Competitive ELISA based on pH-responsive persistent luminescence nanoparticles for autofluorescence-free biosensor determination of ochratoxin A in cereals.

An accurate and sensitive competitive enzyme-linked immunosorbent assay (ELISA) based on persistent luminescence nanoparticles Zn2GeO4:Mn2+, Eu3+ (ZGME) was developed for detecting ochratoxin A (OTA), a powerfully toxic mycotoxin usually found in grains. As a signal output element of autofluorescence-free biosensors, ZGME can be integrated into ELISA with glucose oxidase (GOx)-binding OTA molecules due to its excellent pH-responsive persistent luminescence. In the absence of OTA, the OTA-GOx conjugate was captured by the anti-OTA monoclonal antibody (anti-OTA mAb) pre-coated on the 96-well plate. The results indicate a decrease in the pH value of the solution, which triggered the quenching of ZGME luminescence due to GOx-dependent gluconic acid production. The presence of OTA inhibited the binding of OTA-GOx on the plate, thus decreasing the production of gluconic acid and increasing the persistent luminous intensity of ZGME. Under the optimized concentrations of anti-OTA mAb and OTA-GOx, quantitative determination of OTA was achieved by plotting the increase or decrease in persistent luminescence intensity of ZGME at 535 nm. In this study, the linear range was from 0.1 µg L-1 to 63 µg L-1, and the limit of detection (LOD) was as low as 0.045 µg L-1. In five food samples (corn grit, brown rice, soybean, rice, and wheat), the results exhibited good stability and repeatability, with a recovery range from 81.3% to 94.4% and a relative standard deviation (RSD) of less than 4.2%. Hence, the established method provides a sensitive, accurate, and autofluorescence-free approach for the determination of OTA in different grain samples.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Cascade amplified colorimetric immunoassay based on an integrated multifunctional composite with catalytic coordination polymers for prostate specific antigen detection.

Coordination polymers (CPs) have been extensively investigated for a variety of applications because of their tunable structures and properties. In this work, we demonstrated the potential of catalytic CPs in the fabrication of an integrated multifunctional composite for establishing a cascade amplified immunoassay. For this purpose, an Fe(iii)-based CP (FeCP) with peroxidase-like activity was employed as a model of catalytic CPs to simultaneously integrate glucose oxidase (GOx) and the anti-prostate specific antigen (anti-PSA) antibody through a self-adaptive inclusion process. This leads to the formation of a dual-functional anti-PSA/GOx@FeCP composite with cascade catalytic activity and capture ability to target the antigen. Benefiting from the shielding effect of FeCPs as a host, a significantly improved stability against harsh environments can be achieved for the loaded GOx and anti-PSA antibody in the composite. On this basis, by utilizing anti-PSA/GOx@FeCPs as a detection antibody, a colorimetric immunoassay based on the cascade catalysis of GOx and FeCPs as a signal amplified enhancer was developed for the detection of PSA. Under optimal conditions, satisfactory detection results have been achieved in both buffered aqueous solutions and serum samples. We believe that this study will open up a new avenue for the rational design and fabrication of multifunctional composites while offering a new cascade amplification strategy for PSA detection.

Screen-Printed Glucose Sensors Modified with Cellulose Nanocrystals (CNCs) for Cell Culture Monitoring.

Glucose sensors are potentially useful tools for monitoring the glucose concentration in cell culture medium. Here, we present a new, low-cost, and reproducible sensor based on a cellulose-based material, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidized-cellulose nanocrystals (CNCs). This novel biocompatible and inert nanomaterial is employed as a polymeric matrix to immobilize and stabilize glucose oxidase in the fabrication of a reproducible, operationally stable, highly selective, cost-effective, screen-printed glucose sensor. The sensors have a linear range of 0.1-2 mM (R2 = 0.999) and a sensitivity of 5.7 ± 0.3 µA cm-2∙mM-1. The limit of detection is 0.004 mM, and the limit of quantification is 0.015 mM. The sensor maintains 92.3 % of the initial current response after 30 consecutive measurements in a 1 mM standard glucose solution, and has a shelf life of 1 month while maintaining high selectivity. We demonstrate the practical application of the sensor by monitoring the glucose consumption of a fibroblast cell culture over the course of several days.

The effects of glucose and glucose oxidase on the Uv-vis spectrum of gold nanoparticles: A study on optical biosensor for saliva glucose monitoring.

In this study we have investigated the effect of glucose and glucose oxidase (GOD) on the absorption spectrum of gold nanoparticles (Au NPs) with 10-13 nm diameter, in order to improve optical methods of glucose monitoring using surface plasmon resonance of these particles. Different concentrations of glucose solution in water were prepared in the range of human saliva intensity. Two procedures are applied to study glucose effects on the particles; mixing the glucose to the nanocolloid and then adding the GOD, and reversely mixing the glucose and GOD solutions and then pouring in to the nanocolloid. Two different results were obtained that are analyzed based on optical properties. In each method, the effect of glucose and GOD on the size and Uv-vis spectrum of gold nanoparticles has been investigated. Results were interpreted by the physical concept of surface Plasmon resonance (SPR) of Au NPs. This study can open new insight about optical glucose sensing based on surface plasmon of metal nanoparticles.

Multi-attribute PAT for UF/DF of Proteins-Monitoring Concentration, particle sizes, and Buffer Exchange.

Ultrafiltration/diafiltration (UF/DF) plays an important role in the manufacturing of biopharmaceuticals. Monitoring critical process parameters and quality attributes by process analytical technology (PAT) during those steps can facilitate process development and assure consistent quality in production processes. In this study, a lab-scale cross-flow filtration (CFF) device was equipped with a variable pathlength (VP) ultraviolet and visible (UV/Vis) spectrometer, a light scattering photometer, and a liquid density sensor (microLDS). Based on the measured signals, the protein concentration, buffer exchange, apparent molecular weight, and hydrodynamic radius were monitored. The setup was tested in three case studies. First, lysozyme was used in an UF/DF run to show the comparability of on-line and off-line measurements. The corresponding correlation coefficients exceeded 0.97. Next, urea-induced changes in protein size of glucose oxidase (GOx) were monitored during two DF steps. Here, correlation coefficients were ≥ 0.92 for static light scattering (SLS) and dynamic light scattering (DLS). The correlation coefficient for the protein concentration was 0.82, possibly due to time-dependent protein precipitation. Finally, a case study was conducted with a monoclonal antibody (mAb) to show the full potential of this setup. Again, off-line and on-line measurements were in good agreement with all correlation coefficients exceeding 0.92. The protein concentration could be monitored in-line in a large range from 3 to 120 g L- 1. A buffer-dependent increase in apparent molecular weight of the mAb was observed during DF, providing interesting supplemental information for process development and stability assessment. In summary, the developed setup provides a powerful testing system for evaluating different UF/DF processes and may be a good starting point to develop process control strategies. Graphical Abstract Piping and instrumentation diagram of the experimental setup and data generated by the different sensors. A VP UV/Vis spectrometer (FlowVPE, yellow) measures the protein concentration. From the data of the light scattering photometer (Zetasizer, green) in the on-line measurement loop, the apparant molecular weight and z-average are calculated. The density sensor (microLDS) measures density and viscosity of the fluid in the on-line loop.

Antibacterial potential of Swiss honeys and characterisation of their bee-derived bioactive compounds.

BACKGROUND: Antibacterial activity of honey is not only crucial characteristic in selection of honey for medical usage but also an important honey quality marker. The aim of the study was to characterise the antibacterial potential of 29 honey samples representing the main types of multi-floral blossom and honeydew honeys produced in Switzerland. Antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa was expressed as a minimum inhibitory and bactericidal concentrations (MIC and MBC). Furthermore, the content of bee-derived glucose oxidase (GOX) and its enzymatic product, H2 O2 , were also evaluated. RESULTS: All honey samples successfully met basic defined criteria (moisture and hydroxymethylfurfural (HMF)) tested in this study. Honeydew honeys were the most effective honey samples and generated the highest levels of H2 O2 . A strong significant correlation was found between the overall antibacterial activity and the level of H2 O2 among all honey samples. Interestingly, the content of GOX in honey samples did not correlate with their antibacterial activity as well as H2 O2 production capacity. A weak antibacterial activity was determined in five floral honeys, most likely due to increased enzymatic activity of pollen-derived catalase. CONCLUSION: This study showed that antibacterial effect of Swiss honey samples is associated mainly with H2 O2 . © 2019 Society of Chemical Industry.

pH-sensitive response of a highly photoluminescent MoS2 nanohybrid material and its application in the nonenzymatic detection of H2O2.

The mechanism behind the variation in the photoluminescence (PL) of a MoS2 nanohybrid material with pH was investigated. Highly fluorescent MoS2 quantum dots dispersed across MoS2 nanosheets (MoS2 QDNS) were synthesized by a hydrothermal route in the presence of NaOH. Upon reducing the pH from 13 to 6.5, the PL intensity was markedly quenched. The removal of dangling sulfur atoms by adding mineral acids could be a plausible mechanism for this PL quenching, together with the inner filter effect and Förster resonance energy transfer due to the resulting species. A label-free turn-on fluorescence sensor for H2O2 was developed using this hybrid material. The PL of the acidified MoS2 QDNS at pH 6.5 increased (i.e., recovered) linearly with the concentration of H2O2. The dynamic range of the sensor was found to be 2-94 µM with a limit of detection (LOD) of 2 µM. This sensing strategy was also extended for the detection of glucose by appending glucose oxidase (GOx) as a catalyst. In the presence of GOx, glucose oxidizes to gluconic acid and H2O2, so the original level of glucose can be estimated by determining the H2O2 present. The absence of a complicated enzyme immobilization step is the prime advantage of the present glucose sensor. The current work exemplifies the utility of MoS2-based nanoparticle systems in the biological sensor domain. Graphical abstract.

Detection of Single Proteins with a General Nanopore Sensor.

Single protein sensing based on solid-state nanopores is promising but challenging, because the fast translocation velocity of a protein is beyond the bandwidth of nanopore instruments. To decelerate the translocation speed, here, we employed a common protein cross-link interaction to achieve a general and robust nanopore sensing platform for single-molecule detection of protein. Benefiting from the EDC/NHS coupling interaction between nanopore and proteins, a 10-fold decrease in speed has been achieved. The clearly distinguishable current signatures further reveal that the anisotropic translocation of a protein, which are horizontal, vertical, and flipping transit inside nanopore confinement. This strategy provides a general platform for rapid detection of proteins as well as exploring fundamental protein dynamics at the single-molecule level.

In Vitro Sugar Interference Testing With Amperometric Glucose Oxidase Sensors.

BACKGROUND: Electrochemical enzymatic glucose sensors are intended to measure blood or interstitial fluid glucose concentrations. One class of these glucose sensors are continuous glucose monitors (CGMs), indicated for tracking and trending of glucose concentrations in interstitial fluid and as an adjunct to blood glucose testing. Currently approved CGMs employ a glucose oxidase (GOx) electrochemical detection scheme. Potential interfering agents can impact the accuracy of results obtained by glucose sensors, including CGMs. METHODS: Seven sugars, seven sugar alcohols, and three artificial sweeteners were in vitro screened for interference with amperometric glucose oxidase (GOx) sensors at concentrations greater than physiologic concentrations. Galactose was investigated further at physiologically relevant concentrations using a custom amperometric system. Furthermore, glucose and galactose calibration experiments were conducted to facilitate multiple enzyme kinetic analysis approaches (Michaelis-Menten and Hill equation) to understand the potential source and mechanism of interference from galactose. RESULTS: Under in vitro testing, except for galactose, xylose and mannose, all screened compounds exhibited interference bias, expressed in mean absolute relative difference (MARD), of ⩽ 20% even at concentrations significantly higher than normal physiologic concentrations. Galactose exhibited, CGM-dependent, MARD of 47-72% and was subjected to further testing. The highest recorded mean relative difference (MRD) was 6.9 ± 1.3% when testing physiologically relevant galactose concentrations (0.1-10 mg/dL). Enzyme kinetic analysis provided calculations of maximum reaction rates ( imax ), apparent Michaelis constants ( Kmapp ), and Hill equation h parameters for glucose and galactose substrates for the enzymes in the CGMs. CONCLUSION: Under the conditions of in vitro screening, 14 of the 17 compounds did not exhibit measuarable interference. Galactose exhibited the highest interference during screening, but did not substantially interfere with CGMs under the conditions of in vitro testing at physiologically relevant concentrations. Enzyme kinetic analysis conducted with galactose supported the notion that (1) the reactivity of GOx enzyme toward nonglucose sugars and (2) the presence of enzymatic impurities (such as galactose oxidase) are two potential sources for sugar interference with GOx glucose sensors, and thus, should be considered during device development.