Search

Immobilizing enzymes into microcarriers is a strategy to improve their long-term stability and reusability, hindered by (UV) light irradiation. However, in such approaches, enzyme-substrate interaction is mediated by diffusion, often at slow kinetics. In contrast, enzyme-linked self-propelled motors can accelerate this interaction, frequently mediated by the convection mechanism. This work reports on a new photosensitive polymeric Janus micromotor (JM) for UV-light protection of enzymatic activity and efficient degradation of substrates accelerated by the JMs. The JMs were assembled with UV-photosensitive modified chitosan, co-encapsulating fluorescent-labeled proteins and enzymes as models and magnetite and platinum nanoparticles for magnetic and catalytic motion. The JMs absorbed UV light, protecting the enzymatic activity and accelerating the enzyme-substrate degradation by magnetic/catalytic motion. Immobilizing proteins in photosensitive JMs is a promising strategy to improve the enzyme's stability and hasten the kinetics of substrate degradation, thereby enhancing the enzymatic process's efficiency.

We report enzyme-powered upconversion-nanoparticle-functionalized Janus micromotors, which are prepared by immobilizing uricase asymmetrically onto the surface of silicon particles, to actively and rapidly detect uric acid. The asymmetric distribution of uricase on silicon particles allows the Janus micromotors to display efficient motion in urine under the propulsion of biocatalytic decomposition of uric acid and simultaneously detect uric acid based on the luminescence quenching effect of the UCNPs modified on the other side of SiO 2 . The efficient motion of the motors greatly enhances the interaction between UCNPs and the quenching substrate and improves the uric acid detection efficiency. Overall, such a platform using uric acid simultaneously as the detected substrate and motion fuel offers considerable promise for developing multifunctional micro/nanomotors for a variety of bioassay and biomedical applications.

A sensitive detection of carbohydrate antigen 15-3 (CA15-3) levels may allow for early diagnosis and monitoring the treatment of breast cancer, but this can only be made in routine clinical practice if low-cost immunosensors are available. In this work, we developed a sandwich-type electrochemical immunosensor capable of rapid detection of CA15-3 with an ultra-low limit of detection (LOD) of 0.08 fg mL -1 within a wide linear concentration range from 0.1 fg mL -1 to 1 µg mL -1 . The immunosensor had a matrix of a layer-by-layer film of Au nanoparticles and reduced graphene oxide (Au-rGO) co-electrodeposited on screen-printed carbon electrodes (SPCE). The high sensitivity was achieved by using secondary antibodies (Ab 2 ) labeled with horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H 2 O 2 ) as signal amplifiers, and hydroquinone (HQ) was used as an electron mediator. The immunosensor was selective for CA15-3 in human serum and artificial saliva samples, robust, and stable to permit storage at 4 °C for more than 30 days. With its high performance, the immunosensor may be incorporated into future point-of-care (POC) devices to determine CA15-3 in distinct biological fluids, including in blood and saliva samples., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A novel approach for trace detection of fipronil with a molecularly imprinted electrochemiluminescence sensor (MIECLS) is proposed. The sensitivity is significantly improved via signal amplification of the enzymatic reaction of horseradish peroxidase (HRP) released from encapsulated liposomes which linked onto the template molecules after rebinding. The molecularly imprinted polymer membrane was prepared through the electropolymerization of monomers with fipronil as a template. After the elution of the template molecules, the analyte fipronil was reabsorbed into the cavities. HRP-encapsulated liposomes were linked to the target molecules by light-triggered click reaction. The higher the concentration of the target was, the more HRP-encapsulated liposomes were present on the molecularly imprinted polymer (MIP) sensor. Then, HRP was liberated from liposomes, and the catalytic degradation of hydrogen peroxide (H 2 O 2 ) by HRP occurs, which changed the electrochemiluminescence intensity of luminol significantly. The change of the ∆ECL intensity was linearly proportional to the logarithm of the fipronil concentration ranging from 1.00 × 10 -14 to 1.00 × 10 -9  mol/L, and the detection limit was 7.77 × 10 -16  mol/L. The recoveries obtained ranged from 95.7 to 105.8% with RSD < 5%. The sensitivity of the detection was significantly improved, and the analysis process was simplified in that the incubation step required in the conventional method was avoided. The sensor proposed provides a feasible platform for ultra-trace amount determination., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Polyaniline (PANI) was functionalized by thiol-ene click chemistry to obtain carboxylic acid-tethered polyaniline (PCOOH). The versatility of PCOOH as an immobilization matrix was demonstrated by constructing four different biosensors for detection of metabolites and cancer biomarker. Immobilization efficiency of PCOOH was investigated by surface plasmon resonance and fluorescence microscopic analysis which revealed dense immobilization of biomolecules on PCOOH as compared to conventional PANI. A sandwich electrochemical biosensor was constructed using PCOOH for detection of liver cancer biomarker, α-fetoprotein (AFP). The sensor displayed sensitivity of 15.24 µA (ng mL -1 ) -1  cm -2 , with good specificity, reproducibility (RSD 3.4%), wide linear range (0.25-40 ng mL -1 ) at - 0.1 V (vs. Ag/AgCl), and a low detection limit of 2 pg mL -1 . The sensor was validated by estimating AFP in human blood serum samples where the AFP concentrations obtained are consistent with the values estimated using ELISA. Furthermore, utilization of PCOOH for construction of enzymatic biosensor was demonstrated by covalent immobilization of glucose oxidase, uricase, and horseradish peroxidase (HRP) for detection of glucose, uric acid, and H 2 O 2 , respectively. The biosensors displayed reasonable sensitivity (50, 148, 127 µA mM -1  cm -2 ), and linear ranges (0.1-5, 0.1-6, 0.1-7 mM) with a detection limit of 10, 1, and 8 µM for glucose, uric acid, and H 2 O 2 , respectively. The present study demonstrates the capability of PCOOH to support and enable oxidation of H 2 O 2 generated by oxidase enzymes as well as HRP enzyme catalyzed reduction of H 2 O 2 . Thus, PCOOH offers a great promise as an immobilization matrix for development of high-performance biosensors to quantify a variety of other disease biomarkers. Carboxylic acid-tethered polyaniline synthesized by thiol-ene click chemistry was used as matrix to construct four different electrochemical biosensors for detection of cancer biomarker α-fetoprotein, glucose, uric acid, and H 2 O 2 ., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

An electroanalytical platform capable to take and dilute the sample has been designed in order to fully integrate the different steps of the analytical process in only one device. The concept is based on the addition of glass-fiber pads for sampling and diluting to an electrochemical cell combining a paper-based working electrode with low-cost connector headers as counter and reference electrodes. In order to demonstrate the feasibility of this all-in-one platform for biosensing applications, an enzymatic sensor for glucose determination (requiring a potential as low as -0.1 V vs. gold-plated wire by using ferrocyanide as mediator) was developed. Real food samples, such as cola beverages and orange juice, have been analyzed with the bioelectroanalytical lab-on-paper platform. As a proof-of-concept, and trying to go further in the integration of steps, sucrose was successfully detected by depositing invertase in the sampling strip. This enzyme hydrolyzes sucrose into fructose and glucose, which was determined using the enzymatic biosensor. This approach opens the pathway for the development of devices applying the lab-on-paper concept, saving costs and time, and making possible to perform decentralized analysis with high accuracy., (© 2021. The Author(s).)

Versatile sensing matrixes are essential for the development of enzyme-immobilized optical biosensors. A novel three-dimensional titanium dioxide nanotubes/alginate hydrogel scaffold is proposed for the detection of sweat biomarkers, lactate, and glucose in artificial sweat. Hydrothermally synthesized titanium dioxide nanotubes were introduced to the alginate polymeric matrix, followed by cross-linking nanocomposite with dicationic calcium ions to fabricate the scaffold platform. Rapid colorimetric detection (blue color optical signal) was carried out for both lactate and glucose biomarkers in artificial sweat at 4 and 6 min, respectively. The superhydrophilicity and the capillarity of the synthesized titanium dioxide nanotubes, when incorporated into the alginate matrix, facilitate the rapid transfer of the artificial sweat components throughout the sensor scaffold, decreasing the detection times. Moreover, the scaffold was integrated on a cellulose paper to demonstrate the adaptability of the material to other matrixes, obtaining fast and homogeneous colorimetric detection of lactate and glucose in the paper substrate when image analysis was performed. The properties of this new composite provide new avenues in the development of paper-based sensor devices. The biocompatibility, the efficient immobilization of biological enzymes/colorimetric assays, and the quick optical signal readout behavior of the titanium dioxide nanotubes/alginate hydrogel scaffolds provide a prospective opportunity for integration into wearable devices.

Magnetic field-induced synthesis and biocatalysis of magnetic materials have inspired great interest due to the flexible controllability of morphologies and unique magnetoelectrical properties. However, the interaction of the magnetic field and the reaction kinetics during the synthesis of magnetic nanochains has not been revealed. The collective motions in fluids and the multifunctional enhancements for bioreaction of 3D magnetic-controlled nanochains have not been systematically researched. Here, an integrated 3D magnetic control method was reported for the synthesis, collective motion, and multifunctional bioreaction enhancement of peasecod-like nanochains. The interactions of magnetic field and reaction kinetics were rationally controlled to synthesize magnetic nanochains of different morphologies. Collective motions of nanochains under alternating magnetic fields were studied to provide insights into the disturbance on confined fluids. Three mechanisms of reaction enhancement of nanostir, magnetic agent, and nanocatalyst were achieved simultaneously via 3D magnetic-controlled nanochains using a glucose oxidase-horseradish peroxidase multi-enzyme system. The peasecod-like nanochain also exhibited excellent reaction enhancement in cell-free protein synthesis reaction, which is desired for effective high-throughput screening. The integrated 3D magnetic control method through the whole process from fabrication to applications of magnetic nanomaterials could be extended to multifunctional biocatalysis and multi-task biomedicine.

A photoelectrochemical (PEC) biosensor capable of detecting cytokeratin 19 fragment 21-1 (CYFRA21-1) was optimized by taking advantage of the powerful conjugate repeats of horseradish peroxidase and tyramine (HRP-tyramine)-triggered enzymatic biocatalytic precipitation (BCP) on high-performance BiVO 4 /Ag 3 VO 4 /SnS 2 photoelectrodes. Compared with the ubiquitous BCP strategy, we identified a design supporting conjugate repeats generated by HRP and tyramine-triggered immeasurable insoluble precipitates in the presence of hydrogen peroxide and 4-chloro-1-phenol (4-CN), and the steric hindrance improved sensitivity. Moreover, by virtue of BiVO 4 , Ag 3 VO 4 , SnS 2 excellent level matching structure and chemical stability, a heterojunction (BiVO 4 /Ag 3 VO 4 /SnS 2 ) with high light absorption efficiency has been successfully prepared. The novel heterostructure system of BiVO 4 /Ag 3 VO 4 /SnS 2 with high detection current and low background signal exhibited high-performance PEC determination. Generally, the hitherto untapped biosensor resource realized the sensitive detection of CYFRA21-1 with a wide linear range from 50 fg/mL to 200 ng/mL, and a detection limit of 15 fg/mL, which illustrated the potential for biotechnological applications., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Enzyme immobilization plays an essential role in solving the problems of the inherently fragile nature of enzymes. Although prominent stability and reuse of enzymes can be achieved by enzyme immobilization, their bioactivity and catalytic efficiency will be adversely affected. Herein, PdCu hydrogel nanozymes with a hierarchically porous structure were used to immobilize horseradish peroxidase (HRP) to obtain PdCu@HRP. In addition to the improvement of stability and reusability, PdCu@HRP displayed synergistically enhanced activities than native HRP and PdCu hydrogels. Not only the specific interactions between PdCu hydrogel nanozymes and enzymes but also the enrichment of substrates around enzymes by electrostatic adsorption of hydrogels was proposed to expound the enhanced catalytic activity. Accordingly, by taking advantage of the excellent catalytic performance of the PdCu@HRP and the glucose oxidase encapsulated in zeolitic imidazolate framework-8, colorimetric biosensing of the carcinoembryonic antigen via catalytic cascade reactions for achieving signal amplification was performed. The obtained biosensor enhanced the detection sensitivity by approximately 6.1-fold as compared to the conventional HRP-based enzyme-linked immunosorbent assay, demonstrating the promising potential in clinical diagnosis.

A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Metal-organic frameworks (MOFs) have recently emerged as excellent hosting matrices for enzyme immobilization, offering superior physical and chemical protection for biocatalytic reactions. However, for multienzyme and cofactor-dependent biocatalysis, the subtle orchestration of enzymes and cofactors is largely disrupted upon immobilizing in the rigid crystalline MOF network, which leads to a much reduced biocatalytic efficiency. Herein, we constructed hierarchically porous MOFs by controlled structural etching to enhance multienzyme and cofactor-dependent enzyme biocatalysis. The expanded size of the pores can provide sufficient space for accommodated enzymes to reorientate and spread within MOFs in their lower surface energy state as well as to decrease the inherent barriers to accelerate the diffusion rate of reactants and intermediates. Moreover, the developed hierarchically porous MOFs demonstrated outstanding tolerance to inhospitable surroundings and recyclability., (© 2020 Wiley-VCH GmbH.)

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

Various organelles (e.g., mitochondria and chloroplasts) have a multicompartment structure, providing superior function of material transformation, selective segregation and energy conversion. Enlightened by the elegant evolution of nature, intended isolation of the biochemical process by cooperative multicompartments in cells has become an appealing blueprint to construct bioreactors. In this study, we develop a "soft separation" way to establish a delicate multicompartment multienzyme system (MMS) with polyphenol-encapsulated enzyme-DNA conjugates, which are anchored on magnetic Janus particles, providing a biomimetic catalysis network with the model cascade reactions in confinement. The well-designed MMS exhibits preferable bioactivity benefitting from the dependable DNA bridges and the oriented immobilization of enzymes, while the polyphenol shell further protects the anchored enzymes from exterior attacks, such as heat and enzymatic degradation. Moreover, by applying the MMS as nanomotors, the asymmetrical distribution of enzymes on Janus particles is found to improve mutual elevation between the self-driven locomotion and enzyme-mediated reactions, delivering enhanced dispersal ability and bioactivity. Owing to the excellent enzymatic activity, promoted stability and satisfying biocompatibility, the assembled MMS is proved to be promising for the in vitro and intracellular sensing of glucose, showing significant potential for biochemical analysis applications.

Formation of a thermally stiffening microemulsion-based gel showing a nanoconfinement effect of carbohydrates in terms of microviscosity and hydrodynamic diameter of the reverse micelle (specifically with sucrose) is reported. The advantage of this gel as an efficient batch bioreactor for entrapped enzymes (horseradish peroxidase and thermophilic α-glucosidase) was shown, and illustrated its potential biocatalytic application at high temperatures.

An ultrasensitive sandwich electrochemiluminescence (ECL) biosensor was designed for determination of concanavalin A (ConA) through the specific carbohydrate-ConA interactions. Three-dimensional porous metal-organic framework (Zn-MOF) was synthesized, which loaded a large amount of luminescent reagents as luminol by encapsulating into its pores to form Zn-MOF@luminol complex. Interestingly, Zn-MOF also acted as the coreactant accelerator in the luminol-H 2 O 2 ECL system. This Zn-MOF@luminol complex was used as the signal probe to achieve a super strong and stable ECL signal. In addition, three-dimensional hierarchical molybdenum disulfide nanoflower and multiwalled carbon nanotubes complex (MoS 2 NF@MWCNTs) with peroxidase-mimicking enzyme property were used as a substrate to modify the glassy carbon electrode to further enhance the ECL signal of luminol by promoting decomposition of H 2 O 2 into reactive oxygen species (ROSs). In addition to the horseradish peroxidase (HRP) catalysis effect on the luminol ECL signal, a triple amplified ConA sandwich ECL sensor with high sensitivity sensor was constructed. The linear range for ConA detection was from 0.5 pg/mL to 100 ng/mL with a detection limit of 0.3 pg/mL (S/N = 3). The recovery test for ConA in human serum samples was performed with satisfactory results. Graphical abstract.

Graphitic carbon nitride (g-C 3 N 4 ) as an outstanding photoresponsive nanomaterial has been widely used in biosensing. Other than the conventional single channel sensing mode, a triple-channel sensing array was developed for high discrimination of proteins based on the photoresponsive g-C 3 N 4 . Besides the photoluminescence and Rayleigh light scattering features of g-C 3 N 4 , we exploit the new photosensitive colorimetry of g-C 3 N 4 as the third channel optical input. The triple-channel optical behavior of g-C 3 N 4 can be synchronously changed after interaction with the protein, resulting in the distinct response patterns related to each specific protein. Such a triple-channel sensing array is demonstrated for highly discriminative and precise identification of nine proteins (hemoglobin, trypsin, lysozyme, cytochrome c, horseradish peroxidase, transferrin, human serum albumin, pepsin, and myoglobin) at 1 μM concentration levels with 100% accuracy. It also can discriminate proteins being present at different concentration and protein mixtures with different content ratios. The practicability of this sensor array is validated by high accuracy identification of nine proteins in human urine samples. This indicates that the array has a great potential in terms of analyzing biological fluids. Graphic abstract .

Fumonisins (FBs) exist widely in crops, foods and feeds. Consumption of FBs contaminated corn can cause oesophageal cancer. So it is necessary to develop sensitivity methods for its detection. Here, we report an enhanced assay for rapid and ultra-sensitive detection of FBs based on nanomagnetic beads (NMBs) and antibody-biotin-streptavidin-HRP. Because antibody-BNHS can bind with several number of streptavidin-HRP, the signal amplification of the catalytical oxidation of TMB was enhanced. The detection limit of sensor was down to 0.21 ng mL -1 with a linear range from 0.31 to 162.42 ng mL -1 . Since NMBs provide a nearly "in solution" reaction, they lead to a shortened reaction time (22 min) than that of flat solid-phase based traditional assay. Furthermore, the recoveries obtained by standard FBs spiked to maize samples were from 100.6 to 107.3%. This enhanced assay supplied a rapid, sensitive and reliable method for detection of FBs in maize., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

A novel nanobody (Nb)-based voltammetric immunosensor coupled with horseradish peroxidase concatemer-modified hybridization chain reaction (HRP-HCR) signal amplifying system is described to realize the rapid and ultrasensitive detection of AFB 1 . To design such an immunoassay, anti-AFB 1 Nbs with smaller molecular size were coated densely onto the surface of Au nanoparticle-tungsten disulfide-multi-walled carbon nanotubes (AuNPs/WS 2 /MWCNTs) functional nanocomposites as an effective molecular recognition element, whereas AFB 1 -streptavidin (AFB 1 -SA) conjugates were ingeniously bound with biotinylated HCR dsDNA nanostructures as the competitor, amplifier, and signal report element. In the presence of AFB 1 targets, a competitive immunoreaction was performed between the analyte and AFB 1 -SA-labeled HCR (AFB 1 -HCR) platform. Upon the addition of SA-modified polyHRP (SA-polyHRP), AFB 1 -HCR nanostructures containing abundant biotins were allowed to cross-link to a quantity of HRP by streptavidin-biotin chemistry for signal amplification and signal conversion. Under optimal conditions, the immunosensor displayed a good linear correlation toward AFB 1 ranging from 0.5 to 10 ng mL -1 with a sensitivity of 2.7 μA • (mL ng -1 ) and an ultralow limit of detection (LOD) of 68 fg mL -1 . The specificity test showed that the AFB 1 immunosensor had no obvious cross-reaction with OTA, DON, ZEN, and FB 1 . The signal of this sensor decreased by 10.18% in 4 weeks indicating satisfactory stability, and its intra- and inter-laboratory reproducibility was 3.42~10.35% and 4.03%~12.11%, respectively. This biosensing system will open up new opportunities for the detection of AFB 1 in food safety and environmental analysis and extend a wide range of applications in the analysis of other small molecules. Graphical abstract.

A biomimetic antibody is described for colorimetric determination of glycoprotein, and horseradish peroxidase (HRP) is used as model analyte. Use is made of oriented surface imprinted inverse opal hydrogel particles functionalized with phenylboronic acid. The inverse opal hydrogel particles were negatively replicated from silica colloidal crystal beads (SCCBs), so that they were endowed with larger specific surface area than the bulk structure. Benefit from that, there were abundant surface molecularly imprinting sites in the hydrogel particles. Because the imprinting sites match the structure of the template molecules, they can recognize HRP with high selectivity and sensitivity. The recognized glycoprotein was bonded with the phenylboronic acid within the sites. The bonded HRP was determined by colorimetry of 3, 3', 5, 5'-tetramethylbenzidine (TMB) single-component solution at 450 nm, and it shows a 16.03 imprinting factor under optimized conditions. Alpha-fetoprotein (AFP) was also investigated and demostrated the value of this strategy in practical applications. The results show that the absorbance increases linearly in the 1-50 ng mL -1 AFP concentration range and has a 1.32 ng mL -1 detection limit. The assay of human serum was realized by the standard addition method. This strategy is promising to open new horizons for glycoprotein assay. Graphical abstract Schematic representation of the preparation of oriented boronate affinity-imprinted inverse opal hydrogel particles for glycoprotein assay.

HRP@ZIF-8 nanocomposite was prepared by in situ encapsulation of horseradish peroxidase (HRP) in the frame of zeolitic imidazolate framework-8 (ZIF-8) with a simple one-pot method. The HRP@ZIF-8 nanocomposite displays outstanding thermal stability and efficiently catalyzes the chemiluminescence (CL) reaction of luminol with hydrogen peroxide (H 2 O 2 ) under near-neutral pH condition (pH 7-8). This CL system has a good response to H 2 O 2 with a linear range of 0.1-100.0 μmol L -1 . The limit of detection (LOD) is 0.06 μmol L -1 H 2 O 2 . By marriage with cholesterol oxidase, cholesterol is determined with a linear range from 0.1 to 100.0 μmol L -1 and a LOD of 0.04 μmol L -1 . The relative standard deviations (RSD) are 1.7% and 2.5%, respectively, in 11 repeated measurements of 50.0 μmol L -1 solutions of H 2 O 2 and cholesterol, indicating excellent precision of the method. The method shows good selectivity and has been applied to the determination of total cholesterol in real serum samples. No significant difference has been observed between the results obtained by this method and the cholesterol oxidase-peroxidase coupling method. Graphical abstract Schematic presentation of in situ one-pot synthesis of horseradish peroxidase@zeolitic imidazolate framework-8 (HRP@ZIF-8) nanocomposite and chemiluminescence determination of cholesterol with HRP@ZIF-8 catalyzing luminol-H 2 O 2 system.

Polymersome nanoreactors encapsulating the enzymes or particulate catalysts attract interest because of their potential use as modular reactors to synthesize complex compounds via a cascade of chemical reactions in a single batch. To achieve these goals, a key requirement is the tunable permeability of the polymersome membrane, which allows the size-selective transportation of reagents and products while protecting the encapsulated catalysts during the chemical reaction. We report here a stimuli-responsive route for controlling the permeability of the polymersomes of the binary blend of poly(ethylene glycol)- b -polystyrene (PEG- b -PS) and poly(ethylene glycol)- b -poly(acrylbenzylborate) (PEG- b -PABB). The presence of H 2 O 2 (1 mM) in the medium (0.1 M PBS, pH 7.4) triggers the oxidation of benzyl borate pendants of PABB to form poly(acrylic acid) (PAA). This transformation results in the perforation of the compartmentalizing membrane of polymersomes by the dissolution of PEG- b -PAA domains embedded in the inert PEG- b -PS matrix. By controlling the composition of the stimuli-responsive block copolymer, the polymersomes of the binary blend exhibit size-selective permeability without losing the structural integrity. Release of fluorescent guests with different sizes (fluorescein, PEG2k- Cm , PEG5k-Rho) can be controlled by tuning the composition (PEG- b -PS/PEG- b -PABB = 100/0-80/20) of blended polymersomes. Selective permeability of the membrane provides protection of the encapsulated enzymes from external proteases present in the medium, resulting in the one-pot synthesis of small molecules via cascades of chemical reactions. The nanoparticular catalysts are also encapsulated within the permeable polymersomes, serving as modular reactors for the conversion of organic compounds via a cascade of reactions.

A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results showed that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A biosensor for hydrogen peroxide (H 2 O 2 ) has been developed based on the use of MoS 2 nanosheets and graphite that are assembled to form a microfiber hybrid structure. The MoS 2 nanosheets are synthesized in situ on a graphite microfiber. The chemical composition and surface morphology of the microfiber hybrid structure has been characterized. The microfiber is shown to display peroxidase-mimicking activity. In the next step, horseradish peroxidase, methylene blue, and chitosan are co-immobilized on the microfiber electrode. The use of MoS 2 nanosheets warrants high electrochemical activity of immobilized enzyme on the electrode surface. The modified microfiber electrode, best operated at a voltage of - 0.3 V (vs. Ag/AgCl), can be used to sense H 2 O 2 with a linear response in the 0.1 to 90 μM concentration range and with a determination limit of 30 nM (at S/N = 3). The good response is attributed to the synergistic enhancement of the synthetic nanozymes (few-layered MoS 2 nanosheets) and immobilized natural horseradish peroxidase (HRP). Grapical abstract.

DNA aptamers that bind to bovine pregnancy-associated glycoproteins (bPAGs) were selected by the systematic evolution of ligands by exponential enrichment (SELEX) procedure coupled to surface plasmon resonance (SPR) and high-throughput sequencing (HTS) technology. After seven rounds of selection using carboxylated magnetic beads (MB) coated with bovine pregnancy-associated glycoproteins 9 (bPAG9) and bovine serum albumin (BSA) as target and counter targets, respectively, two aptamers designated as A1 and A24 showed high affinities to bPAG9 (K d  = 1.04 and 2.5 nM). Moreover, the specificity was determined by testing the non-targets bPAG4, bPAG6, bPAG16, BSA, and ovalbumin (OVA). Results showed that two aptamers demonstrated broad group specificity to bPAG family. Subsequently, a colorimetric sandwich enzyme-linked aptamer assay was developed for ultrasensitive detection of bPAG9 based on hybridization chain reaction (HCR) amplification strategy. The method exhibited a broad determination from 0.134 to 134 ng/mL with a detection limit of 0.037 ng/mL. The method has been successfully applied to determine bPAGs in real samples. The results demonstrate that the developed aptamers could be used as promising molecular probes for the development of pregnancy diagnostic tools. Graphical abstract In this study, we first selected aptamers against bovine pregnancy-associated glycoproteins (bPAGs) as molecular recognition elements and then developed a colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction to detect bPAGs in the serum.The GA can't be deleted, the modified GA can not upload. So themodified GA and figures will be send to CorrAdmin3@spi-global.com.

A ratiometric fluorescent assay is fabricated for the evaluation of alkaline phosphatase (ALP) activity. This assay is composed of ionic liquid-functionalized carbon dots (IL-CDs) with blue fluorescence signal at 470 nm and 2,3-diaminophenazine (DAP) with yellow fluorescence signal at 570 nm. IL-CDs were synthesized via electrochemical method by using ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate) and ultrapure water as precursors. DAP is produced by the oxidation reaction between o-phenylenediamine and H 2 O 2 under the catalysis of horseradish peroxidase. H 2 O 2 is reduced by ascorbic acid which is the hydrolysis product of ascorbic acid 2-phosphate under the catalysis of ALP, finally reducing the amount of DAP. The activity of ALP is evaluated through the ratiometric fluorescent signal between IL-CDs and DAP via Förster resonance energy transfer. Under optimal experimental conditions, this ratiometric fluorescent assay has a response that covers the 0.04 to 3.2 U L -1 (12 to 960 pM) ALP activity. This assay possesses ultralow detection limit of 0.012 U L -1 (3.6 pM) for ALP and high selectivity for ALP among several enzymes. The method was used to measure ALP activity in human serum samples with satisfying results. Graphical abstract Schematic presentation of IL-CDs-based ratiometric fluorescent assay for ALP activity evaluation via FRET strategy between IL-CDs and DAP. This ratiometric fluorescent assay possessed low detection limit of ALP activity (0.012 U L -1 ) and high selectivity among several enzymes.

In this work, we developed a ''naked-eye'' colorimetric and ratiometric fluorescence probe for a very important biomarker of uric acid (UA). The method was based on the oxidation of UA by uricase to allantoin and hydrogen peroxide, and then o-Phenylenediamine (OPD) was oxidized to the yellow-colored 2,3-diaminophenazine (oxOPD) in the presence of horseradish peroxidase (HRP) and hydrogen peroxide. The fluorescence emission of glutathione functionalized Ti 3 C 2 MQDs (GSH-Ti 3 C 2 MQDs) centered at 430 nm overlaps with the UV absorption of oxOPD at 425 nm to a large extent, which facilitates fluorescence resonance energy transfer (FRET) between GSH-Ti 3 C 2 MQDs and oxOPD. With the increase of the UA concentration, the emission at 430 nm of GSH-Ti 3 C 2 MQDs is progressively quenched and the emission at 568 nm of oxOPD was gradually increased. Moreover, the probe we designed is easier to distinguish with color change by naked eye for the detection of UA. This is the first report about the determination of UA by a ''naked-eye'' colorimetric and ratiometric fluorescence method combining GSH-Ti 3 C 2 MQDs and uricase/HRP enzymes. This work enables assays to perform fluorescence and visual detection of biomarker in biological fluids based on Ti 3 C 2 MQDs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

This work presents a novel development that exploits the concept of in-situ gas-separation together with a specific enzymatic colorimetric detection to produce a portable biosensor called "Blood Alcohol Micro-pad" for direct quantitation of ethanol in whole blood. The thin square device (25 mm × 25 mm × 1.8 mm) comprises two layers of patterned filter paper held together with a double-sided mounting tape with an 8-mm circular hole (the headspace). In operation, the reagent is deposited on one layer and covered with sticky tape. Then 8 μL of a blood sample is dispensed onto the opposite layer and covered with sticky tape. Diffusion of ethanol across the 1.6 mm narrow headspace permits selective detection of ethanol by the enzymatic reagents deposited on the opposite layer. This reagent zone contains alcohol oxidase, horseradish peroxidase and 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, as the chromogenic reagent. The color intensity, measured from the recorded digital image, resulting from the enzymatic assay of ethanol, correlates with the concentration of blood alcohol. The results obtained with spiked mice and sheep blood samples, using an external calibration in the range of 1-120 mg dL -1 ethanol, gave recoveries of 93.2-104.4% (n = 12). The "Blood Alcohol Micro-pad" gave good precision with %RSD <1 (50 mg dL -1 ethanol, n = 10) and limit of quantification (10SD of intercept/slope) of 11.56 mg dL -1 . The method was successfully validated against a headspace gas chromatography-mass spectrometric method. It has good potential for development as a simple and convenient blood alcohol sensor for on-site testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel type of enzyme-antibody conjugation using mesoporous silicon nanospheres (MSN) was developed, which amplified the labeling signal and highly increased the sensitivity of enzyme-linked immunosorbent assay (ELISA) for the determination of pesticide and veterinary drug residues in food. First, conjugates were prepared through layer-by-layer immobilization of an enzyme and an antibody on an MSN scaffold. Then the MSN scaffold was employed for labeling and signal amplification to develop a sensitive colorimetric immunoassay through the catalytic oxidation reaction of 5,50-tetramethylbenzidine (TMB). When this MSN-based ELISA was applied to detect chloramphenicol, avermectin, tetracycline and streptomycin in food samples, it provided linear ranges of 0.025 ng ml-1-25 ng ml-1, 0.05 ng ml-1-10 ng ml-1, 0.025 ng ml-1-10 ng ml-1 and 0.05 ng ml-1-25 ng ml-1, respectively, with low detection limits down to 0.011 ng mL-1, 0.134 ng mL-1, 0.015 ng ml-1 and 0.106 ng ml-1, respectively. For avermectin, it provided a 16.7-fold decrease of the limit of detection in contrast to that of standard ELISA without the loss of method specificity and accuracy. This novel immunoassay was hypersensitive, simple and easy-to-use, which made it high potential in applying for the accurate analysis of harmful substances in food.

We designed a new type of MIP-SERS substrate for specific and label-free detection of patulin (PAT), by combining molecular imprinting polymer (MIP) selectivity and SERS technology sensitivity. Initially, the solid substrate of PDMS/AAO was prepared using poly dimethylsiloxane (PDMS) concreted anodized aluminum oxide (AAO) template. Then moderate Au was sputtered on the surface of PDMS/AAO to obtain Au/PDMS/AAO SERS substrate. Based on the HRP enzyme initiated in situ polymerization on the Au/PDMS/AAO, the MIP-SERS substrate was successfully synthesized with selective polymer and high tense of SERS "hot spots". The new MIP-SERS substrate showed strong SERS enhancement effect and good selectivity for PAT. Besides, the results showed that the method owned a linear range from 5 × 10 -10 to 10 -6  M with the limit of detection (LOD) of 8.5 × 10 -11  M (S/N = 3) for PAT. The proposed method also exhibited acceptable reproducibility (relative standard deviation, RSD = 4.7%)，good stability (Raman intensity is above 80% after two weeks) and recoveries from 96.43% to 112.83% with the average RSD of 6.3%. The substrate is easy to use without complex sample pretreatment, which makes it a potential candidate as a rapid and sensitive detection method in food samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

A direct competitive immunosensor for the electrochemical determination of Imidacloprid (IMD) pesticide on gold nanoparticle-modified screen-printed carbon electrodes (AuNP-SPCE) is here reported for the first time. Self-obtained specific monoclonal antibodies are immobilized on the AuNP-SPCE taking advantage of the AuNPs biofunctionalization abilities. In our biosensor design, free IMD in the sample competes with IMD conjugated with horseradish peroxidase (IMD-HRP) for the recognition by the antibodies. After that, 3,3',5,5'-Tetramethylbenzidine (TMB) is enzymatically oxidized by HRP, followed by the oxidized TMB reduction back at the surface of the SPCE. This process gives an associated catalytic current (analytical signal) that is inversely proportional to the IMD amount. The main parameters affecting the analytical signal have been optimized, reaching a good precision (repeatability with a RSD of 6%), accuracy (relative error of 6%), stability (up to one month), selectivity and an excellent limit of detection (LOD of 22 pmol L -1 ), below the maximum levels allowed by the legislation, with a wide response range (50-10000 pmol L -1 ). The detection through antibodies also allows to have an excellent selectivity against other pesticides potentially present in real samples. Low matrix effects were found when analysing IMD in tap water and watermelon samples. The electrochemical immunosensor was also validated with HPLC-MS/MS, the reference method used in official laboratories for IMD analysis, through statistical tests. Our findings make the electrochemical immunosensor as an outstanding method for the rapid and sensitive determination of IMD at the point-of-use., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H 2 O 2 . Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10 -15  M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The immobilization of enzymes on an electrode surface is critical in preserving enzyme activity and providing a sufficient electron transfer pathway for bioelectrocatalysis. Here, we present a novel single-step, cross-linker free immobilization for direct bioelectrocatalysis using an ionic strength induced phase inversion of oppositely charged polyelectrolytes. Cationic poly-guanidinyl-propyl-methacrylate (pGPMA, PG) and anionic inorganic polyphosphate, sodium hexametaphosphate (P6) were used to make an electrostatically condensed phase (PGP6). A mixture of PGP6 and laccase (LAC) from Tramates versicolor or HRP (HRP) from Armoracia rusticana were deposited on the electrode surface and were submerged in DI water to form white porous electrode coatings. Each electrode showed a current generation corresponding to the respective substrates via direct bioelectrocatalysis.

Magnetic and nonmagnetic biochar (MBC & BC) were produced from biosolids under hydrothermal conditions and characterized in order to understand surface chemistry impacts on enzyme immobilization and activity. Peak surface pore size of MBC was 180 nm and that of BC was 17 nm. Despite similar surface area (≈ 49 m 2 /g) MBC immobilized more laccase (99 mg/g) than biochar (31 mg/g). For horseradish peroxidase (HRP), the two biochars had similar immobilization capacity (≈ 65 mg/g). Laccase and HRP on MBC had 47.1 and 18.0% higher specific activity than on BC, respectively. The matrix activity of MBC-laccase (33.3 U/mg support) was 3.7-fold higher than BC-laccase (8.8 U/mg support) and higher than the same amount of free laccase (30.2 U) at pH 3.0 (P < 0.05). Although MBC had its own peroxide oxidation activity (104.1 and 165.9 U/mg biochar at pHs 5&6) this only accounted for 16.7 and 20.4% of the total MBC-H RP activity respectively. After 10 wash cycles, MBC still retained 79.3% and 60.3% of laccase and HRP activity, respectively. Additionally, MBC had lower acute toxicity, suggesting that it is relative benign from an environmental perspective., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We report the development of a dual-enzyme electrochemical biosensor based on microfabricated gold band array electrodes which were first modified by gold foam (Au-foam) in order to dramatically increase the active surface area. The resulting nanostructured Au-foam deposits then served as a highly porous 3D matrix for the electrodeposition of a nanocomposite film consisting of multi walled carbon nanotubes embedded in a chitosan matrix (CS:MWCNT) designed to provide a conducting, biocompatible and chemically versatile surface suitable for the attachment of a wide range of chemically or biologically active agents. Finally, a dual enzyme mixture of glucose oxidase (GOx) and horseradish peroxidase (HRP) was immobilised onto the CS:MWCNT nanocomposite film surface. It is shown that the resulting sensing platform developed demonstrates excellent analytical performance in terms of glucose detection with a sensitivity of 261.8 μA mM -1 cm -2 and a reproducibility standard deviation (RSD) of 3.30% as determined over 7 measurements. Furthermore, long term stability studies showed that the electrodes exhibited an effectively unchanged response to glucose detection after some 45 days. The example of glucose detection presented here illustrates the fact that the particular combination of nanostructured materials employed represents a very flexible platform for the attachment of enzymes or indeed any other bioactive agent and as such may form the basis of the fabrication of a wide range of biosensors. Finally, since the platform used is based on lithographically-deposited gold electrodes on silicon, we note that it is also very suitable for further miniaturisation, mass production and packaging- all of which would serve to reduce production costs.

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H 2 O 2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL -1 . The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.

Easy and visually appealing demonstrations are precious tools for introducing students to the study of enzymes. However, they most often involve purified enzymes and dedicated techniques. Here, we propose a set of demonstrations, which require only fresh horseradish root and consumer chemicals, to help support biochemistry and enzymology courses from high school to the undergraduate level. Horseradish root is a naturally rich source of horseradish peroxidase, an enzyme with many relevant practical applications. Slices of horseradish root are used to demonstrate the characteristic reaction of horseradish peroxidase with hydrogen peroxide, the selective inhibition of this enzyme by dilute hydrochloric acid, its chromogenic reaction with N,N'-diethyl-p-phenylenediamine, and its ability to trigger the chemiluminescent reaction of luminol. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):38-43, 2020., (© 2019 International Union of Biochemistry and Molecular Biology.)

Urinary glucose determination using a glucose test strip is simple and convenient in daily self-monitoring of diabetes. However, diabetic patients exhibit acquired impaired color vision (ICV), which results in the inability to discriminate between hues. Even with the assistance of a color chart, it is still not easy for these patients to read the urinary glucose results with the naked eye. In this study, a smartphone camera using an image-based colorimetric detection method was successfully developed for quantitative analysis of urine glucose. A horseradish peroxidase-hydrogen peroxide-3,3'5,5'-tetramethylbenzidine (HRP-H 2 O 2 -TMB) system was optimized for a reliable and gradual color fading process via a glucose oxidase (GOD) catalyzed oxidation reaction. The color changes of the peroxidase-H 2 O 2 enzymatic reactions in the 96-well microplate were captured by a smartphone RGB camera with subsequent detection of red, green, and blue (RGB) intensities decreasing at each image pixel. The highly quantitative relationships between the glucose concentrations and the color characteristic values of the blue channel of the captured images were successfully established. The high accuracy of this method was demonstrated in urine glucose measurements with a linear response over the 0.039 mg mL -1 to 10.000 mg mL -1 glucose concentration range and a 0.009 mg mL -1 detection limit. The method has great potential as a point-of-need platform for diabetic patients with defective color vision and features high accuracy and low cost., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.

Immobilized enzymes play significant roles in many practical applications. However, the enzymes need to be purified before immobilization by conventional immobilizing methods, and the purification process is expensive, laborious, complicated and results in a decrease of the enzymatic activity. So, we present a novel method by a facile one-step targeted immobilization of an enzyme without a purification process from complex samples. For this purpose, a novel molecularly imprinted polymer was prepared via a silane emulsion self-assembly method using boric acid-modified Fe3O4 nanoparticles as magnetic nuclei, horseradish peroxidase as a template, 3-aminopropyltriethoxysilane as a functional monomer and tetraethyl orthosilicate as a crosslinking agent. The molecularly imprinted polymers were characterized using a scanning electron microscope, X-ray photoelectron spectroscope, vibrating sample magnetometer and X-ray diffractometer. The as-prepared and characterized materials were employed to immobilize horseradish peroxidase from a crude extract of horseradish. Moreover, the immobilized horseradish peroxidase was employed to develop visual sensors for the detection of glucose and sarcosine. This study demonstrated that the molecularly imprinted polymers prepared via the silane emulsion self-assembly method can facilely immobilize horseradish peroxidase from a crude extract of horseradish without any purification process. The developed visual method based on the immobilized horseradish peroxidase shows great potential applications for the visual detection of glucose and sarcosine.

Copper is involved in Alzheimer's disease (AD) where it appears to affect the aggregation of amyloid-β (Aβ) and to catalyze the production of reactive oxygen species (ROS). Oxidative stress apparently produces Aβ dimers that are covalently linked through two tyrosine residues. Such dityrosine cross-links are considered as potential markers of the disease and seem to be implicated in the pathological disorder. In the present study, pure o,o'-dityrosine (diY) was prepared enzymatically (with horseradish peroxidase; HRP), which was subsequently used to construct calibration lines aimed at quantifying nanomolar amounts of diY in reaction mixtures by fluorescence spectroscopy. Hence, diY concentrations down to 67 nM could be determined, which allowed to find that ca. 3% of dityrosine-bridged dimers of Aβ(1-40) were produced after 3 days at 37 °C in the presence of copper and dihydrogen peroxide. These cross-linked dimers in the presence of copper(II) ions completely inhibit the typical aggregation of Aβ, since β sheets could not be detected applying the usual Thioflavin T (ThT) method. Furthermore, the use of a potent Cu(II) chelator, such as the ATCUN tripeptide, L-histidyl-L-alanyl-L-histidine (HAH), efficiently prevented the copper-mediated generation of ROS and the associated dityrosine-bridged Aβ dimers, suggesting that such metal chelators may find future applications in the field of anti-AD drug design.

A colorimetric assay is described for simultaneous detection of multiple analytes related to food safety. It is based on the use of a sandwich aptasensor and terminal deoxynucleotidyl transferase (TdT) which produces a primer for subsequent rolling circle amplification (RCA). Two split aptamer fragments (Apt1 and Apt2) are firstly immobilized, Apt1 on gold nanoparticles (AuNPs), and Apt2 on magnetic beads (MBs). They are then used in a sandwich aptasensor. In the presence of analyte, two probes could specifically recognize target and form a ternary assembly, and the magnetic beads also act to separate rapidly and enrich the target. Then, the extension of template-free DNA is triggered by TdT at the exposed 3'-hydroxy terminals of Apt1. This produces polyA sequences that serve as primers for subsequent RCA. The product of RCA is hybridized with a complementary horse radish peroxidase (HRP) DNA probe. HRP catalyzes the H 2 O 2 -mediated oxidation of tetramethylbenzidine (TMB) and forms a blue chromogenic product. After magnetic separation, the absorption values of the blue product in the supernatant are measured at a wavelength of 600 nm. Based on this dual amplification mechanism, the assay was applied to multiplexed determination of enrofloxacin (ENR), lead(II), Escherichia coli O157:H7 and tropomyosin. Exemplarily, ENR is detectable at concentrations down to 2.5 pg mL -1 with a linear range that extends from 1 pg mL -1 to 1 μg·mL -1 . The assay was validated by analysis of spiked fish samples. Recoveries range between 87.5 and 92.1%. Graphical abstractSchematic representation of a TdT-RCA based aptasensor for multiple analytes related to food safety. It makes use of sandwich aptasensors and TdT-produced universal primer-triggered RCA reaction. dATP: deoxyadenosine triphosphate, TdT: Terminal Deoxynucleotidyl Transferase, RCA: rolling circle amplification, TMB: 3,3',5,5'-Tetramethylbenzidine.

A lectin magnetic separation (LMS) method for Staphylococcus aureus (S. aureus) was developed with the aim to improve the efficiency of magnetic nanoparticles and to expand the scope of bacterial recognition. Poly(ethylene glycol) (PEG)-mediated magnetic nanoparticles modified with streptavidin (MNP-PEG-SA) were synthesized and then applied to a two-step LMS based on the use of wheat germ agglutinin (WGA). Three specific methods for S. aureus detection (suitable for different requirements including detection time and sensitivity) were designed. The new LMS has improved anchoring efficiency (compared to two-step LMS methods) and requires a reduced number of magnetic particles. The Baird-Parker (B-P) method can detect S. aureus with a detection limit of 3 × 10 0  CFU·mL -1 within 15 h; the polymerase chain reaction (PCR) method can be finished within 4 h, with the lowest detection limit (LOD) of 3 × 10 2  CFU·mL -1 . The LOD of HRP-pig IgG-based colorimetric method is 3 × 10 5  CFU·mL -1 , and the method only lasts for 2 h. If combined with specific detection methods, it meets different needs for rapid detection of S. aureus. Graphical abstractSchematic representation of lectin magnetic separation (LMS) based on biotin-wheat germ agglutinin (WGA) and poly (ethylene glycol) (PEG)-mediated streptavidin-modified magnetic nanoparticles (MNP-PEG-SA) and three different quantification strategies (including B-P culture assay, PCR assay, and colorimetric assay) for S. aureus.

Monitoring soluble immune checkpoints in circulating fluids has the potential for minimally-invasive diagnostics and personalised therapy in precision medicine. Yet, the sensitive detection of multiple immune checkpoints from small volumes of liquid biopsy samples is challenging. In this study, we develop a multiplexed immune checkpoint biosensor (MICB) for parallel detection of soluble immune checkpoints PD-1, PD-L1, and LAG-3. MICB integrates a microfluidic sandwich immunoassay using engineered single chain variable fragments and alternating current electrohydrodynamic in situ nanofluidic mixing for promoting biosensor-target interaction and reducing non-specific non-target binding. MICB provides advantages of simultaneous analysis of up to 28 samples in <2 h, requires as little as a single sample drop (i.e., 20 μL) per target immune checkpoint, and applies high-affinity yeast cell-derived single chain variable fragments as a cost-effective alternative to monoclonal antibodies. We investigate the assay performance of MICB and demonstrate its capability for accurate immune checkpoint detection in simulated patient serum samples at clinically-relevant levels. MICB provides a dynamic range of 5 to 200 pg mL-1 for PD-1 and PD-L1, and 50 to 1000 pg mL-1 for LAG-3 with a coefficient of variation <13.8%. Sensitive immune checkpoint detection was achieved with limits of detection values of 5 pg mL-1 for PD-1, 5 pg mL-1 for PD-L1, and 50 pg mL-1 for LAG-3. The multiplexing capability, sensitivity, and relative assay simplicity of MICB make it capable of serving as a bioanalytical tool for immune checkpoint therapy monitoring.

A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at -200 mV vs. Ag pseudo-reference electrode, using H 2 O 2 as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47 pg mL -1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients. The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this work, we present a smartphone-based multiplexed enzymatic biosensor utilizing the unique colorimetric properties of the poly(aniline-co-anthranilic acid) (ANI-co-AA) composite film coupled with horseradish peroxidase (HRP), glucose oxidase (GOx), horseradish peroxidase-glucose oxidase (GOx-HRP) and tyrosinase (Tyr) enzymes. The enzymes are immobilized on the composite polymer film by adsorption and they catalyze a reversible redox color change of the host polymer from green to blue in the presence of their substrate. A smartphone was applied as color detector, for image acquisition and data handling. A ColorLab ® android application, free of charge software application, was used to enable easy and clear display of the sensors' response indicating remarkable changes in the optical features. The results were confirmed by the spectrophotometric measurements. The developed colorimetric enzymatic biosensors were studied and optimized in relation to different experimental parameters. Moreover, the colorimetric enzymatic biosensors were applied to food and pharmaceutical analysis. It has been shown by these studies that the colorimetric biosensors are promising as quick and simple tests for handheld analysis in various fields., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The repeatable immobilization of molecular recognition elements onto particle surfaces has a strong impact on the outcomes of affinity-based assays. In this work, an automatic method for the immobilization of immunoglobulin G (IgG) onto protein A-Sepharose microbeads was established through the flow programming features of the portable lab-on-valve platform using micro-bead injection spectroscopy. The reproducible packing of protein A-microbeads between two optic fibers was feasible, allowing on-column probing of IgG retention. The automation of solutions handling and the precise control of time of IgG interaction with the beads rendered repeatable immobilization cycles, within a short timeframe (<2 min). The proposed method featured the preparation of disposable immunosorbents for downstream analytical applications, such as immunosensing or microenrichment of target analytes. In-situ quantification of IgG@protein A-microbeads was carried out using a horseradish peroxidase-labeled detection IgG. The colorimetric oxidation of 3,3',5,5'-tetramethylbenzidine was monitored on-column. Quantitation of mouse and human IgG immobilized@protein A-microbeads was achieved for loading masses between 0.1 and 0.4 μg per ca. 5.5 mg of sorbent. The implemented detection strategy allowed the quantification of human IgG in certified human serum (ERM ® - DA470k/IFCC) and spiked saliva, yielding recoveries of 102-108% and requiring minimal volume (1-15 μL) from serum and saliva., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fluorescent polymer dots (PDs) with maximum excitation/emission wavelengths of 410/515 nm were prepared in water solution from 1,4-benzoquinone and ethylenediamine. The green fluorescence of these PDs is screened off by the red-colored oxidation product (PPDox, maximum absorption at 510 nm) formed by horseradish peroxidase (HRP)-catalyzed oxidation of p-phenylenediamine (PPD). It causes the reduction of the fluorescence intensity of the PDs due to spectral overlap and an inner filter effect (IFE). If glucose is enzymatically oxidized under the formation of H 2 O 2 , the formed H 2 O 2 can be quantified by the above IFE. The assay for HRP activity and glucose have detection limits of 0.2 U·L -1 and 0.1 μM, respectively. The nanoprobe was further extended to an immunosorbent assay (ELISA) for the determination of insecticidal Cry1Ab/Ac protein with a detection limit of 0.25 ng·mL -1 . The ELISA was applied to rice leaf analysis. Graphic abstract Schematic representation of fluorometrict enzyme-linked immunosorbent assay for Cry1Ab/Ac protein detection based on horseradish peroxidase (HRP)-triggered fluorescence quenching of polymer dots (PDs). Quenching is caused by an inner filter effect (IFE) caused by PPDox, the oxidation product of p-phenylenediamine (PPD).

Mass spectrometry (MS)-based analysis of glycoproteins and glycopeptides requires selective separation strategies to eliminate interferences from more abundant non-glycosylated biomolecules. In this work, we describe a two-phase liquid-liquid extraction method using supramolecular polymeric nanoassemblies that can selectively and efficiently enrich glycopeptides for enhanced MS detection. The polymeric nanoassemblies are made selective for glycopeptides via the incorporation of hydrazide functional groups that covalently bind to glycans. The enrichment efficiency is further enhanced via the incorporation of acidic functional groups that lead to a proximity-assisted catalysis of the hydrazide-glycan conjugation reaction. Our results further demonstrate the value of designer supramolecular nanomaterials for the selective enrichment of modified peptides from complicated mixtures.

The development of an automated miniaturized analytical system that allows for the rapid monitoring of carbamazepine (CBZ) levels in serum and wastewater is proposed. Molecular recognition of CBZ was achieved through its selective interaction with microbeads carrying anti-CBZ antibodies. The proposed method combines the advantages of the micro-bead injection spectroscopy and of the flow-based platform lab-on-valve for implementation of automatic immunosorbent renewal, rendering a new recognition surface for each sample. The sequential (or simultaneous) perfusion of CBZ and the horseradish peroxidase-labelled CBZ through the microbeads is followed by real-time on-column monitoring of substrate (3,3',5,5'-tetramethylbenzidine) oxidation by colorimetry. The evaluation of the initial oxidation rate and also the absorbance value at a fixed time point provided a linear response versus the logarithm of the CBZ concentration. Under the selected assay conditions, a single analysis was completed after only 11 min, with a quantification range between 1.0 and 50 μg L -1 . Detection of CBZ levels in undiluted wastewater samples was feasible after a simple filtration step while good recoveries were attained for spiked certified human serum, analyzed without sample clean-up., (Copyright © 2019 Elsevier B.V. All rights reserved.)