a low-cost and versatile technique that can facilitate the transfer of nanomaterial-based sensors to the market. Herein, an electroactive and self-assembling inkjet-printable ink based on protein-nanomaterial composites and exfoliated graphene is reported. The consensus tetratricopeptide proteins (CTPRs), used to formulate this ink, are engineered to template and coordinate electroactive metallic nanoclusters (NCs), and to self-assemble upon drying, forming stable films. The authors demonstrate that, by incorporating graphene in the ink formulation, it is possible to dramatically improve the electrocatalytic properties of the ink, obtaining an efficient hybrid material for hydrogen peroxide (H2 O2 ) detection. Using this bio-ink, the authors manufactured disposable and environmentally sustainable electrochemical paper-based analyticaldevices (ePADs) to detect H2 O2 , outperforming commercial screen-printed platforms. Furthermore, it is demonstrated that oxidoreductase enzymes can be included in the formulation, to fully inkjet-print enzymatic amperometric biosensors ready to use.

Nanoelectrochemistry reveals how soluble Aβ42 oligomers alter vesicular storage and release of glutamate

Glutamate (Glu) is the major excitatory transmitter in the nervous system. Impairment of its vesicular release by β-amyloid (Aβ) oligomers is thought to participate in pathological processes leading to Alzheimer's disease. However, it remains unclear whether soluble Aβ42 oligomers affect intravesicular amounts of Glu or their release in the brain, or both. Measurements made in this work on single Glu varicosities with an amperometric nanowire Glu biosensor revealed that soluble Aβ42 oligomers first caused a dramatic increase in vesicular Glu storage and stimulation-induced release, accompanied by a high level of parallel spontaneous exocytosis, ultimately resulting in the depletion of intravesicular Glu content and greatly reduced release. Molecular biology tools and mouse models of Aβ amyloidosis have further established that the transient hyperexcitation observed during the primary pathological stage is mediated by an altered behavior of VGLUT1 responsible for transporting Glu into synaptic vesicles. Thereafter, an overexpression of Vps10p-tail-interactor-1a, a protein that maintains spontaneous release of neurotransmitters by selective interaction with t-SNAREs, resulted in a depletion of intravesicular Glu content, triggering advanced-stage neuronal malfunction. These findings are expected to open perspectives for remediating Aβ42-induced neuronal hyperactivity and neuronal degeneration.

Graphene-Based Electrochemical Nano-Biosensors for Detection of SARS-CoV-2

COVID-19, a viral respiratory illness, is caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), which was first identified in Wuhan, China, in 2019 and rapidly spread worldwide. Testing and isolation were essential to control the virus’s transmission due to the severity of the disease. In this context, there is a global interest in the feasibility of employing nano-biosensors, especially those using graphene as a key material, for the real-time detection of the virus. The exceptional properties of graphene and the outstanding performance of nano-biosensors in identifying various viruses prompted a feasibility check on this technology. This paper focuses on the recent advances in using graphene-based electrochemical biosensors for sensing the SARS-CoV-2 virus. Specifically, it reviews various types of electrochemical biosensors, including amperometric, potentiometric, and impedimetric biosensors, and discusses the current challenges associated with biosensors for SARS-CoV-2 detection. The conclusion of this review discusses future directions in the field of electrochemical biosensors for SARS-CoV-2 detection, underscoring the importance of continued research and development in this domain.

In vitro biomedical corrosion and enzyme activity inhibition on modified Cu-Zn-Al bioalloy

Abstract The amperometric biosensor was created using a flat sheet of the Cu-Zn-Al shape memory alloy, with a shiny surface and uniform thickness below 1 mm. The high biocompatibility and a large specific surface area for enzyme loading are evaluated. In vitro biomedical corrosion testing of samples revealed successful immobilization of catalase, which has undergone quasi-reversible electron transfer from the surface and saline solution. A catalase that had been immobilized retained its basic structure and bioactivity and demonstrated a remarkable electrocatalytic response to hydrogen peroxide reduction. The reduction of hydrogen peroxide on the catalase-modified Cu-25.38Zn-3.3Al alloy was investigated using hydrodynamic amperometry in both the absence and presence of l-cysteine and K2[B3O3F4OH] inhibitors. Catalytic reduction currents have increased as a result of the gradual increase in hydrogen peroxide concentration. The study on enzyme activity inhibition has shown a lower corrosion rate of catalase-modified bioalloy than the initial sample because inhibitor ions occupy all sites of the immobilized enzyme.

Electrodeposited Carbonyl Functional Polymers as Suitable Supports for Preparation of the First-Generation Biosensors.

The aim of this electrochemical study was to ascertain which type of electrochemically deposited carbonyl functionalized polymer represents the most suitable electrode substrate for direct covalent immobilization of biological catalysts (enzymes). For this purpose, a triad of amperometric biosensors differing in the type of conductive polymers (poly-vanillin, poly-trans-cinnamaldehyde, and poly-4-hydroxybenzaldehyde) and in the functioning of selected enzymes (tyrosinase and alkaline phosphatase) has been compared for the biosensing of neurotransmitters (dopamine, epinephrine, norepinephrine, and serotonin) and phenyl phosphates (p-aminophenyl phosphate and hydroquinone diphosphate). The individual layers of the polymers were electrochemically deposited onto commercially available screen-printed carbon electrodes (type C110) using repetitive potential cycling in the linear voltammetric mode. Their characterization was subsequently performed by SEM imaging and attenuated total reflectance FTIR spectroscopy. Molecules of enzymes were covalently bonded to the free carbonyl groups in polymers via the Schiff base formation, in some cases even with the use of special cross-linkers. The as-prepared biosensors have been examined using cyclic voltammetry and amperometric detection. In this way, the role of the carbonyl groups embedded in the polymeric structure was defined with respect to the efficiency of binding enzymes, and consequently, via the final (electro)analytical performance.

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (−0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.

Shield, Anchor, and Adhesive Roles of Methylene Blue in Tyrosinase Adsorbed on Carbon Felt for a Flow Injection Amperometric Enzyme Biosensor for Phenolic Substrates and Inhibitors.

Methylene blue (MB) acted as a stabilizer for preventing surface-induced denaturation of tyrosinase (TYR) adsorbed on a carbon felt (CF) surface, which is based on shield and anchor roles preventing the unfavorable conformational change of TYR on the hydrophobic CF surface. Furthermore, MB acted as an effective adhesive for TYR immobilization on CF. The resulting TYR and MB coadsorbed CF (TYR/MB-CF) worked as an excellent working electrode unit in an electrochemical detector in a flow injection amperometric biosensor, which allowed highly sensitive consecutive determination of not only TYR substrates but also competitive inhibitors. Simultaneous adsorption of TYR and MB from their mixed solution was much useful as compared with step-wise separated adsorption of TYR on the MB-adsorbed CF, which suggests that the binding interaction of MB with TYR in the solution phase is important for this phenomenon. Fluorescence and UV-vis spectroscopy revealed that not only electrostatic forces between the cationic MB and anionic amino acid residues of TYR but also hydrophobic interactions via the phenothiazine ring of MB play a principal binding driving force of MB with TYR at the surface of the TYR molecules. Synchronous fluorescence, three-dimensional fluorescence, and circular dichroism (CD) spectroscopy clarified that the conformation and the secondary structure of TYR slightly changed upon the MB binding, implying that MB binding leads to the modification of the original intramolecular bonding in part.

Laccase‐based amperometric biosensor enhanced by Ni/Au/PPy‐COOH multisegmental nanowires for selective dopamine detection

AbstractOne‐dimensional Ni/Au/PPy‐COOH nanowires with multiple segments were synthesized in this study. Smooth surfaces and magnetic properties of nanowires were investigated by scanning transmission electron microscopy (SEM), Energy‐dispersive X‐ray spectroscopy (EDX), and Electron Spin Resonance (ESR) techniques. The nanowires were used to modify the screen‐printed electrode surface and as a micro‐environment for Trametes versicolor laccase. The ability of this enzyme biosensor to detect dopamine change in human biological samples was demonstrated by a wide linear range (0.01–50 μM) and a low LOD (2.265 nM). In addition, the biosensor exhibited excellent selectivity allowing the detection of dopamine in the presence of ascorbic acid, uric acid, L‐Cys, serotonin, and glucose, with high sensitivity of reduction currents obtained at −0.2 V (vs. Ag/AgCl). The proposed biosensor allowed the detection of dopamine in commercial serum and artificial urine with recovery values close to 100 %. It also demonstrated reproducibility, reusability, and long‐term storage stability. The sensitivity, Kmapp, and Imax values of the biosensor were determined as 2.05 μM and 1.03 μA, respectively. The LAC‐Ni/Au/PPy‐COOH/NAF/SPE biosensor is a reliable design for detecting dopamine with a wide linear range.

Amperometric biosensor based on immobilized laccase onto Cys-Ag@Fe3O4 magnetic nanoparticles for selective catechol detection

Amperometric biosensor based on immobilized laccase onto Cys-Ag@Fe3O4 magnetic nanoparticles for selective catechol detection

An Amperometric Acetylcholine Biosensor Based on Co-Immobilization of Enzyme Nanoparticles onto Nanocomposite.

An electrochemical biosensor was fabricated using nanoparticles of acetylcholinesterase (AChE) and choline oxidase (ChO)/Pt nanoparticles (PtNPs)/porous graphene oxide nanosheet (GONS) composite. A pencil graphite electrode (PGE) was used for the electrodeposition of nanocomposite and the determination of acetylcholine (ACh), a neurotransmitter. Various techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectra and cyclic voltammetry (CV) were used for characterization. This biosensor (AChENPs-ChONPs/GONS/PtNPs/PGE) indicated a very short response time (3 s), a lower limit of detection (0.001 µM), good linearity (0.001-200 µM), longer storage stability (6 months) and better reproducibility. The percent analytical recoveries of added acetylcholine in serum (5.0 and 10 µM) were found to be 97.6 ± 0.7 and 96.5 ± 0.3 for the present biosensor. The coefficients of variation were obtained to be 8% and 3.25%, correspondingly. The biosensor was applied to measure the ACh amount in the serum of healthy individuals and patients with Alzheimer's disease. The number of interferents had no effect on the biosensor at their physiological concentrations.

Quantification of Lactose in Lactose-Free and Low-Lactose Milk and Milk Products by BIOMILK 300/3000 Lac, Collaborative Study: Final Action 2020.09.

In April 2020, the BIOMILK 300 LAC method for the quantification of lactose in lactose-free and low-lactose dairy products was adopted as First Action Official Method of AnalysisSM2020.09 by AOAC INTERNATIONAL. To test the reproducibility of the method, as the last step toward the Final Action status, a collaborative study was organized by BIOLAN Microbiosensores. Fifteen collaborators within the European Union took part in this study, where nine different samples were sent as duplicate blind test portions for lactose determination. The data obtained were used to determine method repeatability and reproducibility and also to validate the new version of the biosensor. The test method is based on the direct enzymatic recognition-electrochemical detection of trace levels of lactose over a wide range of dairy samples by means of the BIOMILK 300 biosensor, in less than 5 min, and without intricate sample pretreatments. All samples resulted in a repeatability relative standard deviation (RSDr) of <8.1% and a reproducibility relative standard deviation (RSDR) of 14% maximum, meeting requirements from Standard Method Performance Requirement (SMPR®) 2018.09. The method has proved to give reproducible results for the quantification of lactose in lactose-free and low-lactose dairy products. On the basis of these results, the enzymatic amperometric biosensor method developed by BIOLAN Microbiosensores was adopted as a Final Action Official Method in July 2022.

A Sensitive Amperometric Biosensor Based on Carbon Dot 3-Chloropropyl-trimethoxysilane Modified Electrode for Detection of Neurotransmitter Dopamine

Dopamine (DA) is an important electroactive neurotransmitter. The concentration of DA in the body of a healthy person is approximately 1.0 × 10−7−1.0 × 10−3 M. A decrease in dopamine concentration is associated with Parkinson’s disease. Thus, it is important to determine the amount of dopamine in early diagnosis of Parkinson’s disease. Different methods such as immunoassay, flow injection analysis (FIA), high performance liquid chromatography (HPLC) etc could be used for determination of dopamine but they are expensive and have long determination times, and pre analytic processes. In this study, a tyrosinase based amperometric biosensor was developed with carbon paste electrode modified with carbon nano dot 3-Chloropropyl-trimethoxysilane (CDs-CPTMS) for determination of the amount of dopamine. CDs-CPTMS was synthesized for the first time. Determination of dopamine was carried out by the reduction of dopamine-o-quinone at −0.15 V versus Ag/AgCl. The effect of temperature, pH, and substrate concentration on the dopamine response of the prepared biosensor and interference effect were investigated. There was no interference effect of uric acid and ascorbic acid. The designed biosensor has wide working range (0.001–0.01 μM and 0.01−0.1 μM), low limit of detection, very good reproducibility and shelf life. In addition, the preparation of the biosensor is practical and cost-effective.

Electrochemical detection of lactate produced by foodborne presumptive lactic acid bacteria

The detection of lactate is an important indicator of the freshness, stability, and storage stability of products as well as the degree of fermentation in the food industry. In addition, it can be used as a diagnostic tool in patients' healthcare since it is known that the lactate level in blood increases in some pathological conditions. Thus, the determination of lactate level plays an important role in not only the food industry but also in health fields. As a result, biosensor technologies, which are quick, cheap, and easy to use, have become important for lactate detection. In the current study, amperometric lactate biosensors based on lactate oxidase immobilization (with Nafion 5% wt) were designed and the limit of detection, linear range, and sensitivity values were determined to be 31μM, 50-350μM, and 0.04μAμM-1cm-2, respectively. Then, it was used for the measurement of lactic acid that produced by six different and morphologically identified presumptive lactic acid bacteria (LAB) which are isolated from different naturally fermented cheese samples. The biosensors were then used to successfully perform lactate measurements within 3min for each sample, even though a few of them were out of the limit of detection. Thus, electrochemical biosensors should be used as an alternative and quick solutions for the measurement of lactate metabolites rather than the traditional methods which require long working hours. This is the first study to use a biosensor to measure lactate produced by foodborne LAB in a real sample.

The Platinization of Graphite Composites Turns Widespread and Low-Cost Materials into Hydrogen Peroxide Sensors and High-Value Biosensor Transducers

Electrochemical microsensors and biosensors have been widely used in many fields, in particular neurochemical monitoring, because of their features. Usually, hydrogen peroxide (HP), obtained as a by-product of an enzymatic reaction, is the detected compound on transducers made of precious metals, in particular platinum. The over-time increase in the price of platinum and its alloys requires the use of miniaturizable low-cost supports that can be suitably modified with the deposition of Pt particles; among them, graphite is the most widespread. In the present paper, carbon-composition resistors (CCRs) and pencil leads (PLs) of different diameters (0.3, 0.5 and 2.0 mm), mainly made up of graphite, clay and some other components were used as carbonaceous support for the deposition of platinum. Platinizations were carried out by means of cyclic voltammetry (CV) and constant potential amperometry (CPA) techniques. On the platinized supports, hydrogen peroxide (HP) and ascorbic acid (AA) sensitivity were assessed in order to verify the possibility of using them as transducers of amperometric biosensors. All the used protocols determined the occurrence of HP monitoring, not appreciable on carbonaceous surfaces. We chose 0.3 mm Ø PLs for the construction of glucose biosensors by appropriately modifying the platinum surface layering, a permselective polymer, an enzyme booster, the glucose oxidase (GOx) enzyme and a containing network. The biosensor constructed in this way demonstrated a behavior comparable to that obtained using classic platinum wires.

High-performance amperometric biosensor for flow injection analysis consisting of a replaceable lactate oxidase-based mini-reactor and a silver amalgam screen-printed electrode

L-lactic acid (LA) is an essential compound in the winemaking process; furthermore, it is considered a significant biomarker for the diagnosis of a wide range of diseases, including cancer. Therefore, a cost-effective and reliable biosensing system for FIA is developed here for rapid and selective determination of LA. The biosensor architecture includes an easily replaceable lactate oxidase (LOx)-based mini-reactor connected in front of a silver amalgam screen-printed electrode acting as a transducer. The mini-reactor represents a tube filled with mesoporous silica powder (SBA−15) coated with covalently immobilized LOx. The principle of LA determination is based on the amperometric monitoring of oxygen consumption by its four-electron reduction at −900 mV vs. Ag pseudo-reference electrode, which avoids the interferences from commonly interfering compounds. The application of a strategy of a spatially separated biorecognition and detection part allowed for the immobilization of a notably large amount of enzyme (one mini-reactor contains c.a. 270 μg of the LOx), which, in turn, ensured excellent operational (93.8% of the initial signal after 350 measurements) and storage (96.9% of the initial biosensor response after 7 months) stability. The newly developed high-performance biosensor was successfully tested for the quantification of LA in saliva, wine, and dairy products.

Screen-printed biosensor based on electro-polymerization of bio-composite for nitrate detection in aqueous media

ABSTRACT Bacillus sp. possessing a periplasmic nitrate reductase was used as a recognition element to develop a nitrate biosensor. The bacteria was embedded within a polyaniline (PANI) electro-conductive matrix via electro-polymerization on miniaturized carbon screen-printed electrodes (SPE) at 100 mV/s and scan rate from −0.35 V to + 1.7 V. Surface medication of SPE was verified via Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). The optimal bacterial density was OD600 1.2. To enhance the biosensors performance, Bacillus sp. was (1) grown in riboflavin (RF) inducing media as an endogenous redox mediator and (2) exposed to different gamma radiation doses as a physical method to increase electron transfer. Results show a link between exposing cells to gamma irradiation stress, this was evident by electron spin resonance (ESR) and changes in FTIR spectrum, in addition to the increase in catalase enzyme. The nitrate limit of detection (LOD) was 0.5–25 mg/L for non-irradiated RF induced immobilized cells and LOD was 0.5–75 mg/L nitrate for 2 kGy gamma irradiated cells. The prepared biosensor showed acceptable reproducibility and multiple usages after storage at 4°C over 3 months. Low cost and simple preparation allow the biosensor to be mass-produced as a disposable device. Bacillus sp. and its endogenous redox mediator immobilized within polyaniline are good candidates for the improvement of amperometric biosensors for the quantification of nitrate in aqueous solutions.

Redox-labelled detection probe enabled immunoassay for simultaneous detection of multiple cancer biomarkers.

Some of the cancer biomarkers often lack specificity and sensitivity; thus, simultaneous detection of multiple biomarkers can make the diagnosis more accurate. Also, simple sensing system without utilization of extra reagents like mediator or substrate during detection event is desirable for point-of-care testing. To address this, mediator and substrate-free amperometric biosensor for simultaneous detection of cancer biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) have been demonstrated by designing two different redox-labelled detection probes. Colloidal nanoparticles of polyaniline-pectin conjugated with AFP antibody along with ferrocene and silver nanoparticles conjugated with CEA antibody along with anthraquinone were used as redox probes to bind with AFP and CEA during the detection event. Sensor constructed using carboxylic acid tethered polyaniline as immobilization matrix displayed 5 times wider linear range than conventional polyaniline for AFP and CEA detection by sandwich electrochemical assay. The detection limit was 30pgmL-1 for AFP and 80pgmL-1 for CEA. The biosensor displayed appropriate sensitivity, good specificity, and negligible cross-reactivity between the two targets. The proposed sensor was used to determine APF and CEA in human blood serum. The strategy demonstrated can be further extended for detection of panel of cancer biomarkers by designing appropriate redox probes.

Laccase modified GO/TiS2 nanocomposite based amperometric biosensor for (-)-epicatechin detection

In this study, 2-dimensional graphene oxide-titanium disulfide (GO/TiS2) nanolayers were synthesized, and the surface and size properties of nanolayers were investigated by transmission electron microscopy (TEM) technique. The nanolayers were used to modify the screen-printed electrode surface and applied as a micro-medium for Trametes versicolor laccase. The ability of this enzyme biosensor to detect the (-)-epicatechin has been demonstrated with a wide linear range (0.1–250 µM), LOD (0.073 µM), and sensitivity (0.3264 µAµM−cm−2). In addition, the biosensor exhibited excellent selectivity allowing selective detection of (-)-epicatechin in the presence of other polyphenolic compounds and high reducing currents sensitivity (vs Ag/AgCl) examined at −0.6 V. The proposed biosensor showed reproducibility, reusability, long-term storage stability, and applicability in actual food samples with recovery values close to 100 %. Also, Kmapp and Imax values ​​of the biosensor were determined as 2.245 µM and 0.757 µA, respectively.

Microbial Biofuel Cells: Fundamental Principles, Development and Recent Obstacles.

This review focuses on the development of microbial biofuel cells to demonstrate how similar principles apply to the development of bioelectronic devices. The low specificity of microorganism-based amperometric biosensors can be exploited in designing microbial biofuel cells, enabling them to consume a broader range of chemical fuels. Charge transfer efficiency is among the most challenging and critical issues while developing biofuel cells. Nanomaterials and particular redox mediators are exploited to facilitate charge transfer between biomaterials and biofuel cell electrodes. The application of conductive polymers (CPs) can improve the efficiency of biofuel cells while CPs are well-suitable for the immobilization of enzymes, and in some specific circumstances, CPs can facilitate charge transfer. Moreover, biocompatibility is an important issue during the development of implantable biofuel cells. Therefore, biocompatibility-related aspects of conducting polymers with microorganisms are discussed in this review. Ways to modify cell-wall/membrane and to improve charge transfer efficiency and suitability for biofuel cell design are outlined.

Amperometric Biosensor Strip With Carbon Nanotube and Ketone Body 3-Hydroxybutyrate Dehydrogenase

We propose an amperometric biosensor strip for the detection of ketone body 3-hydroxybutyrate (3HB) in serum. The biosensor strip has a small volume ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\approx 2 ~\mu \text{L}$ </tex-math></inline-formula> ) capillary chamber. The working electrode consists of carbon nanotubes (CNTs) and 3HB dehydrogenase (3HBDH). The CNTs are dispersed in aqueous media with sodium cholate (SC) surfactant and mixed with HBDH solution. Ultraviolet–visible, electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) demonstrated that the CNTs were well dispersed in aqueous solution. Any type of CNT enhances the current owing to an enzymatic reaction without an electron transfer mediator. A small amount of HBDH/CNT solution is dipped into a small-area (1.5 mm2) electrode and sealed with a coversheet to make the reaction chamber. The sensor can detect 3HB in human serum within the range of 0.05–4.4 mM, which can cover physiological use. The response time is 15 s.