High-Throughput µPAD with Cascade Signal Amplification through Dual Enzymes for arsM in Paddy Soil.

The arsM gene is a critical biomarker for the potential risk of arsenic exposure in paddy soil. However, on-site screening of arsM is limited by the lack of high-throughput point-of-use (POU) methods. Here, a multiplex CRISPR/Cas12a microfluidic paper-based analytical device (µPAD) was constructed for the high-throughput POU analysis of arsM, with cascade amplification driven by coupling crRNA-enhanced Cas12a and horseradish peroxidase (HRP)-modified probes. First, seven crRNAs were designed to recognize arsM, and their LODs and background signal intensities were evaluated. Next, a step-by-step iterative approach was utilized to develop and optimize coupling systems, which improved the sensitivity 32 times and eliminated background signal interference. Then, ssDNA reporters modified with HRP were introduced to further lower the LOD to 16 fM, and the assay results were visible to the naked eye. A multiplex channel microfluidic paper-based chip was developed for the reaction integration and simultaneous detection of 32 samples and generated a recovery rate between 87.70 and 114.05%, simplifying the pretreatment procedures and achieving high-throughput POU analysis. Finally, arsM in Wanshan paddy soil was screened on site, and the arsM abundance ranged from 1.05 × 106 to 6.49 × 107 copies/g; this result was not affected by the environmental indicators detected in the study. Thus, a coupling crRNA-based cascade amplification method for analyzing arsM was constructed, and a microfluidic device was developed that contains many more channels than previous paper chips, greatly improving the analytical performance in paddy soil samples and providing a promising tool for the on-site screening of arsM at large scales.

Automatic ultrasensitive lateral flow immunoassay based on a color-enhanced signal amplification strategy.

Lateral flow immunoassays (LFIAs) are an essential and widely used point-of-care test for medical diagnoses. However, commercial LFIAs still have low sensitivity and specificity. Therefore, we developed an automatic ultrasensitive dual-color enhanced LFIA (DCE-LFIA) by applying an enzyme-induced tyramide signal amplification method to a double-antibody sandwich LFIA for antigen detection. The DCE-LFIA first specifically captured horseradish peroxidase (HRP)-labeled colored microspheres at the Test line, and then deposited a large amount of tyramide-modified signals under the catalytic action of HRP to achieve the color superposition. A limit of detection (LOD) of 3.9 pg/mL and a naked-eye cut-off limit of 7.8 pg/mL were achieved for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein. Additionally, in the inactivated virus detections, LOD equivalent to chemiluminescence (0.018 TCID50/mL) was obtained, and it had excellent specificity under the interference of other respiratory viruses. High sensitivity has also been achieved for detection of influenza A, influenza B, cardiac troponin I, and human chorionic gonadotrophin using this DCE-LFIA, suggesting the assay is universally applicable. To ensure the convenience and stability in practical applications, we created an automatic device. It provides a new practical option for point-of-care test immunoassays, especially ultra trace detection and at-home testing.

Encapsulation of an enzyme-immobilized smart polymer membrane in a metal-organic framework for enhancement of catalytic performance.

Encapsulation of enzymes within porous materials has shown great promise for protecting enzymes from denaturation, increasing their tolerance to harsh environments and promoting their industrialization. However, controlling the conformational freedom of the encapsulated enzymes to enhance their catalytic performance remains a great challenge. To address this issue, herein, following immobilization of GOx and HRP on a thermo-responsive porous poly(styrene-maleic-anhydride-N-isopropylacrylamide) (PSMN) membrane, a GOx-HRP@PSMN@HZIF-8 composite was fabricated by encapsulating GOx-HRP@PSMN in hollow ZIF-8 (HZIF-8) with liposome (L) as the sacrificial template. The improved conformational freedom for enzymes arising from the hollow cavity formed in ZIF-8 through the removal of L enhanced the mass transfer and dramatically promoted the catalytic activity of the composite. Interestingly, at high temperature, the coiled PN moiety in PSMN provided the confinement effect for GOx-HRP, which also significantly boosted the catalytic performance of the composites. Compared to the maximum catalytic reaction rates (Vmax) of GOx-HRP@PSMN@LZIF-8, the free enzyme and GOx-HRP@ZIF-8, the Vmax of the GOx-HRP@PSMN@HZIF-8 composite exhibited an impressive 17.8-fold, 10.8-fold and 6.0-fold enhancement at 37 °C, respectively. The proposed composites successfully demonstrated their potential as catalytic platforms for the colorimetric detection of glucose in a cascade reaction. This study paves a new way for overcoming the current limitations of immobilizing enzymes in porous materials and the use of smart polymers for the potential fabrication of enzyme@polymer@MOF composites with tunable conformational freedom and confinement effect.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Kinetic detection of hydrogen peroxide in single horseradish peroxidase-concentrated silica particle using confocal fluorescence microspectroscopic measurement.

In the present study, we propose a scheme for detecting H2O2 by using horseradish peroxidase (HRP) adsorbed onto single silica particles and fluorescence microspectroscopy. When the silica particles were immersed in an HRP solution, the HRP concentration in the silica particles increased by a factor of 690 compared to that in the bulk aqueous solution because HRP was adsorbed on the silica surface. When a single particle containing HRP was added to a mixed solution of H2O2 and Amplex Red, fluorescence from resorufin, which was produced by the reaction of HRP, H2O2, and Amplex Red, was observed. The fluorescence from the resorufin in the particles increased after a single particle was added to the solution, and the release of resorufin was observed. As the concentration of H2O2 (CH2O2) decreased, the time it takes for fluorescence intensity to reach its maximum was shorter. The detection limit for H2O2 in the present system was 980 nM. The reaction behavior of a single silica particle was evaluated using a spherical diffusion model, which explains the approximate concentration change of resorufin in the silica particle. The proposed method has the advantages of simple sample preparation and detection, low sample consumption, and a short detection time.

Characterization of nanozyme kinetics for highly sensitive detection.

Nanozymes have been widely used as enzyme substitutes. Based on a comprehensive literature survey of 261 publications, we report the significant differences in the Michaelis-Menten constants (Km) between peroxidase-mimicking nanozymes and horseradish peroxidase (HRP). Further, these differences were not considered in more than 60% of the publications for analytical developments. As a result, nanozymes' catalytic activity is limited, resulting in a potentially higher limit of detection (LOD). We used a peroxidase-mimicking Au@Pt nanozyme, which has Km for TMB comparable with HRP and three orders of magnitude higher Km for H2O2. Using the Au@Pt nanozyme as a label for immunoassays, non-optimized nanozyme substrate concentrations led to 30 times higher LOD compared to optimized conditions. The results confirm the necessity of measuring nanozymes' kinetic parameters and the corresponding adjustment of substrate concentrations for highly sensitive detection.

Visual detection of H2O2 and glucose by HBcAb-HRP fluorescence-enhanced CdTe QDs/CDs ratiometric fluorescence sensing platform.

This study presents the development of a sensitive and simple enhanced ratiometric fluorescence sensing platform in the consist of CdTe quantum dots (QDs), carbon dots (CDs), and hepatitis B core antibody labeled with horseradish peroxidase (HBcAb-HRP) for the visual analysis of H2O2 and glucose. The sulfur atoms in HBcAb-HRP have a strong affinity for Cd(II), which effectively enhances the fluorescence intensity of the CdTe QDs due to the generation of more radiative centers at the CdTe/Cd-SR complex. In the presence of H2O2, the Cd-S bonds are oxidized to form disulfide products and results in linear fluorescence quenching, while CDs maintain stable. Becasue glucose can be converted into H2O2 with the aid of glucose oxidase, this sensing platform can also be used for analyzing glucose. The detection limits for H2O2 and glucose are 2.9 µmol L-1 with RSD of 2.6% and 1.6 µmol L-1 with RSD of 2.4% respectively. In addition, under UV lamp irradiation, the orange-yellow CdTe QDs gradually quench with increasing H2O2 and glucose, while the blue CDs remain unchanged. A color change from orange-yellow to blue enables a visual semi-quantitative determination of H2O2 in commercial contact lens solution and glucose in human serum without any pretreatment. Thus, this CdTe QDs/CDs ratiometric sensing platform has significant potential for the rapid analysis of H2O2 and glucose in actual application.

Highly Light-Harvesting MOF-on-MOF Heterostructure: Cascading Functionality to Flexible Photogating of Organic Photoelectrochemical Transistor and Bienzyme Cascade Detection.

Recently, organic photoelectrochemical transistor (OPECT) bioanalysis has become a prominent technique for the high-performance detection of biomolecules. However, as a sensitive index of the OPECT, the dynamic regulation transconductance (gm) is still severely deficient. Herein, this work reports a new photosensitive metal-organic framework (MOF-on-MOF) heterostructure for the effective modulation of maximum gm and natural bienzyme interfacing toward choline detection. Specifically, the bidentate ligand MOF (b-MOF) was assembled onto the UiO-66 MOF (u-MOF) by a modular assembly method, which could facilitate the charge separation and generate enhanced photocurrents and offer a biophilic environment for the immobilization of choline oxidase (ChOx) and horseradish peroxidase (HRP) through hydrogen-bonded bridges. The transconductance of the OPECT could be flexibly altered by increased light intensity to maximal value at zero gate bias, and sensitive choline detection was achieved with a detection limit of 0.2 µM. This work reveals the potential of MOF-on-MOF heterostructures for futuristic optobioelectronics.

A self-assembled 3D nanoflowers based nano-ELISA platform for the sensitive detection of pyridaben.

Biomimetic methods are invariably employed to synthesize hybrid organic-inorganic multilevel structure nanoflowers with self-assembly processes in aqueous solutions, which is an ideal way to meet the challenges of immobilizing antibodies or enzymes in nanomaterial based enzyme-linked immunosorbent assay (nano-ELISA). In this study, we developed protein-inorganic hybrid 3D nanoflowers composed of bovine serum albumin (BSA), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (IgG-HRP) and copper(Ⅱ) phosphate (BSA-(IgG-HRP)-Cu3(PO4)2) using a self-assembly biomimetic method. The preparation process avoided the use of any organic solvent and protein immobilization did not require covalent modifications. Additionally, the unique hierarchical structure enhances the thermal and storage stability of HRP. The BSA-(IgG-HRP)-Cu3(PO4)2 hybrid 3D nanoflower was then applied to a nano-ELISA platform for pyridaben detection, achieving a 50% inhibition concentration of 3.90 ng mL-1. The nano-ELISA achieved excellent accuracy for pyridaben detection. Such a novel BSA-(IgG-HRP)-Cu3(PO4)2 hybrid 3D nanoflower provide an excellent reagent for small molecule immunoassay.

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery.

In the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained systems proved successful enzyme deposition on all the presented materials. The immobilization of horseradish peroxidase on all the tested supports occurred with an efficiency above 70%. However, for multi-walled carbon nanotubes and hybrids made of chitosan, magnetic nanoparticles, and selenium ions, it reached up to 90%. For these materials, the immobilization yield exceeded 80%, resulting in high amounts of immobilized enzymes. The produced system showed the same optimal pH and temperature conditions as free enzymes; however, over a wider range of conditions, the immobilized enzymes showed activity of over 50%. Finally, a reusability study and storage stability tests showed that horseradish peroxidase immobilized on a hybrid made of chitosan, magnetic nanoparticles, and selenium ions retained around 80% of its initial activity after 10 repeated catalytic cycles and after 20 days of storage. Of all the tested materials, the most favorable for immobilization was the above-mentioned chitosan-based hybrid material. The selenium additive present in the discussed material gives it supplementary properties that increase the immobilization yield of the enzyme and improve enzyme stability. The obtained results confirm the applicability of these nanomaterials as useful platforms for enzyme immobilization in the contemplation of the structural stability of an enzyme and the high catalytic activity of fabricated biocatalysts.

Tyramine-Invertase Bioconjugate-Amplified Personal Glucose Meter Signaling for Ultrasensitive Immunoassay.

Highly sensitive and facile detection of low levels of protein markers is of great significance for the early diagnosis and efficacy monitoring of diseases. Herein, aided by an efficient tyramine-signal amplification (TSA) mechanism, we wish to report a simple but ultrasensitive immunoassay with signal readout on a portable personal glucose meter (PGM). In this study, the bioconjugates of tyramine and invertase (Tyr-inv), which act as the critical bridge to convert and amplify the protein concentration information into glucose, are prepared following a click chemistry reaction. Then, in the presence of a target protein, the sandwich immunoreaction between the immobilized capture antibody, the target protein, and the horseradish peroxidase (HRP)-conjugated detection antibody is specifically performed in a 96-well microplate. Subsequently, the specifically loaded HRP-conjugated detection antibodies will catalyze the amplified deposition of a large number of Tyr-inv molecules onto adjacent proteins through highly efficient TSA. Then, the deposited invertase, whose dosage can faithfully reflect the original concentration of the target protein, can efficiently convert sucrose to glucose. The amount of finally produced glucose is simply quantified by the PGM, realizing the highly sensitive detection of trace protein markers such as the carcinoembryonic antigen and alpha fetoprotein antigen at the fg/mL level. This method is simple, cost-effective, and ultrasensitive without the requirement of sophisticated instruments or specialized laboratory equipment, which may provide a universal and promising technology for highly sensitive immunoassay for in vitro diagnosis of diseases.

Exploitation of Luminescent Lanthanide Nanoparticles for a Sensitivity-Enhanced ELISA Detection Method.

A new detection method based on the photoluminescence properties of dye-sensitized lanthanide nanoparticles (Ln NPs) was developed for enzyme-linked immunosorbent assays (ELISAs). In this method, the horseradish peroxidase (HRP) enzyme catalyzes the oxidation of phenol derivatives in the presence of hydrogen peroxide, providing dimers that are able to interact with the Ln NP surface and to efficiently photosensitize the Ln ions. Due to the very long emission lifetime of Ln, the time-gated detection of Ln NP luminescence allows the elimination of background noise due to the biological environment. After a comparison of the enzyme-catalyzed oxidation of various phenol derivatives, methyl 4-hydroxyphenyl acetate (MHPA) was selected as the most promising substrate, as the highest Ln emission intensity was observed following its HRP-catalyzed oxidation. After a meticulous optimization of the conditions of both the enzymatic reaction and the Ln sensitization (buffer, pH, concentration of the reactants, NP type, etc.), this new detection method was successfully implemented in a commercial insulin ELISA kit as a proof-of-concept, with an increased sensitivity compared to the commercial detection method.

Development of a new affinity chromatography method for purification of horseradish peroxidase enzyme.

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC50 and Ki values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 ± 0.054 µM, respectively, while the IC50 and Ki values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 ± 9.90 µM, respectively. It was found that the IC50 and Ki values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 ± 43.78 µM, respectively, whereas the IC50 and Ki values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 ± 68.80 µM, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44 kDa. The optimum pH was 8.0, the optimum temperature was 15°C, and the optimum ionic strength was 0.4 M for the purified HRP enzyme.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

Exploration of the structural mechanism of hydrogen (H2)-promoted horseradish peroxidase (HRP) activity via multiple spectroscopic and molecular dynamics simulation techniques.

Horseradish peroxidase (HRP) is an enzyme that is widely used in various fields. In this study, the effects of molecular hydrogen (H2) on the activity and structural characteristics of HRP were investigated by employing multiple spectroscopic techniques, atomic force microscopy (AFM) and molecular dynamics (MD) simulations. The results demonstrated that H2 could enhance HRP activity, especially in 1.5 mg/L hydrogen-rich water (HRW). The structural analysis results showed that H2 might alter HRP activity by affecting the active sites, secondary structure, hydrogen bonding network, CS groups, and morphological characteristics. The MD results also confirmed that H2 could increase the FeN bond distance in the active site, affect the secondary structure, and increase the number of hydrogen bonds. The MD results further suggested that H2 could increase the number of salt bridges, and lengthen the SS bonds in HRP. This study primarily revealed the mechanism by which H2 enhances the HRP activity, providing insight into the interactions between gas and macromolecular proteins. However, some of the results obtained via MD simulations still need to be verified experimentally. In addition, our study also provided a new convenient strategy to enhance enzyme activity.

Patterning of Protein-Sequestered Liquid-Crystal Droplets Using Acoustic Wave Trapping.

Development of spatially organized structures and understanding their role in controlling kinetics of multistep chemical reactions are essential for the successful design of efficient systems and devices. While studies that showcase different types of methodologies for the spatial organization of various colloidal systems are known, design and development of well-defined hierarchical assemblies of liquid-crystal (LC) droplets and subsequent demonstration of biological reactions using such assemblies still remain elusive. Here, we show reversible and reconfigurable one-dimensional (1D) assemblies of protein-bioconjugate-sequestered monodisperse LC droplets by combining microfluidics with noninvasive acoustic wave trapping technology. Tunable spatial geometries and lattice dimensions can be achieved in an aqueous medium comprising ≈19 or 62 µm LC droplets. Different assemblies of a mixed population of larger and smaller droplets sequestered with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, exhibit spatially localized enzyme kinetics with higher initial rates of reaction compared with GOx/HRP cascades implemented in the absence of an acoustic field. This can be attributed to the direct substrate transfer/channeling between the two complementary enzymes in close proximity. Therefore, our study provides an initial step toward the fabrication of LC-based devices for biosensing applications.

A new strategy to build electrochemical enzymatic biosensors using a nanohybrid material based on carbon nanotubes and a rationally designed schiff base containing boronic acid.

We report a nanohybrid material obtained by non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with the new ligand (((1E,1'E)-(naphthalene-2,3-diylbis(azaneylylidene))bis(methaneylylidenedene)) bis(4-hydroxy-3,1-phenylene))diboronic acid (SB-dBA), rationally designed to mimic some recognition properties of biomolecules like concanavalin A, for the development of electrochemical biosensors based on the use of glycobiomolecules as biorecognition element. We present, as a proof-of-concept, a hydrogen peroxide biosensor obtained by anchoring horseradish peroxidase (HRP) at a glassy carbon electrode (GCE) modified with the nanohybrid prepared by sonication of 2.0 mg mL-1 MWCNTs and 0.50 mg mL-1 SB-dBA in N,N-dimethyl formamide (DMF) for 30 min. The hydrogen peroxide biosensing was performed at -0.050 V in the presence of 5.0 × 10-4 M hydroquinone. The analytical characteristics of the resulting biosensor are the following: linear range between 0.175 µM and 6.12 µM, detection limit of 58 nM, and reproducibility of 2.0 % using the same nanohybrid (6 biosensors), and 9.0 % using three different nanohybrids. The sensor was successfully used to quantify hydrogen peroxide in enriched milk and human blood serum samples and in a commercial disinfector.

Registration of Functioning of a Single Horseradish Peroxidase Macromolecule with a Solid-State Nanopore.

Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.

Development of a Progesterone-Receptor-Based Pseudo-immunoassay for Multi-detection of Progestins in Milk and Studying Its Recognition Mechanism.

The residues of progestins in milk are dangerous to consumers, but an immunoassay capable of multi-determining progestins in milk has not been reported thus far. In this study, the ligand binding domain of the human progesterone receptor was expressed and its intermolecular interactions with the commonly used steroid hormones were studied. The docking results showed that the receptor fragment only recognized progestins and did not recognize other steroid hormones. Then, it was used as recognition material to develop a pseudo-direct competitive enzyme-linked immunosorbent assay for multi-determination of five progestins in milk. Because biotinylated horseradish peroxidase was combined with streptavidinated horseradish peroxidase to enhance the signal, the sensitivities for the five progestins (IC50 of 0.029-0.097 ng/mL) were improved 96-143-fold in comparison to the use of the conventional horseradish peroxidase signal system (IC50 of 3.0-12.5 ng/mL). This method showed negligible cross-reactivities to other steroid hormones, consistent with the docking results. This was the first paper developing a progesterone-receptor-based method for detection of progestins, and this method exhibited generally better performance than all of the previously reported immunoassays for progestins.