Application Of Bioassay Research Articles

SERS Sensor for Acetylcholine Detection Based on Covalent Organic Framework Hybridized Gold Nanoparticles As Nanozymes.

An innovative and potent nanozyme with reductase-like activity was developed by integrating the in situ synthesis of gold nanoparticles (Au NPs) onto the surface of a covalent organic framework (COF). Based on the reductase-like activity of the COF-hybridized Au NPs, this nanozyme could efficiently catalyze the reduction of 4-nitrophenol (4-NPH). Moreover, the prepared nanohybrid was utilized as an excellent surface-enhanced Raman scattering (SERS) substrate for highly sensitive SERS detection by combining the excellent adsorption properties of COFs and the large number of Raman hotspots between the high-density Au NPs. For the first time, 4-NPH was used as an SERS marker to detect the electron receptor acetylcholine (Ach), with high sensitivity; Ach acted as an inhibitor in this catalytic reaction. The linear range of Ach was 1.0 pM to 10 nM, the correlation coefficient was 0.993, the detection limit was approximately 0.3 pM with a signal-to-noise ratio (s/n) of 3, and the acceptable recovery in the serum was 97.2% to 104.5%. The results from this study show that the nanozyme-SERS system has great potential for chemical and bioassay applications.

A novel controllable nanocyclic plasma coupled array in SERS trace detection of multi-component pollutants

The development and design of a novel, uniform and highly active local electromagnetic field enhanced structure is crucial for expanding Surface-enhanced Raman Scattering (SERS) applications. In this study, we developed Ag ring-coupled nanoarrays (Ag RCNAs) with controllable nanogaps using a substrate rotary evaporation coating technique with self-assembled polystyrene (PS) microspheres as templates. This straightforward and cost-effective method efficiently prepares plasma-coupled nanoarrays. Ag RCNAs demonstrated high sensitivity in detecting organic dyes, our prepared Ag RCNAs showed high sensitivity (with the limit of detection of 10−8 M), high signal reproducibility (with the relative standard deviation of 6.73 %). Furthermore, Ag RCNAs showed remarkable sensitivity to a broad spectrum of dyes in river water, indicating the large-area uniform and highly active circular-ring-shaped nanogaps can realize highly sensitive detection of various pollutants. This approach offers advantages in electromagnetic field enhancement, tunable nanogaps, uniformity, reproducibility, and recyclability, making it promising for applications in environmental monitoring, bioassays, food safety, and medical diagnostics.

The ‘ABC’ of split-nanoluciferase HIF heterodimerization bioassays: Applications, Benefits & Considerations

Hypoxia-inducible factors (HIF) are interesting targets for multiple therapeutic indications. While HIF activation is desired for the treatment of anemia-related and ischemic diseases, HIF inhibition is of tremendous interest to anti-cancer drug development. Different signaling events within the HIF pathway are being targeted by drug discovery programs, with a special interest in HIF-selective (possibly also HIF1/2 isoform-selective) compounds. In this study, we applied recently developed cell-based split-nanoluciferase HIF heterodimerization assays to study the effects of compounds, targeting HIF activity by various mechanisms of action. This study shows that the application of similar or diverse assay protocols allows to detect various influences on HIF heterodimerization as a key signaling event in the oxygen sensing pathway: increased HIF heterodimerization (roxadustat, MG-132), decreased HIF heterodimerization (PX-478, ibuprofen) and direct (HIF isoform-selective) heterodimerization inhibiting effects (PT-2385). Changes in treatment time and in the assay protocol allowed to assess direct and indirect effects on HIFα-HIFβ heterodimerization. In addition to the evaluation of applications of these new bioassays regarding pharmacological characterizations, benefits and considerations are discussed related to the use of cellular, luminescent-based bioassays. Briefly, benefits include the bidirectional nature of the biological readout, the upstream mechanism of detection, the differentiation between HIF1 and HIF2 effects and the simulation of various conditions. Specific and general considerations include cell-based, technical and disease/drug-related aspects (e.g., non-specific effects, color interference). In summary, the versatility of these bioassays offers benefits in widespread applications regarding drug discovery and pharmacological characterization of various therapeutics, applying either the same or optimized experimental protocols.

Excitation/emission-enhanced heterostructure photonic crystal array synergizing with "DD-A" FRET entropy-driven circuit for high-resolution and ultrasensitive analysis of ctDNA

Circulating tumor DNA (ctDNA) is an emerging biomarker of liquid biopsy for cancer. But it remains a challenge to achieve simple, sensitive and specific detection of ctDNA because of low abundance and single-base mutation. In this work, an excitation/emission-enhanced heterostructure photonic crystal (PC) array synergizing with entropy-driven circuit (EDC) was developed for high-resolution and ultrasensitive analysis of ctDNA. The donor donor−acceptor FÖrster resonance energy transfer ("DD-A" FRET) was integrated in EDC based on the introduction of simple auxiliary strand, which exhibited higher sensitivity than that of traditional EDC. The heterostructure PC array was constructed with the bilayer periodic nanostructures of nanospheres. Because the heterostructure PC has the adjustable dual photonic band gaps (PBGs) by changing nanosphere sizes, and the "DD-A" FRET can offer the excitation and emission peak with enough distance, it helps the successful matches between the dual PBGs of heterostructure PC and the excitation/emission peaks of "DD-A" FRET; thus, the fluorescence from EDC can be enhanced effectively from both of excitation and emission processes on heterostructure PC array. Besides, high-resolution of single-base mutation was obtained through the strict recognition of EDC. Benefiting from the specific spectrum-matched and synergetic amplification of heterostructure PC and EDC with "DD-A" FRET, the proposed array obtained ultrasensitive detection of ctDNA with LOD of 12.9 fM, and achieved the analysis of mutation frequency as low as 0.01%. Therefore, the proposed strategy has the advantages of simple operation, mild conditions (enzyme-free and isothermal), high-sensitivity, high-resolution and high-throughput analysis, showing potential in bioassay and clinical application.

Adaption and application of cell-based bioassays to whole-water samples

The increasing presence of contaminants of emerging concern in wastewater and their potential environmental risks require improved monitoring and analysis methods. Direct toxicity assessment (DTA) using bioassays can complement chemical analysis of wastewater discharge, but traditional in vivo tests have ethical considerations and are expensive, low-throughput, and limited to apical endpoints (mortality, reproduction, development, and growth). In vitro bioassays offer an alternative approach that is cheaper, faster, and more ethical, and can provide higher sensitivity for some environmentally relevant endpoints. This study explores the potential benefits of using whole water samples of wastewater and environmental surface water instead of traditional solid phase extraction (SPE) methods for in vitro bioassays testing. Whole water samples produced a stronger response in most bioassays, likely due to the loss or alteration of contaminants during SPE sample extraction. In addition, there was no notable difference in results for most bioassays after freezing whole water samples, which allows for increased flexibility in testing timelines and cost savings. These findings highlight the potential advantages of using whole water samples in DTA and provide a framework for future research in this area.

Integrative assessment of in situ combined bioremediation strategies applied to remediate soils spilled with sewage sludges

Landfills and waste disposal sites in the Basque Country are summarized in the inventory of soils that either currently support or have supported potentially polluting activities or facilities (Law 4/2015). Notably, “Landfill 17,” located in Gernika-Lumo, has been receiving, for decades, sewage sludges from the local wastewater treatment plant (WWTP) as agricultural amendment. In order to decontaminate and recover soil functionality, a combination of bioremediation (which involved bioagumentation and phyto- and vermitechnologies) and complementary bioremediation strategy (i.e., promotion and maintenance of the native vegetation) was implemented in situ. Physicochemical and ecotoxicological characterization were achieved. Furthermore, an ecotoxicological assessment of the soils upon flora and fauna was carried out through the application of different bioassays and biomarkers. Additionally, an integrative biomarker response (IBR/n) index was calculated to provide a holistic view of the soil general status. Critical pollutants [Cd, Cr, Ni, Pb, benzo(a)pyrene, and dieldrin] were observed in most of the treated sites. Microbial parameters did not present remarkable differences among sites. However, plant indicators pointed the non-treated site (MN8) as the unhealthiest. This was also observed in earthworms’ immune system, where cytotoxicity appears when exposed to non-treated soils. In conclusion, this field study showed that the combination of bioaugmentation, phytoremediation with native species, and vermiremediation is highly useful in eliminating mixed contamination, improving soil health, and ultimately restoring ecosystem functionality and biodiversity.

Application of bioassays and physicochemical analysis for assessing groundwater quality of selected tube wells in a CKDu impacted area

Groundwater from tube and dug wells are primarily used for drinking purposes by rural communities inhabiting Chronic Kidney Disease of unknown aetiology (CKDu) impacted areas in Sri Lanka. Groundwater safety assessment using targeted physicochemical analysis alone is not adequate to capture interactive effects of chemical mixtures in the groundwater. This study explored for the first time combined use of physicochemical analysis and bioassays with root meristems of Allium cepa and erythrocytes of Cyprinus carpio to assess groundwater quality of selected tube wells in Medawachchiya (a CKDu impacted area), Sri Lanka covering pre-monsoon, monsoon and postmonsoon seasons. The pH, turbidity, arsenic, cadmium, lead, mercury, fluoride, chloride, nitrate and nitrite levels in the groundwater complied with Sri Lanka standards for potable drinking water in all seasons. Yet, alkalinity and total hardness levels in the groundwater exceeded national drinking water standards (nearly up to two folds) mostly in the monsoon season. Bioassays revealed genotoxic effects of the groundwater of some tube wells in pre-monsoon and monsoon seasons. Observed genotoxicity may be associated with synergistic effects of contaminants including undetected and unknown chemicals in the groundwater depending on the season. The results support the use of bioassays as complementary tools for evaluating groundwater quality of CKDu hotspots considering consumer safety.

Magnetic-Actuated Jumping of Droplets on Superhydrophobic Grooved Surfaces: A Versatile Strategy for Three-Dimensional Droplet Transportation.

On-demand droplet transportation is of great significance for numerous applications. Although various strategies have been developed for droplet transportation, out-of-surface three-dimensional (3D) transportation of droplets remains challenging. Here, a versatile droplet transportation strategy based on magnetic-actuated jumping (MAJ) of droplets on superhydrophobic grooved surfaces (SHGSs) is presented, which enables 3D, remote, and precise manipulation of droplets even in enclosed narrow spaces. To trigger MAJ, an electromagnetic field is utilized to deform the droplet on the SHGS with the aid of an attached magnetic particle, thereby the droplet acquires excess surface energy. When the electromagnetic field is quickly removed, the excess surface energy is partly converted into kinetic energy, allowing the droplet to jump atop the surface. Through high-speed imaging and numerical simulation, the working mechanism and size matching effect of MAJ are unveiled. It is found that the MAJ behavior can only be observed if the sizes of the droplets and the superhydrophobic grooves are matched, otherwise unwanted entrapment or pinch-off effects would lead to failure of MAJ. A regime diagram which serves as a guideline to design SHGSs for MAJ is proposed. The droplet transportation capacities of MAJ, including in-surface and out-of-surface directional transportation, climbing stairs, and crossing obstacles, are also demonstrated. With the ability to remotely manipulate droplets in enclosed narrow spaces without using any mechanical moving parts, MAJ can be used to design miniaturized fluidic platforms, which exhibit great potential for applications in bioassays, microfluidics, droplet-based switches, and microreactions.

Highly sensitive detection of aflatoxin B1 byCRISPR/Cas12a-assisted single nanoparticle counting

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated protein (Cas) have gained extensive applications in bioassays. However, CRISPR-based detection platforms are often hampered by limited analytical sensitivity, while nucleic acid-based amplification strategies are usually indispensable for additional signal enhancement with potential risks of amplification leakages. To address these challenges, an amplification-free CRISPR-based bioassay of aflatoxin B1 (AFB1) was proposed by applying single nanoparticle counting. Single-particle mode inductively coupled plasma mass spectrometry (Sp-ICPMS) has been regarded as a sensitive tool for nanoparticle counting since one nanoparticle can generate considerable signals above backgrounds. With AFB1, activator strands were introduced to initiate the trans-cleavage of CRISPR/Cas12a for cutting the nanoparticles-tagged-magnetic beads, which were transduced to nanoparticle count signals after separation. Finally, a pico-mole level limit-of-detections (LODs) with moderate selectivity was achieved. Certified reference materials (CRMs) analysis and recovery tests were conducted with promising results. To our best knowledge, this is the first report of the single particle counting-based CRISPR/Cas12a biosensing study.

Cytogenotoxicity of raw and treated dairy manure slurry by two-stage chemical and electrocoagulation: An application of the Allium cepa bioassay

Livestock farming is an essential agricultural practice. However, the improper management of livestock wastes and discharge of untreated or partially treated livestock manure slurry poses significant environmental problems. In this study, we aimed to compare the cytogenotoxic potential of untreated and treated dairy manure slurry treated with a two-stage chemical and electrocoagulation (EC) using the Allium cepa bioassay. The A. cepa bioassay is a well-established standard tool for assessing the cytogenotoxic effects of environmental contaminants, especially those that are occurred as complex contaminant mixtures. The dairy manure slurry was subjected to chemical treatment utilizing polyaluminum chloride (PAC) and cationic polyacrylamide (CPAM) at optimized conditions, followed by EC utilizing either aluminum (Al) or steel anodes. The treated and untreated samples were then evaluated for their potential cytogenotoxicty using the A. cepa bioassay, by measuring the nuclear abnormalities (NAs) and chromosomal aberrations (CAs), along with the mitotic indices (MIs). Our findings revealed a significant reduction in cytogenotoxic indicators in the treated liquid fraction compared to the untreated dairy manure slurry. Specifically, the frequency of total NAs showed a significant reduction from 154 ‰ to 37 ‰ when the dairy manure slurry was treated with chemical coagulation followed by EC utilizing an Al anode. Moreover, the MI exhibited a significant improvement from 7 ‰ to 123 ‰, suggesting the mitigation of toxic effects. These results collectively demonstrate the effectiveness of the two-stage chemical and EC treatment under optimal conditions in treating diary manure slurry while reducing its cytogenotoxicity for living systems. The A. cepa bioassay proved to be a sensitive and reliable method for assessing the toxicity of the treated samples. The efficient solid–liquid separation and the reduction of toxicity in the liquid fraction for biological systems achieved through this treatment process highlight its potential for sustainable management of livestock waste and the preservation of water quality. Nevertheless, further studies are required to assess the toxicity of solid fraction.

Dendritic quinary PtRhMoCoFe high-entropy alloy as a robust immunosensing nanoplatform for ultrasensitive detection of biomarker

Recently, high-entropy alloys have superior physicochemical properties as compared to conventional alloys for their glamorous “cocktail effect”. Nevertheless, they are scarcely applied to electrochemical immunoassays until now. Herein, uniform PtRhMoCoFe high-entropy alloyed nanodendrites (HEANDs) were synthesized by a wet-chemical co-reduction method, where glucose and oleylamine behaved as the co-reducing agents. Then, a series of characterizations were conducted to illustrate the synergistic effect among multiple metals and fascinating structural characteristics of PtRhMoCoFe HEANDs. The obtained high-entropy alloy was adopted to build a electrochemical label-free biosensor for ultrasensitive bioassay of biomarker cTnI. In the optimized analytical system, the resultant sensor exhibited a dynamic linear range of 0.0001–200 ng mL−1 and a low detection limit of 0.0095 pg mL−1 (S/N = 3). Eventually, this sensing platform was further explored in serum samples with satisfied recovery (102.0 %). This research renders some constructive insights for synthesis of high-entropy alloys and their expanded applications in bioassays and bio-devices.

The application of bioassay using Daphnia magna for the evaluation of hospital and pharmaceutical industry wastewater containing Active Pharmaceutical Ingredients (APIs) toxicity

Several groups of Contaminants of Emerging Concern (CECs) including Active Pharmaceutical Ingredients (APIs) can influence ecosystem even at low levels of exposure. The concentration of APIs often belongs to the class of micropollutants, so that toxicity tests should be developed using sensitive test organisms. In this research we developed a potential sensitive Daphnia magna to evaluate the toxicity of APIs-containing wastewater from the total of 7 hospitals and pharmaceutical industries in Greater Bandung, West Java, Indonesia. Physico-chemical parameters that were analyzed including pH, BOD5, COD, TSS, Total N, and Phenol, according to the Ministry of Environment Regulation No. 5/2014. To determine the LC50 values, the Whole Effluent Toxicity (WET) method was applied. The results showed that the maximum concentration of pH, BOD5, COD, TSS, Total N, and Phenol of the pharmaceutical industries WWTP effluents were 7.9, 293 mgL-1, 418 mgL-1, 37 mgL-1, 10.7 mgL-1, and 0.188 mgL-1, respectively. These values have fulfilled the effluent standards. However, based on the acute toxicity testing using WET method, it was found that the minimum LC50 value of those WWTP effluents was 6.51% or equal to the TUa value of 15.35. Based on the TUa toxicity class, this effluent was categorized to be high acute toxic (10 ≤ TUa < 100).

A paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for multiplexed ultrasensitive detection of cancer biomarkers.

Conventional affinity-based colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the detection of biomarkers. However, rapid point-of-care (POC) detection of multiple cancer biomarkers by conventional ELISA is limited by long incubation time, large reagent volume, and costly instrumentation along with low sensitivity due to the nature of colorimetric methods. Herein, we have developed a reusable and cost-effective paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for ultrasensitive and high-throughput multiplexed detection of disease biomarkers within an hour without using specialized instruments. A piece of pre-patterned chromatography paper placed in the PMMA polymer pond facilitates rapid protein immobilization to avoid intricate surface modifications of polymer and can be changed with a fresh paper layer to reuse the device. Reagents can be simply delivered from the top PMMA layer to multiple microwells in the middle PMMA layer via flow-through microwells, thereby increasing the efficiency of washing and avoiding repeated manual pipetting or costly robots. Quantitative colorimetric analysis was achieved by calculating the brightness of images scanned by an office scanner or a smartphone camera. Sandwich-type immunoassay was performed in the PiPP hybrid device after the optimization of multiple assay conditions. Limits of detection of 0.32 ng mL-1 for carcinoembryonic antigen (CEA) and 0.20 ng mL-1 for prostate-specific antigen (PSA) were obtained, which were about 10-fold better than those of commercial ELISA kits. We envisage that this simple but versatile hybrid device can have broad applications in various bioassays in resource-limited settings.

Application of multi-species microbial bioassay for toxicity risk assessment LumiMARA luminometric tests and chemical analyses to assess water quality in the świętokrzyskie voivodeship

The area of Świętokrzyskie voivodeship is located within the range of several macro-regions, the parent rocks are highly differentiated, which affects the diversity of soil and water conditions. and water conditions. The weakest soils occur in the central and northern parts, the best soils in the eastern and southern parts of the Świętokrzyskie Voivodeship. The study presents selected physicochemical properties of groundwaters captured by borehole wells and spring waters in selected areas of the and spring waters in selected areas of the swietokrzyskie voivodeship. The analysed water was subjected to toxicity analysis using multispecies LumiMARA test (Microbial Assay for Risc Assessment) and physicochemical parameters were determined - pH, carbonate hardness and total hardness, conductivity, OWO and chlorides. The water samples for the study were collected at different times of the year 2021- January, April, July, November. The conductivity of the tested waters, regardless of the source of origin, varies by location. The aim of the study was to analyze the quality of water due to ground and water conditions and very high interest and use of water from the studied sources by the inhabitants of the regions. The study showed that the analysed waters contained toxic substances in concentrations exceeding the WHO recommendations. Chemical analyses were reflected in ecotoxicological analyses, which showed toxic effects on individual bacterial strains and allowed the waters to be classified into individual toxicity classes.

Establishment and application of bioassay- and molecular marker-based methods for monitoring fluvalinate resistance of Varroa mites

The Varroa mite, Varroa destructor, is an ectoparasite that infests honey bees. The extensive use of acaricides, including fluvalinate, has led to the emergence of resistance in Varroa mite populations worldwide. This study's objective is to monitor fluvalinate resistance in field populations of Varroa mites in Korea through both bioassay-based and molecular marker-based methods. To achieve this, a residual contact vial (RCV) bioassay was established for on-site resistance monitoring. A diagnostic dose of 200 ppm was determined based on the bioassay using a putative susceptible population. In the RCV bioassay, early mortality evaluation was effective for accurately discriminating mites with the knockdown resistance (kdr) genotype, while late evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 indicated potential resistance development in four populations. As an alternative approach, quantitative sequencing was employed to assess the frequency of the L925I/M mutation in the voltage-gated sodium channel (VGSC), associated with fluvalinate kdr trait. While the mutation was absent in 2020 Varroa mite populations, it emerged in 2021, increased in frequency in 2022, and became nearly widespread across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation further underscores the urgent need to replace fluvalinate with alternative acaricides. A few novel VGSC mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Application of the Aliivibrio fischeri bacterium bioassay for assessing single and mixture effects of antibiotics and copper.

The Aliivibrio fischeri bioassay was successfully applied in order to evaluate the acute effect of sulfamethoxazole (SMX), ciprofloxacin (CIP), chlortetracycline (CTC) and copper (Cu), alone or in binary, ternary, and overall mixture. The toxicity results are reported in terms of both effective concentrations, which inhibited 50% of the bacterium bioluminescence (EC50%), and in Toxic Units (TUs). The TUs were compared with predicted values obtained using the Concentration Addition model (CA). Finally, the toxicity of water extracts from a soil contaminated by the three antibiotics (7mg Kg-1 each) in the presence/absence of copper (30mg Kg-1) was also evaluated. Copper was the most toxic chemical (EC50: 0.78mg L-1), followed by CTC (EC50: 3.64mg L-1), CIP (96mg L-1) and SMX (196mg L-1). Comparing the TU and CA values of the mixtures, additive effects were generally found. However, a synergic action was recorded in the case of the CIP+Cu co-presence and antagonistic effects in the case of CTC+Cu and the ternary mixture (containing each antibiotic at 0.7mg L-1), were identified. Soil water extracts did not show any toxicity, demonstrating the buffering ability of the soil to immobilize these chemicals.

Synthesis and Modification of Magnetic Nanoparticles for Biosensing and Bioassay Applications: A Review

Biosensors are analytical devices that use biological interactions to detect and quantify single molecules, clinical biomarkers, contaminants, allergens, and microorganisms. By coupling bioreceptors with transducers, such as nucleic acids or proteins, biosensors convert biological interactions into electrical signals. Electrochemical and optical transductions are the most widely used methods due to their high detection capability and compatibility with miniaturization. Biosensors are valuable in analytical chemistry, especially for health diagnostics, as they offer simplicity and sensitivity. Despite their usefulness, challenges persist in immobilizing biorecognition elements on the transducer surface, leading to issues such as loss of sensitivity and selectivity. To address these problems, the introduction of nanomaterials, in particular magnetic nanoparticles (MNPs) and magnetic beads, has been implemented. MNPs combine their magnetic properties with other interesting characteristics, such as their small size, high surface-to-volume ratio, easy handling, and excellent biocompatibility, resulting in improved specificity and sensitivity and reduced matrix effects. They can be tailored to specific applications and have been extensively used in various fields, including biosensing and clinical diagnosis. In addition, MNPs simplify sample preparation by isolating the target analytes via magnetic separation, thus reducing the analysis time and interference phenomena and improving the analytical performance of detection. The synthesis and modification of MNPs play a crucial role in adjusting their properties for different applications. This review presents an overview of the synthesis and surface modifications of magnetic nanoparticles and their contributions to the development of biosensors and bioassays for their applications across different areas. The future challenges of MNP synthesis and integration in assays are focused on their stability, multiplex detection, simplification and portability of test platforms, and in vivo applications, among other areas of development.

Imaging and Simulation of Ruthenium Derivative Coating Microbeads at the Opaque Electrode with Electrogenerated Chemiluminescence.

Electrogenerated chemiluminescence (ECL) imaging is gaining increasing attention in various fields because of its high sensitivity, low background, and good temporal and spatial resolution. However, ECL imaging of microsized objects at the opaque electrode via top-view configuration is challenged with the reactants' diffusion and light propagation. Here, we imaged and numerically simulated ruthenium derivative coating polystyrene microbeads (Ru1-PS@MB) at the glassy carbon electrode (GCE) via top-view configuration by ECL imaging. The ruthenium derivative (bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis (hexafluorophosphate), Ru1), a typical ECL reagent, was covalently linked onto the surface of aminated PS@MBs via the amide reaction. "Strong emission in edge and weak emission in center" phenomena for fluorescence (FL) and ECL emissions were obtained from Ru1-PS@MB on GCE. Z-Stack imaging of the microsized Ru1-PS@MB luminescence was performed on GCE in the presence of tri-n-propylamine (TPA). It is found that the clear luminescence range of Ru1-PS@MB perpendicular to the electrode surface in ECL image is slightly smaller than that in the FL image. The bigger was the diameter of the microbeads (from 5 to 18 μm), the larger was the ECL luminescence range of Ru1-PS@MB perpendicular to the electrode surface (from 5 to 7 μm). Our findings, which are also supported by numerical simulation, provide insights into the ECL imaging of microsized objects at the electrode surface, which will raise promising ECL applications in bioassays and cell imaging at the microscale level.

Cell-inspired design of cascade catalysis system by 3D spatially separated active sites

Cells possess isolated compartments that spatially confine different enzymes, enabling high-efficiency enzymatic cascade reactions. Herein, we report a cell-inspired design of biomimetic cascade catalysis system by immobilizing Fe single atoms and Au nanoparticles on the inner and outer layers of three-dimensional nanocapsules, respectively. The different metal sites catalyze independently and work synergistically to enable engineered and cascade glucose detection. The biomimetic catalysis system demonstrates ~ 9.8- and 2-fold cascade activity enhancement than conventional mixing and coplanar construction systems, respectively. Furthermore, the biomimetic catalysis system is successfully demonstrated for the colorimetric glucose detection with high catalytic activity and selectivity. Also, the proposed gel-based sensor is integrated with smartphone to enable real-time and visual determination of glucose. More importantly, the gel-based sensor exhibits a high correlation with a commercial glucometer in real samples detection. These findings provide a strategy to design an efficient biomimetic catalysis system for applications in bioassays and nanobiomedicines.

H2 O2 -Induced Persistent Luminescence Signal Enhancement Applied to Biosensing.

Persistent luminescence nanoparticles (PLNPs) are innovative materials able to emit light for a long time after the end of their excitation. Thanks to this property, their detection can be separated in time from the excitation, making it possible to obtain images with a high signal-to-noise ratio. This optical property can be of particular interest for the development of in vitro biosensors. Here, wereport the unexpected effect of hydrogen peroxide (H2 O2 ) on the signal intensity of ZnGa2 O4 :Cr3+ (ZGO) nanoparticles. In the presence of H2 O2 , the signal intensity of ZGO can be amplified. This signal amplification can be used to detect and quantify H2 O2 in various media, using non-functionalized ZGO nanoparticles. This small molecule can be produced by several oxidases when they react with their substrate. Indeed, the quantification of glucose, lactic acid, and uric acid is possible. The limit of detection could be lowered by modifying the nanoparticles synthesis route. These optimized nanoparticles can also be used as new biosensor to detect larger molecules such as antigen, using the appropriate antibody. This unique property, i.e., persistent luminescence signal enhancement induced by H2 O2 , represents a new way to detect biomolecules which could lead to a very large number of bioassay applications.