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Brain glucose is an important biomarker of Alzheimer's disease (AD) and has a high specificity especially for early AD. Activatable magnetic resonance imaging (MRI) contrast agents (CAs) serve as a robust technology in the early diagnosis of many diseases; however, there is a lack of glucose-specific MRI CAs. To address this issue, in this work, we synthesized a novel MRI CA (ZIF-8/GOx@MnO

Aberrant cerebral glucose metabolism is related to many brain diseases, especially brain tumor. However, it remains challenging to measure the dynamic changes in cerebral glucose. Here, we developed a near-infrared (NIR) optical transducer to sensitively monitor the glucose variations in cerebrospinal fluid in vivo. The transducer consists of an oxygen-sensitive nanoparticle combined with glucose oxidase (GOx), yielding highly sensitive NIR phosphorescence in response to blood glucose change. We demonstrated long-term continuous glucose monitoring by using the NIR transducer. After subcutaneous implantation, the glucose transducer provides a strong luminescence signal that can continuously monitor blood glucose fluctuations for weeks. By using the NIR emission of the transducer, we further observed abnormal dynamic changes in cerebrospinal fluid glucose and quantitatively assessed cerebral glucose uptake rates in transgenic mice bearing brain tumors. This study provides a promising method for the diagnosis of various metabolic diseases with altered glucose metabolism.

Self-powered sensors do not require a power supply and are easy to miniaturize, which have potential for constructing wearable, portable, and real-time detection devices. However, it is challenging for the detection of low abundant targets due to the low output power density of fuel cells and much interference of complex biological environment. Herein, a new kind of photocatalytic zinc-air battery-based self-powered electrochemical sensor (ZAB-SPES) was constructed for the detection of microRNA let-7a (miRNA let-7a) by combining magnetic nanobeads (MBs) with a metal-organic framework loaded with glucose oxidase (MOFs@GOX). Poly(1,4-di(2-thienyl))benzene (PDTB) was used as the photocathode material, and the proposed ZAB-SPES had a high power density of 22.8 μW/cm

The biomineralized metal-organic frameworks (MOFs) as protective layers help enhance the robustness of enzymes for biocatalysis. Despite great efforts, it is still challenging to develop a recyclable system with high payload and tolerance to harsh conditions. Here, we report a facile surface charge-independent strategy based on Zn-based coordination polymer (ZnCP) for nondestructive immobilization of enzyme. The ZnCP outcompetes most of the previously reported MOFs, in terms of high-payload enzyme packaging. Moreover, benefiting from the hydrophilicity of ZnCP, the entrapped enzymes (e.g., positive cytochrome C and negative glucose oxidase) maintained high catalytic activity, resembling their native counterparts. Notably, compared with ZIF-8, such enzyme-incorporated ZnCP (enzyme@ZnCP) is more tolerant to acidic pH, which imparts the enzyme with good recyclability, even in acid species-generated catalytic reactions, thus broadening its application in biocatalysis. The feasibility of enzyme@ZnCP for protein packaging, enzyme cascade catalysis, and biosensing was also validated. Altogether, enzyme@ZnCP demonstrates high enzyme payload, operational stability, and preservation of enzymatic activity, affording a versatile platform to accommodate bioactive enzyme for biocatalysis and biosensing.

The development of modern technologies has acclimatized biosensors to complicated applicable scenarios with integrated properties as a whole instead of the pursuit of a single-point breakthrough. Here, we targeted a few concerns in the development of enzyme-based biosensors, including stability, analyte enrichment, and signal transduction, and developed a general biosensing model utilizing enzymes, aggregation-induced emission (AIE) luminogens, and stimuli-responsive framework materials as the units. We propose such proof-of-concept of glucose biosensors by coencapsulating glucose oxidase and AIE-type gold nanoclusters into acid-sensitive zeolite imidazolate framework (ZIF)-8 nanocrystals. The acid-activated degradation of ZIF-8 bridges the molecular signals produced by the enzyme-catalytic reaction of glucose and the photon signals generated by ZIF-8-induced AIE effects of gold nanoclusters, resulting in the "turn-off" model nanoprobes for glucose detection with high selectivity. After embedding the nanoprobes into hollow-out tapes, the formed paper biosensors can conveniently detect glucose with the help of a smartphone.

Point-of-care testing (POCT) with the advantages of simplicity, rapidity, portability, and low-cost is of great importance to improve healthcare, especially in resource-limited settings and home healthcare settings. Moreover, it is a great challenge to quantitative POCT of multiplexed biomarkers within a single accessible assay but provides enhanced diagnostic accuracy and improved diagnostic efficiency. Herein, a smartphone optical device has been designed for POCT of glucose and cholesterol in metabolic syndrome patients using a ratiometric fluorescent sensor. The sensing system of Ag NPs/UiO-66-NH2 and o-phenylenediamine presents a dual-emission response to H2O2 (the main product of glucose and cholesterol catalyzed by glucose oxidase and cholesterol oxidase) on account of the inner filter effect, resulting in an increase in the response of the fluorescence intensity ratio (F555 nm/F425 nm) accompanied by a distinguishable color transition from blue to yellow green. After compositing probes with a flexible substrate, the obtained test strip can be integrated with a smartphone-based portable platform to read RGB values for accurate testing of glucose and cholesterol with both detection limits of 10 μmol L-1, which are hundreds of times lower than their concentrations in human serum. With the advantages of low-cost, ease of operation, and broad adaptability, this smartphone optical device holds great potential for portable detection of numerous targets in personalized healthcare and clinical diagnosis.

The mesh-type USB piezoelectric ultrasonic transducer (USB-PUT) used in household humidifiers and inhalation therapy devices is very cheap, small, and energy saving. It holds great promise for sonochemistry. However, the microtapered apertures in the center of the stainless steel substrate of mesh-type USB-PUT can lead to rapid atomization of solution, leakage of solutions containing surfactants and organic solvent through the apertures, and high background emission. Herein, we design a new type of USB-PUT by replacing the meshed stainless steel substrate with an apertureless stainless steel substrate. We have found that this apertureless USB-PUT can not only induce intense sonochemiluminescence (SCL) but can also enable sensitive luminol SCL detection of hydrogen peroxide which is practically impossible using mesh-type PUT because of the strong background SCL emission. By using this apertureless device to induce SCL and using smart phone as a detector, a visual hydrogen peroxide SCL detection method with a linear range of 0.5-50 μM and a detection limit of 0.32 μM is established. Moreover, the device can achieve the detection of glucose oxidase (GOD) activity and glucose by enzymatic conversion of glucose to hydrogen peroxide. The linear range of GOD detection is 1-200U/L with a detection limit of 0.86 U/L. The linear range of glucose detection is 0.5-70 μM with a detection limit of 0.43 μM. The cheap (a few dollars) and user-friendly apertureless USB-PUT is promising for sonochemistry applications and chemical education.

The electrochemiluminescence (ECL) ratiometric assay is usually based on two different ECL luminophores, and the choice of two suitable luminophores and shared co-reactant makes its construction challenging. The single-emitter-based ECL ratio mode could overcome the limitation of two luminophores and simplify the construction process, so it is an ideal choice. In this work, CdTe quantum dots (CdTe QDs) were modulated using black phosphorus (BP) nanosheet to simultaneously emit the cathodic and anodic ECL signals, and H2O2 and tripropylamine (TPrA) served as the cathodic and anodic co-reactants, respectively. MicroRNA-126 (miRNA-126) was selected as the template target to exploit the application of BP-CdTe QDs in the single-emitter-based ECL ratio detection. Through the target recycling triggering rolling-circle amplification (RCA) reaction, a large amount of glucose oxidase (GOx)-modified single strand 1 was introduced. GOx catalyzed glucose to produce H2O2 in situ, which acted as a dual-role moderator to quench the anodic ECL emission with TPrA as the co-reactant while enhancing the cathodic emission, thereby realizing the ratiometric detection of miRNA-126 with a low detection limit of 29 aM (S/N = 3). The dual-ECL-emitting BP-CdTe QDs with TPrA-H2O2 as dual co-reactant provide a superior ECL ratio platform involving enzyme catalytic reaction, expanding the application of single-emitter-based ratio sensing in the diverse biological analysis.

Accurate sweat glucose detection is a promising alternative to invasive finger-prick blood tests, allowing for self-monitoring of blood glucose with good patient compliance. In this study, we have developed a tandem catalytic system, termed as a luminescent "nanochip" (LAON), which was composed of gold nanoparticles (AuNPs) and N-(aminobutyl)-N-(ethylisoluminol) (ABEI)-engineered oxygen-doped carbon nitride (O-g-C3N4), for chemiluminescent detection of sweat glucose. The LAON exhibits dual catalytic activity of glucose oxidase and peroxidase and can not only oxidize glucose to generate H2O2 but catalyze H2O2-mediated luminol chemiluminescence, resulting in sensitive detection of glucose. We identify that the LAON can precisely detect glucose with a detection limit of 0.1 μM, enabling us to measure glucose levels in different biological samples. Particularly, the LAON is capable of sensitively and accurately monitoring dynamic changes in sweat glucose during exercise. Therefore, the LAON provides an alternative approach to supersede invasive blood tests and may improve the management of diabetes mellitus.

At present, enzyme-mediated signal amplification strategies have been widely applied in photoelectrochemical (PEC) biosensing systems, while the introduction of natural enzymes onto the surface of photoelectrodes inevitably obstructs the electron transfer due to their insulating properties as proteins, leading to severe damage to photocurrent. In this work, the PdPt bimetallic nanozymes with the efficient peroxidase-like activity were used as alternatives to natural enzymes and amplified PEC biosensing signals via their efficient enzymatic reaction and remarkable enhancement in photocurrent. As a result, photoactive CdS nanorods modified with PdPt bimetallic nanozymes showed a boosted PEC performance compared with the pristine CdS nanorods due to the localized surface plasmon resonance effect and Schottky junction. On the basis of the as-prepared CdS/PdPt photoelectrode, a sensitive split-type glucose oxidase-mediated PEC immunoassay for carcinoembryonic antigen (CEA) detection was successfully constructed. Along with the sandwich immunocomplexing, the subsequently produced hydrogen peroxide (H2O2) can oxidize 4-chloro-1-naphthol into insoluble precipitates to inhibit photocurrent and simultaneously trigger the bio-etching of CdS to further restrain photocurrent signals due to the excellent peroxidase-mimicking activity of PdPt nanozymes. Owing to the synergistic signal amplification fulfilled by PdPt nanozymes, an ultrasensitive immunoassay of CEA was realized with a wider linear range from 1 to 5000 pg/mL and a low detection limit of 0.21 pg/mL, opening a new avenue for building ultrasensitive PEC biosensors with nanozymes.

Colorimetric glucose sensors using enzyme-coronated gold nanoparticles have been developed for high-throughput assays to monitor the blood glucose levels of diabetic patients. Although those sensors have shown sensitivity and wide linear detection ranges, they suffer from poor selectivity and stability in detecting blood glucose, which has limited their practical use. To address this limitation, herein, we functionalized glucose-oxidase-coronated gold nanoparticles with an erythrocyte membrane (EM-GOx-GNPs). Because the erythrocyte membrane (EM) selectively facilitates the permeation of glucose via glucose transporter-1 (GLUT1), the functionalization of GOx-GNPs with EM improved the stability, selectivity (3.3- to 15.8-fold higher), and limit of detection (LOD). Both membrane proteins, GLUT1 and aquaporin-1 (AQP1), on EM were shown to be key components for selective glucose detection by treatment with their inhibitors. Moreover, we demonstrated the stability of EM-GOx-GNPs in high-antioxidant-concentration conditions, under long-term storage (∼4 weeks) and a freeze-thaw cycle. Selectivity of the EM-GOx-GNPs against other saccharides was increased, which improved the LOD in phosphate-buffered saline and human serum. Our results indicated that the functionalization of colorimetric glucose sensors with EM is beneficial for improving selectivity and stability, which may make them candidates for use in a practical glucose sensor.

Bioorthogonal chemistry has been considered as a powerful tool for biomolecule labeling due to its site specificity, moderate reaction conditions, high yield, and simple post-treatment. Covalent coupling is commonly used to modify quantum dots (QDs) with bioorthogonal functional group (azide or cycloalkyne), but it has a negative effect in the decrease of QDs' quantum yield and stability and increase of QDs' hydrodynamic diameter. To overcome these disadvantages, we propose a novel method for the preparation of two kinds of clickable QDs by the strong interaction of -SH with metal ions. One system involves azide-DNA-functionalized QDs, which are used for bioconjugation with dibenzocyclooctyne (DBCO)-modified glucose oxidase (GOx) to form a GOx-QDs complex. After bioconjugation, the stability of QDs was improved, and the activity of GOx was also enhanced. The GOx-QDs complex was used for rapid detection of blood glucose by spectroscopy, naked eye, and paper-based analytical devices. The second system involves DBCO-DNA-functionalized QDs, which are used for an in situ bioorthogonal labeling of HeLa cells through metabolic oligosaccharide engineering. Therefore, these clickable QDs based on DNA functionalization can be applied for rapid and effective labeling of biomolecules of interest.

Diabetes is a metabolic syndrome associated with hyperglycemia, hypertension, atherosclerosis, and endothelial dysfunction. Applying the mechanical stretch on cells to simulate blood circulation while monitoring the cell glucose metabolism in a high-glucose environment is important for better comprehension of the underlying mechanisms of atherosclerosis caused by diabetes. Herein, we developed a facile strategy integrating zeolitic imidazolate framework-8-encapsulated glucose oxidase (GOx@ZIF-8) and an gold (Au) stretchable electrode (Au SE) to construct a flexible and stretchable glucose sensor (GOx@ZIF-8/Au SE) for investigating the glucose metabolism mechanism of stretched endothelial cells in hyperglycemia. The encapsulation of GOx with ZIF-8 prevents the aggregation and detachment of GOx from the sensing interface and endows the biosensor with high stability. Additionally, the Au SE with inherent stretchability can act as an integrated platform for mechanical stimulation as well as for transient signal sensing during the mechanotransduction process. Moreover, this flexible and stretchable glucose sensor is successfully used for monitoring the glucose metabolism of mechanically stimulated cells in hyperglycemia, and it was found for the first time that the glucose utilization ability of cells under static conditions is higher than that in the stretched state. This facile and straightforward method paves a promising route for designing a stable enzyme-based stretchable biosensor for detecting the underlying mechanisms of atherosclerosis caused by diabetes.

Capture, analysis, and inactivation of circulating tumor cells (CTCs) have emerged as important issues for the early diagnosis and therapy of cancer. In this study, an all-in-one sensing device was developed by integrating magnetic metal-organic framework (magMOF) nanoparticles (NPs) and TiO2 nanotube arrays (TiNTs). The magMOF NPs are composed of a magnetic Fe3O4 core and a MIL-100(Fe) shell, which is loaded with glucose oxidase (GOD) and provides an intensive starvation therapy by catalyzing the consumption of cellular nutrients, thus accelerating the generation of intracellular iron ions by MIL-100(Fe) dissolution. Importantly, these iron ions not only lead to an intensive Fenton-like reaction but also establish an excellent correlation of electrochemical intensities with cancer cell numbers. Owing to the intracellular magMOF NPs, the CTCs were magnetically collected onto TiNTs. The exogenous ·OH radicals generated by TiNT photocatalysis trigger iron ions to be rapidly released out and subsequently detected via differential pulse voltammetry using TiNTs as the electrode. An excellent correlation of differential pulse voltammetry intensities with CTC numbers is obtained from 2 to 5000 cell mL-1. This nanoplatform not only paves a way to combine starvation therapy agents with Fenton-like reaction for chemodynamic therapy but also opens up new insights into the construction of all-in-one chips for CTC capture and diagnosis.

A novel luminol electrochemiluminescence (ECL) sensing platform was developed based on the closed indium tin oxide bipolar electrode (ITO BPE) for detecting the biomarkers of squamous cell carcinomas named cytokeratin 19 fragments (CYFRA 21-1). A strategy of in situ-generated coreactant H2O2 and O2 was proposed to enhance luminol ECL intensity. CaO2, possessing a high capacity of self-supplying H2O2 and O2, was encapsulated by ZIF-67, glucose oxidase, and horseradish peroxidase (GOD/HRP-loaded ZIF-67@CaO2). In the presence of glucose, gluconic acid and H2O2 were generated via the catalytic effect of GOD; then, gluconic acid induced the degradation of ZIF-67, leading to the hydrolysis of the unprotected CaO2 to produce both O2 and H2O2. Therefore, the generated coreactants O2 and H2O2, via the oxidation of glucose and the hydrolysis of CaO2, can effectively enhance the luminol ECL behavior. According to the luminol ECL reaction occurring on the anode surface of the ITO BPE, a sandwich immunosensor was fabricated on the anodic ITO BPE and GOD/HRP-loaded ZIF-67@CaO2 as labels incubated with secondary antibodies. Owing to the cathodic reaction rate influencing the anodic reaction rate, Au nanoparticles were electrodeposited on the cathode surface of the ITO BPE, which not only meliorated oxygen reduction but also further enhanced the ECL intensity of luminol. Under optimal experiments, an ECL immunosensor for detecting CYFRA 21-1 with a wider linear range from 0.0075 to 50 ng mL-1 and a detection limit of 1.89 pg mL-1 (S/N = 3) was obtained.

Diabetes mellitus is one of the most common chronic diseases worldwide. Generally, the levels of fasting or postprandial blood glucose and other biomarkers, such as glycated albumin, glycated hemoglobin, and 1,5-anhydroglucitol, are used to diagnose or monitor diabetes progression. In the present study, we developed a sensor to simultaneously detect the glucose levels and glycation ratios of human serum albumin using a lateral flow assay. Based on the specific enzymatic reactions and immunoassays, a spiked glucose solution, total human serum albumin, and glycated albumin were measured simultaneously. To test the performance of the developed sensor, clinical serum samples from healthy subjects and patients with diabetes were analyzed. The glucose level and glycation ratios of the clinical samples were determined with reasonable correlation. The R-squared values of glucose level and glycation ratio measurements were 0.932 and 0.930, respectively. The average detection recoveries of the sensor were 85.80% for glucose and 98.32% for the glycation ratio. The glucose level and glycation ratio in our results were crosschecked with reference diagnostic values of diabetes. Based on the outcomes of the present study, we propose that this novel platform can be utilized for the simultaneous detection of glucose and glycation ratios to diagnose and monitor diabetes mellitus.

The present study demonstrates the use of highly stable single-molecule enzyme nanocapsules (SMENs) instead of traditional native enzyme as biorecognition element in enzyme-based biosensors. The main purpose of this study is to resolve the major obstacle and challenge in the biosensor field, i.e., the poor stability of enzyme-based biosensors, including thermal stability, organic solvent tolerance, long-term operational stability, etc. Highly active and robust SMENs of glucose oxidase (GOx, as a model enzyme) were synthesized (nGOx) using an in situ polymerization strategy in an aqueous environment. The particle-size distribution, transmission electron microscopic (TEM) images, and UV-vis spectral characterization revealed the formation of a thin polymer layer around each enzyme molecule. The polymer shell effectively stabilized the GOx enzyme core while enabling rapid substrate transportation, resulting in a new class of biocatalytic nanocapsules. Multiple covalent attachments between a thin polymer layer and an enzyme molecule strengthened the encapsulated GOx molecule. Encapsulation created a favorable microenvironment to avoid any structural dissociation at high temperature and helped to retain essential water during the organic solvent operation. The present work reports a study implementing nGOx SMENs as highly stable nano(bio)sensors for point-of-care diagnostic applications. Prepared nGOx SMENs manifested significantly improved thermal stability (even at 65 °C) and organic solvent tolerance without any compromise in biocatalytic activity. For example, the native GOx-based biosensor lost its catalytic activity for glucose after 4 h of incubation at high temperature (65 °C), while the nGOx/N-CNTs-Chi/GCE nano(bio)sensor maintained ∼56% of its original catalytic activity for glucose oxidation. The proposed SMENs-based nano(bio)sensors with robust stability in variable working environment could promote the development and applications of biosensors in point-of care diagnostics, biomedical detection, wearable devices, implantable equipment, and biofuel cells.

A chemiluminescence (CL) system with long persistent and intensive emission is essential for accurate CL quantitative analysis and imaging assay. However, with most known CL systems being flash-type, it is still a great challenge to develop long-lasting CL systems. Here, by combining an iron porphyrin metal-organic frameworks (FePorMOFs) based peroxidase mimic with natural glucose oxidase (GOx), an intensive and persistent CL system is presented on the basis of local tandem catalysis and surface diffusion of the nano-/bioenzymes (FePorMOF/GOx). FePorMOF synthesized by iron porphyrin linker and zirconium ion node possesses high peroxidase catalytic activity and stability. Using luminol and glucose as substrate, the FePorMOF/GOx CL system can produce intensive CL emission containing a plateau period of 7.5 h. The strong CL signal is due to the local tandem generation and reaction of H2O2 by GOx and FePorMOF, which avoids the diffusion-limited kinetics and leads to a high catalytic efficiency of the nano-/bioenzymes. On the other hand, the long persistent CL emission is attributed mainly to the enzymatic reaction-controlled H2O2 supply and surface diffusion-controlled CL reaction. The proposed CL system is explored for CL imaging sensing of glucose and homogeneous immunoassay of α-fetoprotein. The nano-/bioenzymes CL system exhibits intensive and long constant CL emission in physiological condition, showing promising applications in real-time bioassay and bioimaging.

Natural enzyme complex with the subunits cooperating with each other could catalyze cascade reactions in biological system but, just like the limitation of free-floating natural enzymes, usually suffer from deactivation in harsh environment such as high temperature. In this study, a purpose-driven design of amphiphilic aerogel working as the enzymes-immobilization substrate to form the multienzyme complex (MEC) was demonstrated. The aerogel was synthesized only by a single polymer poly(vinyl alcohol) (PVA) as well as a surface modulator maleic acid (MA), the incorporation of which tunes the surface wettability. The usage of the amphiphilic aerogel may do favor for multienzyme immobilization, conserving the enzyme conformation as well as stabilizing the enzymes in high temperature. As a typical example, glucose oxidase and hemin were firmly coimmobilized in the aerogel matrix and actively catalyze the cascade reactions of (i) glucose to gluconic acid and (ii) 3,3,5,5-tetramethylbenzidine (TMB) to its oxidized state. The enzymes could resist the degradation under high temperature (70-100 °C) which is witnessed by the rate of decrease in activity was progressively slackened. Taking the advantage of the chromogenic reaction of TMB, a glucose sensor based on aerogel-enzyme composite for glucose detection in whole blood and sweat was established, exhibiting reliable results and satisfactory recovery. The modified aerogel could also withstand multiple physical deformation meantime maintaining good adsorption capacity as well as catalytic performance. The enzymes-loading aerogel model may hopefully contribute to composing sensors based on other analytes.

The continuous detection of glucose is significant for revealing its role in neuron protection and for diagnosis of various diseases. In this study, for the first time, a nonenzymatic online optical detection platform (OODP) for glucose measurement in rat brain utilizing the tandem enzyme activity of V2O5 nanobelts is developed. V2O5 nanobelts were synthesized via a facile solvothermal strategy, and for the first time it is found that the V2O5 nanobelts possess dual enzyme-like activity, i.e., glucose oxidase (GOx)-like and peroxidase-like activity, and can act as a "tandem nanozyme". To investigate the mechanisms of the GOx-like property, we built an adsorption model, and the RPBE density functional calculations indicate that the glucose molecule can be adsorbed on the V2O5 plane. Based on the ability of V2O5 nanobelts to mimick tandem enzymes, a nonenzymatic online optical detection platform (OODP) for the continuous monitoring of glucose in rat brain was designed, which exhibits excellent stability, high selectivity, and a wide linear detection range from 0.2 to 5 mM and records cerebral glucose alterations in the calm/ischemia model. This facile but reliable nonenzymatic online optical glucose measurement compares favorably with natural enzyme-based online electrochemical glucose analytical systems, and its ready adoption by physiologists and pathologists will facilitate the understanding of brain function and the pathogenesis of diabetes.

Abnormal physiological levels of sialic acid (SA) could be used to diagnosis cancer progression stages. In this work, we describe an enzyme-assist-interference-free strategy for Raman selective determination of SA in serum. First, we assemble gold nanoparticles (Au NPs) onto the indium tin oxide glass (ITO) to construct an ITO/Au two-dimension substrate. Through modification of 4-mercaptoboric acid (4-MPBA) onto the surface of ITO/Au, the SA response plate is prepared due to the reversible esterification bond. In this strategy, a sandwich structure is rationally designed as ITO/Au/4-MPBA/SA/4-MPBA/Au to enhance the Raman scattering. The Raman detection linear concentration of SA ranged from 2.5 × 10-7 to 1.5 × 10-6 M, and a limit of detection about 1.2 × 10-7 M could be achieved. Considering the presence of glucose (Glu) in physiological fluid, we introduce glucose oxidase to remove the interference from Glu and realize the accurate determination of SA. The proposed novel Raman rapid method provides an ultrasensitive and interference-free protocol for the early diagnosis of cancer.

A self-powered temperature sensor based on Seebeck effect transduction was designed for photothermal-thermoelectric coupled immunoassay of α-fetoprotein (AFP). In this system, glucose oxidase (GOx)-conjugated detection antibody was first captured onto the microplate by target-induced sandwich-type immunoreaction. Thereafter, the as-generated hydrogen peroxide via the GOx-glucose system oxidized 3,3',5,5'-tetrametylbenzidine (TMB) into photothermal product oxidized TMB (ox-TMB). Under near-infrared (NIR) laser irradiation, the temperature change of ox-TMB was read out in an electrical signal by the flexible thermoelectric module in a 3D-printed integrated detection device. Under optimal conditions, the photothermal-thermoelectric coupled immunoassay exhibited a limit of detection of 0.39 ng mL

We demonstrate the application of open circuit potentiometry (OCP) to measure enzyme turnover kinetics, kturn. The electrode surface will become poised by the addition of a well-behaved redox pair, such as ferrocenemethanol/ferrocenium methanol (FcMeOH/FcMeOH+), which acts as the cosubstrate for the enzymatic process. A measurable change in potential results when an enzyme consumes the one-electron transfer mediator. Glucose oxidase was studied as a test-case, but the method is generalizable across oxidoreductase enzymes that rely on electron transfer mediators. In the presence of glucose and FcMeOH+, glucose oxidase delivers electrons to FcMeOH+, and the potential changes with respect to the Nernst equation. A theoretical model incorporating enzymatic rate expressions into the Nernst equation was derived to explain the observed potential transients, and experimental data fit theory well. A similar experiment was performed using amperometry on ultramicroelectrodes (UMEs). Here, the same enzymatic rate expression may be incorporated into the equation for steady-state flux to an UME to obtain kturn. While similar kinetic information was obtained from the potentiometric and amperometric responses, potentiometry is independent of electrode size and mass transfer effects. Finally, we show how kturn changes as a function of one-electron mediator. Our results may eventually find applications to biosensors, where electrode fouling plagues long-term sensor performance.

High-purity cellulose membranes prepared via a green pathway were used to attempt to enhance their performance of glucose detection compared with that of traditional filter paper. In this work, cellulose-based strips (CBS) for the low concentration of glucose detection have been designed based on a fast, sensitive, and easy enzyme colorimetric assay from porous and high-purity cellulose membranes (CM). Different from the traditional paper-based sensors that were made of commercially available filter paper, the cellulose-based membranes matrix was fabricated by a "green" route in that cellulose was dissolved in an aqueous NaOH/urea solution, and then modified by the sodium periodate oxidation method to immobilize the glucose oxidase (GOX) and horseradish peroxidase (HRP) with Schiff-base reaction. The structure and properties of CM and CBS were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray mapping (EDS), Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), etc. SEM images showed a porous, interpenetrating structure of CM, which benefited the enzyme immobilization and enzymatic reaction. When glucose solution was dropped onto the CBS, the color change from colorless to blue was only 5 min. The limit of detection (LOD) is 0.45 mM in the linear range of glucose from 1 to 11 mM. Moreover, the CBS had also been successfully used for glucose determination in real urine samples, and the color changes can be easily recorded by a simple camera to achieve the semiquantitative detection of glucose. This work provided a new design strategy for the cellulose-based functional materials which could be applied in biosensors, drug carriers, and biomedicine.

The chemical reaction in a confined space is known to be accelerated due to a high collision probability; however, the study of this confinement effect in a supersmall space down to femtoliter (fL) is seldom reported. Here, an adjustable volume [from picoliter (pL) to fL] of the aqueous phase is retrained at the tip of a nanopipette by an organic solvent so that the confinement effect on the specific activity of glucose oxidase is investigated. The activity is determined by the amount of hydrogen peroxide generated from the reaction between the oxidase and glucose using a nanoelectrode inside the nanopipette. As compared with the activity in bulk solution (82 U/mg), the activity increases up to 7500 U/mg in a 105 fL space. The 2 orders of magnitude increase in the enzymatic activity is the highest amplification in the volume-confined enzyme reaction as reported. A near-exponential drop in the activity is observed with the increase in the space volume, revealing the dominant enhancement in the confined space at the fL level for the first time. The established electrochemical nanopipettes should not only provide a strategy for the study of the enzymatic activity in supersmall confined space but also help understand the confinement effect of enzyme-catalyzed reactions.

Wearable biosensors for real-time and non-invasive detection of biomarkers are of importance in early diagnosis and treatment of diseases. Herein, a high-performance wearable biosensing platform was proposed by combining a three-dimensional hierarchical porous Au hydrogel-enzyme electrode with high biocompatibility, activity, and flexibility and soft-MEMS technologies with high precision and capability of mass production. Using glucose oxidase as the model enzyme, the glucose sensor exhibits a sensitivity of 10.51 μA mM-1 cm-2, a long durability over 15 days, and a good selectivity. Under the mechanical deformation (0 to 90°), it is able to maintain an almost constant performance with a low deviation of

Although single-atom catalysts with high enzyme-like activities have been found, the rational design of highly active peroxidase (POD)-like nanozymes is still a formidable challenge. Herein, highly active POD-like nanozymes were synthesized through loading Pt clusters on the Fe single-atom (FeSA-PtC) nanozymes. The POD-like activity of FeSA-PtC nanozymes is enhanced 4.5-fold and 7-fold, in comparison to that of FeSA and PtC nanozymes, respectively, which is attributed to the unexpected synergistic effect between Fe single atoms and Pt clusters. Based on the outstanding POD-like activity of FeSA-PtC nanozymes, a cascade signal amplification strategy was constructed by combining glucose oxidase for the colorimetric biosensing of prostate-specific antigens, exhibiting satisfactory sensitivity, high selectivity, a low detection limit of 1.8 pg/mL, and practical feasibility in serum sample detection. This work may serve as a tough foundation to guide the design of superior POD-like nanozymes and expand the application in biosensing.

Wearable epidermal sensors that can provide noninvasive and continuous analysis of metabolites and electrolytes in sweat have great significance for healthcare monitoring. This study reports an epidermal sensor that can wirelessly, noninvasively, and potentiometrically analyze metabolites and electrolytes. Potentiometry-based ion-selective electrodes (ISE) are most widely used for detecting electrolytes, such as Na+ and K+. We develop an enzyme-based glucose ISE for potentiometric analysis of sweat glucose. The glucose ISE sensor is obtained by modifying a glucose oxidase layer (GOD) on an H+ ISE sensor. GOD catalyzes glucose to generate H+. The generated H+ passes through the H+ selective membrane to change the potential of the electrode. We have fully examined the limit of detection, detecting range, and stability of our epidermal sensor. Meanwhile, using this epidermal sensor, we can easily analyze the relationship between blood glucose and sweat glucose. The concentration curve of sweat glucose can represent blood glucose concentration, significantly contributing to sports and chronic disease monitoring.

A sustainable three-electrode platform for affordable microfluidic electroanalysis is described. The device can be handmade using common tools and, facilitating broad applicability, is indefinitely reusable through simple surface polishing. Compact prototypes with Pt counter, Pt working, and Ag/AgCl reference electrode disks were combined with silicone lid plates containing a microchannel for electrolyte flow. Redox voltammetry/amperometry of excellent quality was achieved in static and flowing ferricyanide solutions, respectively. Modified with a glucose oxidase surface layer, base plate Pt WEs performed very well as amperometric biosensors for microfluidic blood glucose testing. The electrode system is recyclable, compatible with matching lid plate microchannels, and functionally adaptable regarding the constituent metal and electrode surface modifications. This asset combination makes the device a sustainable detection tool for microfluidic electroanalysis, with applications ranging from direct detection of redox-active analytes to bioreceptor-assisted biosensing. It avoids costly microfabrication with clean-room use, and the accessibility of microfluidic EC (bio)sensing is thus greatly increased, especially for users with restricted budgets.

Aflatoxin B1 (AFB1) contamination is one of the most critical global issues in food safety. The high carcinogenic nature necessitates rapid and specific methods for the determination of AFB1 in foodstuffs at ultratrace levels. Here, we report an enhanced bienzymatic chemiluminescence competitive immunoassay for ultrasensitive and high-throughput determination of AFB1. In this assay, protein G was first coated on the wells of a microplate for recognizing the Fc fragment of anti-AFB1 mAbs to reduce the antibody dosage and guarantee high immunological reaction efficiency. The target AFB1 competed with glucose oxidase labeled AFB1 for the limited anti-AFB1 mAbs in the wells of the microplate. p-Bromophenol was employed as an enhancer to obtain intense and long-lasting chemiluminescence. The utilization of an enhancer and bienzymatic catalysts effectively improved the detection sensitivity. The developed method offered a good linearity over 5 orders of magnitude, a detection limit of 5 pg L-1, and a relative standard deviation of 1.9% for AFB1. The application of the developed method to the analysis of grain samples gave quantitative recoveries from 94.0% to 97.0%. The developed method provides a universal platform for high-throughput, ultrasensitive, and high specific detection of pollutants or nutrients in foods.

Herein, an efficient target-activated enzyme cascade electrocatalysis with low background signal was employed to establish electrochemical biosensor for ultrasensitive detection of thrombin via regulating electrocatalytic efficiency between target-induced hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) and glucose oxidase (GOx). Impressively, only when the target thrombin was introduced, the HRP-mimicking DNAzyme acting simultaneously as electrochemical signal probe would be formed to activate high-efficiency enzyme cascade electrocatalysis for reducing background signal significantly, which could overcome the defect of inevitable high background signal during the detection of target in the traditional cascade electrocatalysis of two existing bioenzymes. In addition, the detection sensitivity could be further improved by regulating the side length of rigid DNA tetrahedron (TDN) scaffold anchored HRP-mimicking DNAzyme and GOx at adjacent vertices for high enzyme cascade electrocatalytic efficiency. Consequently, the proposed biosensor demonstrated a low detection limit down to 0.3 fM for target thrombin, which provided a promising method for ultrasensitive monitoring of biomolecules in sensing analysis and disease diagnosis.

Development of high-performance fiber-shaped wearable sensors is of great significance for next-generation smart textiles for real-time and out-of-clinic health monitoring. The previous focus has been mainly on monitoring physical parameters such as pressure and strains associated with human activities. Development of an enzyme-based non-invasive wearable electrochemical sensor to monitor biochemical vital signs of health such as the glucose level in sweat has attracted increasing attention recently, due to the unmet clinical needs for the diabetic patients. To achieve this, the key challenge lies in the design of a highly stretchable fiber with high conductivity, facile enzyme immobilization, and strain-insensitive properties. Herein, we demonstrate an elastic gold fiber-based three-electrode electrochemical platform that can meet the aforementioned criteria toward wearable textile glucose biosensing. The gold fiber could be functionalized with Prussian blue and glucose oxidase to obtain the working electrode and modified by Ag/AgCl to serve as the reference electrode; and the nonmodified gold fiber could serve as the counter electrode. The as-fabricated textile glucose biosensors achieved a linear range of 0-500 μM and a sensitivity of 11.7 μA mM-1 cm-2. Importantly, such sensing performance could be maintained even under a large strain of 200%, indicating the potential applications in real-world wearable biochemical diagnostics from human sweat.

Artificial self-propelled micromachines have shown great promise in biomedical sciences. In this work, we use Mg/Pt Janus micromotors with self-rejuvenating surfaces to enhance the electrochemical sensing performance and sensitivity toward glucose in human serum. The detection of glucose is based on the glucose oxidase enzyme and ferrocenemethanol shuttle system, where mass transfer was dramatically enhanced by the rapid motion of Mg/Pt Janus micromotors. The obtained chronoamperometric data show that Mg/Pt Janus micromotors play a synergistic role in enhancing the current response at millimolar concentrations of glucose in human serum. The current signals increased with the corresponding increase in amount of micromotors introduced. Furthermore, a linear relationship between current signal and glucose concentration was established, while the limit of detection improved when mobile Mg/Pt Janus micromachines were used. Glucose detection enhanced by micromachines may pave the way for their future applications in biomedicine and medical diagnostic devices. Agency for Science, Technology and Research (A*STAR) This work was supported by the project Advanced Functional Nanorobots (reg. CZ.02.1.01/0.0/0.0/15_003/0000444 financed by the EFRR). The authors acknowledge A*STAR Grant SERC A1783c0005 (Singapore). L.K. acknowledges the Scholarship Fund from the China Scholarship Council (CSC).

Hepatocytes help to maintain glucose homeostasis in response to a variety of signals, including pancreatic hormones such as insulin. Insulin is released from the pancreas with variable dynamics, yet the role that these play in regulating glucose metabolism in the liver is still unclear. In this study, a modular microfluidic system was developed to quantitatively measure the effect of insulin dynamics on glucose consumption by a human hepatocarcinoma cell line, HepG2. A microfluidic bioreactor that contained 106 HepG2 cells was cultured for up to 10 days in an incubator. For glucose consumption experiments, the bioreactor was removed from the incubator and connected with reagents for an enzymatic glucose assay. The mixed components were then delivered into a droplet-based microfluidic system where the intensity of the fluorescent product of the enzyme assay was used to quantify the glucose concentration. By optimizing the mixing time of the reagents, the dynamic range of the enzymatic assay was adjusted to 0–12 mM glucose and had a time resolution of 96 ± 12 s. The system was used to observe rapid changes in insulin-induced glucose consumption from HepG2 cells. This assay format is versatile and can be expanded to measure a variety of hepatic metabolites, such as lactate, pyruvate, or ketone bodies, which will enable the correlation of pancreatic hormone dynamics to liver metabolism.

In this work, a rigid DNA tetrahedron (TDN) scaffold was synthesized to precisely control the interenzyme distance by randomly anchoring two pairs of horseradish peroxidase (HRP)/glucose oxidase (GOx) at the vertices of TDN, which could not only avoid the drawbacks of poor controllability and biocompatibility from traditional scleroid skeletons, but also overcome the defect of imprecise regulation for interenzyme distance caused by DNA origami. Impressively, by varying the side length of TDN scaffold, the interenzyme distance was precisely regulated, thus, an optimal TDN scaffold with highest catalytic efficiency was acquired and subsequently applied for constructing an ultrasensitive biosensor for DNA detection with a low detection limit of 3 fM. This strategy paved an avenue for developing new reliable scaffold to precisely regulate the catalytic efficiency of enzyme cascade reaction with ultimate applications in bioanalysis and clinical diagnosis.

Continuous glucose monitoring (CGM) allows type I and II diabetes patients to track changes in their glucose levels, allowing detection of impending hypoglycemia or hyperglycemia. Polymer dots (Pdots) are candidates for use in implanted CGM systems due to their exceptional brightness, photostability, sensitivity, and biocompatibility. However, Pdot glucose transducers are oxygen-dependent, and changes in tissue oxygen levels affect their measurement accuracy. Here, we describe an external ratiometric calibration method that corrects for changes in tissue oxygen levels to improve measurement accuracy. This method uses the ratio of oxygen concentrations inside and outside the Pdot glucose transducer as an indicator of glucose concentration to correct for signal deviations caused by tissue oxygen fluctuations. A second oxygen-sensitive Pdot that is not conjugated with glucose oxidase is used to measure the oxygen concentration outside the Pdot glucose transducer. We describe the theoretical basis for this approach and demonstrate its effectiveness experimentally in a subcutaneous mouse implant model. This external ratiometric system achieves higher accuracy glucose measurements than previous Pdot-based CGM systems and comparable accuracy to current commercial CGM systems, demonstrating the utility of the external ratiometric calibration strategy.