Modulating the growth of cysteine-capped cadmium sulfide quantum dots with enzymatically produced hydrogen peroxide

Cysteine (CSH) readily stabilizes cadmium sulfide quantum dots (CdS QDs) that grow in aqueous buffered solutions.The oxidation of CSH by hydrogen peroxide (H2O2) at room temperature yields cystine (CSSC),which is less efficient in stabilizing CdS QDs compared to CSH.Herein,we demonstrate that such oxidation causes a decrease in the formation rate of CSH-capped CdS QDs from Cd2+ and S2-ions.For the first time,we combined the oxidation of CSH with the glucose oxidase (GOx)-assisted biocatalytic oxidation of D-glucose,which leads to a buildup of H2O2 in the reaction mixture.The enzymatically modulated in situ growth of CdS QDs was monitored using two techniques:fluorescence spectroscopy and photoelectrochemical (PEC) analysis.This system enables quantification of GOx and glucose in human serum.     

Multiresponsive Supramolecular Theranostic Nanoplatform Based on Pillar[5]arene and Diphenylboronic Acid Derivatives for Integrated Glucose Sensing and Insulin Delivery

A closed‐loop “smart” insulin delivery system with the capability to mimic pancreatic cells will be highly desirable for diabetes treatment. This study reports a multiple stimuli‐responsive insulin delivery platform based on an explicit supramolecular strategy. Self‐assembled from a well‐designed amphiphilic host–guest complex formed by pillar[5]arene and a diphenylboronic acid derivative and loaded with insulin and glucose oxidase, the obtained insulin‐GOx‐loaded supramolecular vesicles can selectively recognize glucose, accompanied by the structure disruption and efficient release of the entrapped insulin triggered by the high glucose concentration as well as the in situ generated H₂O₂ and acid microenvironment during the GOx‐promoted specific oxidation of glucose into gluconic acid. Moreover, such a “smart” supramolecular theranostic nanoplatform is able to function as both a glucose sensor and a controlled insulin delivery actuator. In vivo experiments further demonstrate that this smart supramolecular nanocarrier shows fast response to hyperglycemic circumstances and can effectively regulate the glucose levels in a mouse model of type I diabetes.     

Glucose Oxidase‐Instructed Multimodal Synergistic Cancer Therapy

Over the past 3 years, glucose oxidase (GOx) has aroused great research interest in the context of cancer treatment due to its inherent biocompatibility and biodegradability, and its unique catalytic properties against β‐d ‐glucose. GOx can effectively catalyze the oxidation of glucose into gluconic acid and hydrogen peroxide. This process depletes oxygen levels, resulting in elevated acidity, hypoxia, and oxidative stress in the tumor microenvironment. All of these changes can be readily harnessed to develop a multimodal synergistic cancer therapy by combining GOx with other therapeutic approaches. Herein, the representative studies of GOx‐instructed multimodal synergistic cancer therapy are introduced, and their synergistic mechanisms are discussed systematically. The current challenges and future prospects to advance the development of GOx‐based nanomedicines in this cutting‐edge research area are highlighted.     

Programmable NIR‐II Photothermal‐Enhanced Starvation‐Primed Chemodynamic Therapy using Glucose Oxidase‐Functionalized Ancient Pigment Nanosheets

Chemodynamic therapy (CDT) has attracted considerable attention recently, but the poor reaction kinetics restrict its practical utility in clinic. Herein, glucose oxidase (GOx) functionalized ancient pigment nanosheets (SrCuSi₄O₁₀, SC) for programmable near‐infrared II (NIR‐II) photothermal‐enhanced starvation primed CDT is developed. The SC nanosheets (SC NSs) are readily exfoliated from SC bulk suspension in water and subsequently functionalized with GOx to form the nanocatalyst (denoted as SC@G NSs). Upon laser irradiation, the photothermal effect of SC NSs can enhance the catalytic activity of GOx for NIR‐II photothermal‐enhanced starvation therapy, which effectively eliminates intratumoral glucose and produces abundant hydrogen peroxide (H₂O₂). Importantly, the high photothermal‐conversion efficiency (46.3%) of SC@G NSs in second biological window permits photothermal therapy of deep‐seated tumors under the guidance of NIR‐II photoacoustic imaging. Moreover, the acidity amplification due to gluconic acid generation will in turn accelerate the degradation of SC NSs, facilitating the release of strontium (Sr) and copper (Cu) ions. Both the elevated H₂O₂ and the released ions will prime the Cu²⁺/Sr²⁺‐H₂O₂ reaction for enhanced CDT. Thus, a programmable NIR‐II photothermal‐enhanced starvation primed CDT is established to combat cancer with minimal side effects.     

Enhanced Photocatalytic H2‐Production Activity of CdS Quantum Dots Using Sn2+ as Cocatalyst under Visible Light Irradiation

Herein, oil‐soluble CdS quantum dots (QDs) are first prepared through a solvent‐thermal process. Then, oil‐soluble CdS QDs are changed into water‐soluble QDs via ligand exchange using mercaptopropionic acid as capping agent at pH 13. The photocatalytic performance is investigated under the visible light irradiation using glycerol as sacrificial agent and Sn²⁺ as cocatalyst. No H₂‐production activity is observed for oil‐soluble CdS QDs. Water‐soluble CdS QDs exhibit significantly enhanced hydrogen evolution rate. When the concentration of cocatalyst Sn²⁺ increases to 0.2 × 10^?3 m , the rate of hydrogen evolution reaches 1.61 mmol g^?1 h^?1, which is 24 times higher than that of the pristine water‐soluble CdS QDs. The enhanced H₂‐production efficiency is attributed to the adsorption of Sn²⁺ ions on the surface of CdS QDs that are further reduced to Sn atoms by photogenerated electrons. The in situ generated Sn atoms serve as photocatalytic cocatalyst for efficient hydrogen generation.     

Aqueous synthesis of CdSe and CdSe/CdS quantum dots with controllable introduction of Se and S sources

A new and convenient route is developed to synthesize CdSe and core–shell CdSe/CdS quantum dots (QDs) in aqueous solution. CdSe QDs are prepared by introducing H₂Se gas into the aqueous medium containing Cd²⁺ ions. The synthesized CdSe QDs are further capped with CdS to form core–shell CdSe/CdS QDs by reacting with H₂S gas. The gaseous precursors, H₂Se and H₂S, are generated on-line by reducing SeO₃ ^2? with NaBH₄ and the reaction between Na₂S and H₂SO₄, and introduced sequentially into the solution to form CdSe and CdSe/CdS QDs, respectively. The synthesized water-soluble CdSe and CdSe/CdS QDs possess high quantum yield (3 and 20 %) and narrow full-width-at-half-maximum (43 and 38 nm). The synthesis process is easily reproducible with simple apparatus and low-toxic chemicals. The relatively standard deviation of maxima fluorescence intensity is only 2.1 % (n = 7) for CdSe and 3.6 % (n = 7) for CdSe/CdS QDs. This developed route is simple, environmentally friendly and can be readily extended to the large-scale aqueous synthesis of QDs.     

Recent Advances in Glucose‐Oxidase‐Based Nanocomposites for Tumor Therapy

Glucose oxidase (GOx) can react with intracellular glucose and oxygen (O₂) to produce hydrogen peroxide (H₂O₂) and gluconic acid, which can cut off the nutrition source of cancer cells and consequently inhibit their proliferation. Therefore, GOx is recognised as an ideal endogenous oxido‐reductase for cancer starvation therapy. This process can further regulate the tumor microenvironment by increasing the hypoxia and the acidity. Thus, GOx offers new possibilities for the elaborate design of multifunctional nanocomposites for tumor therapy. However, natural GOx is expensive to prepare and purify and exhibits immunogenicity, short in vivo half‐life, and systemic toxicity. Furthermore, GOx is highly prone to degrade after exposure to biological conditions. These intrinsic shortcomings will undoubtedly limit its biomedical applications. Accordingly, some nanocarriers can be used to protect GOx from the surrounding environment, thus controlling or preserving the activity. A variety of nanocarriers including hollow mesoporous silica nanoparticles, metal–organic frameworks, organic polymers, and magnetic nanoparticles are summarized for the construction of GOx‐based nanocomposites for multimodal synergistic cancer therapy. In addition, current challenges and promising developments in this area are highlighted.     

Glucose oxidase-modified carbon-felt-reactor coupled with peroxidase-modified carbon-felt-detector for amperometric flow determination of glucose

Glucose oxidase (GOx) and horseradish peroxidase (HRP) were covalently immobilized on a porous carbon-felt (CF) by using cyanuric chloride (CC) as a linking reagent. The resulting GOx-modified-CF (GOx-ccCF) was used as column-type enzyme reactor and placed on upstream of the HRP-ccCF-based H₂O₂ flow-detector to fabricate amperometric flow-biosensor for glucose. Sensor setting conditions and the operational conditions were optimized, and the analytical performance characteristics of the resulting flow-biosensor were evaluated. The chemical modification of the GOx via CC was found to be effective to obtain larger catalytic activity as compared with the physical adsorption. Under the optimized conditions (i.e., volume ratio of the GOx-ccCF-reactor to the HRP-ccCF-detector is 1.0; applied potential is − 0.12 V vs. Ag/AgCl; carrier pH is 6.5; and carrier flow rate is 4.3 ml/min), highly selective and quite reproducible peak current responses toward glucose were obtained: the RSD for 30 consecutive injections of 3 mM glucose was 1.04%, and no serious interferences were observed for fructose, ethanol, uric acid, urea and tartaric acid for the amperometric measurements of glucose. The magnitude of the cathodic peak currents for glucose was linear up to 5 mM (sensitivity, 6.38 ± 0.32 μA/μM) with the limit detection of 9.4 μM (S/N = 3, noise level, 20 nA). The present GOx-ccCF-reactor and HRP-ccCF-detector-coupled flow-glucose biosensor was utilized for the determination of glucose in beverages and liquors, and the analytical results by the sensor were in fairly good agreement with those by the conventional spectrophotometry.     

Nanocatalysts‐Augmented and Photothermal‐Enhanced Tumor‐Specific Sequential Nanocatalytic Therapy in Both NIR‐I and NIR‐II Biowindows

The tumor microenvironment (TME) has been increasingly recognized as a crucial contributor to tumorigenesis. Based on the unique TME for achieving tumor‐specific therapy, here a novel concept of photothermal‐enhanced sequential nanocatalytic therapy in both NIR‐I and NIR‐II biowindows is proposed, which innovatively changes the condition of nanocatalytic Fenton reaction for production of highly efficient hydroxyl radicals (?OH) and consequently suppressing the tumor growth. Evidence suggests that glucose plays a vital role in powering cancer progression. Encouraged by the oxidation of glucose to gluconic acid and H₂O₂ by glucose oxidase (GOD), an Fe₃O₄/GOD‐functionalized polypyrrole (PPy)‐based composite nanocatalyst is constructed to achieve diagnostic imaging‐guided, photothermal‐enhanced, and TME‐specific sequential nanocatalytic tumor therapy. The consumption of intratumoral glucose by GOD leads to the in situ elevation of the H₂O₂ level, and the integrated Fe₃O₄ component then catalyzes H₂O₂ into highly toxic ?OH to efficiently induce cancer‐cell death. Importantly, the high photothermal‐conversion efficiency (66.4% in NIR‐II biowindow) of the PPy component elevates the local tumor temperature in both NIR‐I and NIR‐II biowindows to substaintially accelerate and improve the nanocatalytic disproportionation degree of H₂O₂ for enhancing the nanocatalytic‐therapeutic efficacy, which successfully achieves a remarkable synergistic anticancer outcome with minimal side effects.     

Cu2+‐Modified Metal–Organic Framework Nanoparticles: A Peroxidase‐Mimicking Nanoenzyme

The synthesis and characterization of UiO‐type metal–organic framework nanoparticles (NMOFs) composed of Zr⁴⁺ ions bridged by 2,2′‐bipyridine‐5,5′‐dicarboxylic acid ligands and the postmodification of the NMOFs with Cu²⁺ ions are described. The resulting Cu²⁺‐modified NMOFs, Cu²⁺‐NMOFs, exhibit peroxidase‐like catalytic activities reflected by the catalyzed oxidation of Amplex‐Red to the fluorescent Resorufin by H₂O₂, the catalyzed oxidation of dopamine to aminochrome by H₂O₂, and the catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂. Also, the Cu²⁺‐NMOFs mimic NADH peroxidase functions and catalyze the oxidation of dihydronicotinamide adenine dinucleotide, NADH, to nicotinamide adenine dinucleotide, NAD⁺, in the presence of H₂O₂. The Cu²⁺‐NMOFs‐catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂ is used to develop a glucose sensor by monitoring the H₂O₂ formed by the aerobic oxidation of glucose to gluconic acid in the presence of glucose oxidase. Furthermore, loading the Cu²⁺‐NMOFs with fluorescein and activating the catalyzed generation of chemiluminescence in the presence of luminol/H₂O₂ yield an efficient chemiluminescence resonance energy transfer (CRET) process to the fluorescein reflected by the activation of the fluorescence of the dye (λ = 520 nm, CRET efficiency 35%).     

A Multifunctional Cascade Bioreactor Based on Hollow‐Structured Cu2MoS4 for Synergetic Cancer Chemo‐Dynamic Therapy/Starvation Therapy/Phototherapy/Immunotherapy with Remarkably Enhanced Efficacy

The unique tumor microenvironment (TME) facilitates cancer proliferation and metastasis, and it is hard to cure cancer completely via monotherapy. Herein, a multifunctional cascade bioreactor based on hollow mesoporous Cu₂MoS₄ (CMS) loaded with glucose oxidase (GOx) is constructed for synergetic cancer therapy by chemo‐dynamic therapy (CDT)/starvation therapy/phototherapy/immunotherapy. The CMS harboring multivalent elements (Cu^1+/2+, Mo^4+/6+) exhibit Fenton‐like, glutathione (GSH) peroxidase‐like and catalase‐like activity. Once internalized into the tumor, CMS could generate ·OH for CDT via Fenton‐like reaction and deplete overexpressed GSH in TME to alleviate antioxidant capability of the tumors. Moreover, under hypoxia TME, the catalase‐like CMS could react with endogenous H₂O₂ to generate O₂ for activating the catalyzed oxidation of glucose by GOx for starvation therapy accompanied with the regeneration of H₂O₂. The regenerated H₂O₂ can devote to Fenton‐like reaction for realizing GOx‐catalysis‐enhanced CDT. Meanwhile, the CMS under 1064 nm laser irradiation shows remarkable tumor‐killing ability by phototherapy due to its excellent photothermal conversion efficiency (η = 63.3%) and cytotoxic superoxide anion (·O₂^?) generation performance. More importantly, the PEGylated CMS@GOx‐based synergistic therapy combined with checkpoint blockade therapy could elicit robust immune responses for both effectively ablating primary tumors and inhibiting cancer metastasis.     

A Glucose/Oxygen‐Exhausting Nanoreactor for Starvation‐ and Hypoxia‐Activated Sustainable and Cascade Chemo‐Chemodynamic Therapy

Fenton reaction‐mediated chemodynamic therapy (CDT) can kill cancer cells via the conversion of H₂O₂ to highly toxic HO?. However, problems such as insufficient H₂O₂ levels in the tumor tissue and low Fenton reaction efficiency severely limit the performance of CDT. Here, the prodrug tirapazamine (TPZ)‐loaded human serum albumin (HSA)–glucose oxidase (GOx) mixture is prepared and modified with a metal–polyphenol network composed of ferric ions (Fe³⁺) and tannic acid (TA), to obtain a self‐amplified nanoreactor termed HSA–GOx–TPZ–Fe³⁺–TA (HGTFT) for sustainable and cascade cancer therapy with exogenous H₂O₂ production and TA‐accelerated Fe³⁺/Fe²⁺ conversion. The HGTFT nanoreactor can efficiently convert oxygen into HO? for CDT, consume glucose for starvation therapy, and provide a hypoxic environment for TPZ radical‐mediated chemotherapy. Besides, it is revealed that the nanoreactor can significantly elevate the intracellular reactive oxygen species content and hypoxia level, decrease the intracellular glutathione content, and release metal ions in the tumors for metal ion interference therapy (also termed “ion‐interference therapy” or “metal ion therapy”). Further, the nanoreactor can also increase the tumor’s hypoxia level and efficiently inhibit tumor growth. It is believed that this tumor microenvironment‐regulable nanoreactor with sustainable and cascade anticancer performance and excellent biosafety represents an advance in nanomedicine.     

Inorganic–Organic Hybrid Nanoprobe for NIR‐Excited Imaging of Hydrogen Sulfide in Cell Cultures and Inflammation in a Mouse Model

Hydrogen sulfide (H₂S) is an important gaseous signaling agent mediated by many physiological processes and diseases. In order to explore its role in biological signaling, much effort has been focused on developing organic fluorescent probes to image H₂S. However, these downconversion H₂S probes are impractical for bio‐imaging beyond a certain depth because of the short tissue penetration of UV/visible light (as an excitation source). In most circumstance, these probes are also not suitable for long‐term assay due to photo‐bleaching. Herein, a new design to detect H₂S based on the coumarin‐hemicyanine (CHC1)‐modified upconversion nanophosphors is reported. This inorganic–organic integrated nanoprobe is demonstrated to display a fast response time with a large ratiometric upconversion luminescence (UCL) enhancement, and extraordinary photo‐stability. CHC1‐UCNPs not only can be used for ratiometric UCL monitoring of pseudo‐enzymatic H₂S production in living cells, but can also be used to identify the risk of endotoxic shock through ratiometric UCL imaging of tissue and measurement of endogenous H₂S levels in plasma. The first ratiometric UCL H₂S nanoprobe reported here may be further developed as the next‐generation diagnostic tool for the detection of inflammatory‐related diseases.     

A novel method for fabrication of CdS quantum dot-sensitized solar cells

In this report, a novel and facile in situ gas–solid reaction method has been developed for the deposition of CdS quantum-dots (QDs) on a mesoscopic TiO₂ film. In the approach, cadmium nitrate solution was first coated on mesoporous TiO₂ films, and subsequently transformed into CdS QDs (with size about 2–3 nm) by reaction with hydrogen sulfide (H₂S) gas generated in a closed container at room temperature. Different from the conventional solution techniques, this method offers new opportunities for rapid and facile deposition of CdS QD-coated TiO₂ films without the introduction of the by-products. With the CdS QDs-decorated TiO₂ active electrodes, the liquid and solid solar cells were fabricated with power conversion efficiencies (PCEs) of 1.90 and 0.80%, respectively.     

Monolayer Silane‐Coated,Water‐Soluble Quantum Dots

A one‐step method to produce ≈12 nm hydrodynamic diameter water‐soluble CdSe/ZnS quantum dots (QDs), as well as CdS/ZnS, ZnSe/ZnMnS/ZnS, AgInS₂/ZnS, and CuInS₂/ZnS QDs, by ligand exchange with a near‐monolayer of organosilane caps is reported. The method cross‐links the surface‐bound silane ligands such that the samples are stable on the order of months under ambient conditions. Furthermore, the samples may retain a high quantum yield (60%) over this time. Several methods to functionalize aqueous QD dispersions with proteins and fluorescent dyes have been developed with reaction yields as high as 97%.     

Photocatalytic Activity of Protein‐Conjugated CdS Nanoparticles

Colloidal CdS nanoparticles are conjugated with a variety of proteins, including enhanced yellow fluorescent protein, tobacco etch virus protease (TEV), lysozyme, and bacterial cytochrome P450 CYP152A1, and the photochemical properties of the resulting conjugates are analyzed by EPR spectroscopy and hydroxyl radical‐specific fluorimetric assay. While irradiation of bare CdS colloids leads to photogeneration of hydroxyl and superoxide radicals, it is surprisingly observed that coating of the CdS particles with proteins effectively suppresses the production of these radical species and instead leads to increased formation of a long‐lived reactive oxygen species, most likely H₂O₂. A mechanism for the observed results is suggested. The empirical results are capitalized on for the assembly of a CdS–TEV nanohybrid, which shows significantly higher performance as a photocatalytic mediator for fatty acid hydroxylation by CYP152A1 than bare CdS nanoparticles.     

Control over the Size Effect in the Spectroscopic Properties of Zn<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript>Cd<Subscript>1 – <Emphasis Type="Italic">x</Emphasis></Subscript>S Colloidal Quantum Dots

Approaches are developed for controlling the size effect in the absorption and photoluminescence properties of colloidal CdS and Zn_0.5Cd_0.5S quantum dots (QDs) in a gelatin matrix and thioglycolic acid. We analyze spectroscopic manifestations of dark Ostwald ripening and size-selective photoetching and demonstrate that the illumination of molten colloidal CdS and Zn_0.5Cd_0.5S QD solutions at a wavelength lying at the optical absorption edge in the presence of hydrogen peroxide as a catalyst for photoetching leads to a blue shift of the optical absorption and photoluminescence spectra of the QDs. Analysis of TEM images of QD samples leads us to conclude that photoetching reduces the average QD size as a consequence of the photodissolution of the CdS and Zn_0.5Cd_0.5S compounds. We demonstrate that, by contrast, heat treatment of the synthesized CdS and Zn_0.5Cd_0.5S QD sols at a temperature between 75 and 90°C leads to an increase in nanocrystal size through recrystallization, which shows up as a redshift of the optical absorption and photoluminescence spectra of the corresponding samples.     

Microfabricated electrophoresis chips for simultaneous bioassays of glucose, uric acid, ascorbic acid, and acetaminophen

A micromachined capillary electrophoresis chip is described for simultaneous measurements of glucose, ascorbic acid, acetaminophen, and uric acid. Fluid control is used to mix the sample and enzyme glucose oxidase (GOx). The enzymatic reaction, a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, occurs along the separation channel. The enzymatically liberated neutral peroxide species is separated electrophoretically from the anionic uric and ascorbic acids in the separation/reaction channel. The three oxidizable species are detected at the downstream gold-coated thick-film amperometric detector at different migration times. Glucose can be detected within less than 100 s, and detection of all electroactive constituents is carried out within 4 min. Measurements of glucose in the presence of acetaminophen, a neutral compound, are accomplished by comparing the responses in the presence and absence of GOx in the running buffer. The reproducibility of the on-chip glucose measurements is improved greatly by using uric acid as an internal standard. Factors influencing the performance, including the GOx concentration, field strength, and detection potential, are optimized. Such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate), as well as for the introduction of high-speed clinical microanalyzers based on multichannel chips.     

Invasion and Defense Interactions between Enzyme‐Active Liquid Coacervate Protocells and Living Cells

The design and construction of mutual interaction models between artificial microsystems and living cells have the potential to open a wide range of novel applications in biomedical and biomimetic technologies. In this study, an artificial form of invasion‐defense mutual interactions is established in a community of glucose oxidase (GOx)‐containing liquid coacervate microdroplets and living cells, which interact via enzyme‐mediated reactive oxygen species (ROS) damage. The enzyme‐containing coacervate microdroplets, formed via liquid–liquid phase separation, act as invader protocells to electrostatically bind with the host HepG2 cell, resulting in assimilation. Subsequently, the glucose oxidation in the liquid coacervates initiates the generation of H₂O₂, which serves as an ROS resource to block cell proliferation. As a defense strategy, introduction of catalase (CAT) into the host cells is exploited to resist the ROS damage. CAT‐mediated decomposition of H₂O₂ leads to the ROS scavenging and results in the recovery of cell viability. The results obtained in the current study highlight the remarkable opportunities for the development of mutual interacting communities on the interface of artificial protocells/living cells. They also provide a new approach for engineering cellular behaviors through exploiting artificial nonliving microsystems.     

Precipitation of an insoluble product on enzyme monolayer electrodes for biosensor applications: characterization by Faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance analyses.

Precipitation of an insoluble, insulating product on monolayer-functionalized electrodes enables the development of new electrochemical biosensors. Faradaic impedance spectroscopy and cyclic voltammetry are used to probe the electron-transfer resistance at the conductive support upon the accumulation of the insoluble product on the electrode surface. Similarly, microgravimetric quartz crystal microbalance, QCM, analyses were used to assay the formation of the precipitate on the electrode. A horseradish peroxidase, HRP, monolayer electrode is used to analyze H2O2 via the biocatalyzed oxidation of 4-chloro-1-naphthol (1) and the precipitation of the insoluble product (2). A bienzyme-layered electrode consisting of HRP and glucose oxidase, GOx, is used to sense glucose. Biocatalyzed oxidation of glucose by O2, in the presence of GOx, yields H2O2, and the generated hydrogen peroxide effects the formation of the insoluble product (2) in the presence of HRP. The insoluble product accumulated on the electrode, and the extent of the resulting electron-transfer resistance, correlated with the amounts of H2O2 or glucose, and appropriate calibration curves are extracted.