Application Of Bioassay Research Articles

Ligands engineering of gold nanoclusters with enhanced photoluminescence for deceptive information encryption and glutathione detection

Gold nanoclusters (Au NCs) have appeared as an essential alternative to traditional quantum dots and fluorescent molecules for the development of intelligent stimuli-responsive photoluminescence (PL), but the low PL emission of Au NCs restricts their broad applications. Herein, we reported a simple yet effective strategy for preparing Au NCs with high PL by ligands engineering of 4-hydroxy-2-mercapto-6-methylpyrimidine (MTU) and L-Arginine (Arg). Owing to the rigidified shell and the ligand-to-metal charge transfer (LMCT) effects, it was found that the assembly of Arg ligand on MTU-protected Au NCs (Arg/MTU-Au NCs) led to a significantly enhanced PL in the alkaline solution up to 30 times. Moreover, utilizing the tunable LMCT, the Arg/MTU-Au NCs displayed rapid responses to multi-type ionic interaction in a reversible manner, such as H+/OH− and Cu2+/glutathione (GSH) pairs. Inspired by these intriguing ions-responsive LMCT and the associated switchable PL emission, the Arg/MTU-Au NCs were successfully used as excellent stimuli-responsive PL probes for intriguing deceptive information encryption and biosensing as well. This work would provide new insight into regulating the PL emission of Au NCs by ligands engineering and advance their potential applications in information encryption and bioassay.

Fungal bioassays for environmental monitoring.

Environmental pollutants are today a major concern and an intensely discussed topic on the global agenda for sustainable development. They include a wide range of organic compounds, such as pharmaceutical waste, pesticides, plastics, and volatile organic compounds that can be found in air, soil, water bodies, sewage, and industrial wastewater. In addition to impacting fauna, flora, and fungi, skin absorption, inhalation, and ingestion of some pollutants can also negatively affect human health. Fungi play a crucial role in the decomposition and cycle of natural and synthetic substances. They exhibit a variety of growth, metabolic, morphological, and reproductive strategies and can be found in association with animals, plants, algae, and cyanobacteria. There are fungal strains that occur naturally in soil, sediment, and water that have inherent abilities to survive with contaminants, making the organism important for bioassay applications. In this context, we reviewed the applications of fungal-based bioassays as a versatile tool for environmental monitoring.

Rational Design of Conducting Polymer-Derived Tubular Carbon Nanoreactors for Enhanced Enzyme-like Catalysis and Total Antioxidant Capacity Bioassay Application.

The design of void-confined tubular nanostructures has aroused significant interest for catalytic applications because of their distinct microenvironment to modulate the reaction kinetics. Herein, we propose a facile wrapping-pyrolysis strategy to confine Fe0 nanoparticles (Fe NPs) inside N-doped carbon nanotubes (Fe@NC NTs) derived from Fe2O3@polypyrrole (PPy) core-sheath nanofibers (NFs). The resultant Fe@NC NTs can act as efficient enzyme mimics and exhibit a significantly higher peroxidase (POD)-like catalytic activity than unconfined Fe NPs and bare NC NTs. Kinetic experiments demonstrate that the optimized void structure benefits the affinity with the POD substrates and achieves excellent catalytic efficiency. The mechanism study reveals that the generation of •OH from H2O2 endows Fe@NC NTs with excellent POD-like performance. Furthermore, we develop a total antioxidant capacity (TAC) sensing platform on account of this efficient POD-like system, expanding their applications in the field of food safety and human healthcare.

Potential-resolved differential electrochemiluminescence immunosensor based on novel designed IBPHF for self-correctable detection of AFB1

Electrochemiluminophore system with potential-resolved has exhibited giant potentiality for the application of bioassays in recent years, however, rarely studies toward the novel luminophore with potential-resolved electrochemiluminescence (PRECL) have been reported. In this work, a novel organoboron complex microrod (IBPHF) with PRECL property was synthesized, which emitted two strongest electrochemiluminescence (ECL) signals in the anode region at around 1.5 V and cathode region at around −1.5 V by using tri-n-propylamine (TPA)/K2S2O8 as coreactant. The relevant ECL properties and luminescence mechanisms have been discussed in detail. Moreover, electroactive substrate of three-dimensional porous starch carbon nanomaterials (3D-PSC NPs) were designed to capture antibodies. As a result, the competitive potential-resolved differential electrochemiluminescence immunosensor was fabricated, and greatly enhanced sensitivity was achieved by using IBPHF by using IBPHF as a carrier to label competing antigens. In the presence of aflatoxin B1 (AFB1), due to the competitive mechanism and the specific recognition of antigen–antibody, the well-prepared immunosensor performed a quantitative decreasement in the range from 50 fg/mL to 100 ng/mL with a minimum detectable limit of 16.44 fg/mL by using the intensity difference (△ECL) (ECLanode-ECLcathode). The self-correction of △ECL extremely enhanced the accuracy and reliability of the sensor. Furthermore, the designed sensor achieved excellent detection towards AFB1 in walnut samples, and performed a bright application for trace target detection in future.

Bimetallic CuCo Prussian blue analogue nanocubes induced chemiluminescence of luminol under alkaline solution for uric acid detection in human serum

In this paper, bimetallic CuCo Prussian blue analogue (CuCo PBA) nanocubes, synthesized through a self-assembly method, were found to induce chemiluminescence (CL) of luminol-NaOH since the catalytic oxidation activity of CuCo PBA. The CL intensity of luminol-NaOH system was enhanced about 50 times with the addition of CuCo PBA. When uric acid (UA) was added to the new luminol-NaOH-CuCo PBA system, the CL signal could be reduced significantly. Based on these, an enzyme-free CL sensor was constructed for UA detection with a linear response range from 0.30 to 5.00 μM, and detection limit of 0.16 μM (S/N = 3). The other common species in human serum didn’t affect UA detection. Furthermore, this CL sensor was successfully applied to detect the UA in human serum samples with high sensitivity. This work not only discovered the enzyme-like properties of CuCo PBA, but also broadened its application in CL bioassays.

Dual-Signal-Encoded Barcodes with Low Background Signal for High-Sensitivity Analysis of Multiple Tumor Markers

The suspension array technology (SAT) is promising for high-sensitivity multiplexed analysis of tumor markers. Barcodes as the core elements of SAT, can generate encoding fluorescence signals (EFS) and detection fluorescence signals (DFS) in the corresponding flow cytometer channel. However, the bleed-through effect of EFS in the DFS channel and the reagent-driven non-specific binding (NSB) lead to background interference for ultrasensitive assay of multiple targets. Here, we report an ingenious method to eliminate background interference between barcode and reporter using low-background dual-signal-encoded barcodes (DSBs) based on microbeads (MBs) and quantum dots (QDs). The low-background DSBs were prepared via combination strategy of two signals containing scatter signals and fluorescence signals. Three types of MBs were distinguished by the scattering channel of flow cytometer (FSC vs. SSC) to obtain the scattered signals. Green quantum dots (GQDs) or red quantum dots (RQDs) were coupled to the surface of MBs by sandwich immune structure to obtain the distinguishable fluorescent signals. Furthermore, the amount of conjugated capture antibody on the MB’s surface was optimized by comparing the change of detection sensitivity with the addition of capture antibody. The combination measurements of specificity and NSB in SAT platform were performed by incubating the capture antibody-conjugated MBs (cAb-MBs) with individual QD-conjugated detection antibody (QDs-dAb). Finally, an SAT platform based on DSBs was successfully established for highly sensitive multiplexed analysis of six tumor markers in one test, which suggests the promising tool for highly sensitive multiplexed bioassay applications.

3D Concentric Electrodes-Based Alternating Current Electrohydrodynamics: Design, Simulation, Fabrication, and Potential Applications for Bioassays.

Two-dimensional concentric asymmetric microelectrodes play a crucial role in developing sensitive and specific biological assays using fluid micromixing generated by alternating current electrohydrodynamics (ac-EHD). This paper reports the design, simulation, fabrication, and characterization of fluid motion generated by 3D concentric microelectrodes for the first time. Electric field simulations are used to compare electric field distribution at the electrodes and to analyze its effects on microfluidic micromixing in 2D and 3D electrodes. Three-dimensional devices show higher electric field peak values, resulting in better fluid micromixing than 2D devices. As a proof of concept, we design a simple biological assay comprising specific attachment of streptavidin beads onto the biotin-modified electrodes (2D and 3D), which shows ~40% higher efficiency of capturing specific beads in the case of 3D ac-EHD device compared to the 2D device. Our results show a significant contribution toward developing 3D ac-EHD devices that can be used to create more efficient biological assays in the future.

Self-assembled multiprotein nanostructures with enhanced stability and signal amplification capability for sensitive fluorogenic immunoassays

Fundamentally improving the sensing sensitivity of immunoassay remains a huge challenge, which limited further critical applications. Herein we designed a new immunoprobe by integrating biometric unit (antibody) and signal amplification element (enzyme) to form urease-antibody-CaHPO4 hybrid nanoflower (UAhNF) via the biomineralization process. The dual-functional UAhNF enhances the stability of urease in NaCl (10 mmol L−1) and high temperature (60 °C), and also maintains the ability of antibody recognition, fitting greatly well with the need for immunosensor. Using imidacloprid as a model target, the fixed coating antigens are competed with imidacloprid to capture primary antibodies, and the secondary antibody of UAhNF was linked to construct the competitive-type fluorogenic immunoassays. An in-situ etching process of copper nanoparticles initiated by urease is integrated with UAhNF-based immune response for further improving the detection sensitivity. The proposed immunosensor possessed a 50% inhibition concentration value of 0.72 ng mL−1, which is 30-fold lower than conventional enzyme-linked immunosorbent assay. This presented approach provided a versatile sensing tool by varying building blocks, making it practically functional for a variety of bioassay applications.

Ultrasmall phosphatase-mimicking nanoceria with slight self-colour for nonredox nanozyme-based colorimetric sensing

Nanozyme-based colorimetric sensing has attracted significant interest in recent years, and a number of redox-type nanozyme-based colorimetric sensors based on peroxidase and oxidase mimics have been reported. However, conventional redox-type nanozyme-based colorimetric sensing is affected by interference from the endogenous reductants present in actual samples. Herein, we describe the development of a homogeneous nonredox-type nanozyme-based colorimetric sensor that exploits the intrinsic phosphatase-like activity of CeO2. Specifically, colorimetric detection platforms for fluoride ions, zearalenone, and hydrogen peroxide were constructed based on activity inhibition, aptamer-assisted gate control, and plasmonic nanoparticle growth, respectively. Thus, this study reports a versatile route for constructing nonredox nanozyme-based colorimetric sensors that are not affected by interference from endogenous reductants and are thus highly promising for use in food safety and bioassay applications.

Droplet formation of biological non-Newtonian fluid in T-junction generators. I. Experimental investigation.

The extension of microfluidics to many bioassay applications requires the ability to work with non-Newtonian fluids. One case in point is the use of microfluidics with blood having different hematocrit levels. This work is the first part of a two-part study and presents the formation dynamics of blood droplets in a T-junction generator under the squeezing regime. In this regime, droplet formation with Newtonian fluids depends on T-junction geometry; however, we found that in the presence of the non-Newtonian fluid such as red blood cells, the formation depends on not only to the channel geometry, but also the flow rate ratio of fluids, and the viscosity of the phases. In addition, we analyzed the impact of the red blood cell concentration on the formation cycle. In this study, we presented the experimental data of the blood droplet evolution through the analysis of videos that are captured by a high-speed camera. During this analysis, we tracked several parameters such as droplet volume, spacing between droplets, droplet generation frequency, flow conditions, and geometrical designs of the T junction. Our analysis revealed that, unlike other non-Newtonian fluids, where the fourth stage exists (stretching stage), the formation cycle consists of only three stages: lag, filling, and necking stages. Because of the detailed analysis of each stage, a mathematical model can be generated to predict the final volume of the blood droplet and can be utilized as a guide in the operation of the microfluidic device for biochemical assay applications; this is the focus of the second part of this study [Phys. Rev. E 105, 025106 (2022)10.1103/PhysRevE.105.025106].

Multicolour Fluorescence Based on Excitation-Dependent Electron Transfer Processes in o-Carborane Dyads.

Organic materials with excitation wavelength-dependent (Ex-de) emission are highly attractive for anticounterfeiting, optoelectronics and bioassay applications; however, the realization of Ex-de fluorescence, independent of aggregation states, remains a challenge. We herein report a photoinduced electron transfer (PeT) strategy to design Ex-de fluorescence materials by manipulating the relaxation pathways of multiple excited states. As expected, the o-carborane dyad presents a clear Ex-de fluorescence colour in the aggregated states, resulting from the tunable relative intensity of the dual-fluorescence spectra. Taking TP[1]B as an example, the amorphous powders emitted bright blue-violet, white and yellow colours under 390 nm, 365 nm and 254 nm UV illumination, respectively. Importantly, multicolour, flexible and transparent films as well as an anticounterfeiting application using this o-carborane dyad are demonstrated.

Multicolour Fluorescence Based on Excitation‐Dependent Electron Transfer Processes in o‐Carborane Dyads

AbstractOrganic materials with excitation wavelength‐dependent (Ex‐de) emission are highly attractive for anticounterfeiting, optoelectronics and bioassay applications; however, the realization of Ex‐de fluorescence, independent of aggregation states, remains a challenge. We herein report a photoinduced electron transfer (PeT) strategy to design Ex‐de fluorescence materials by manipulating the relaxation pathways of multiple excited states. As expected, the o‐carborane dyad presents a clear Ex‐de fluorescence colour in the aggregated states, resulting from the tunable relative intensity of the dual‐fluorescence spectra. Taking TP[1]B as an example, the amorphous powders emitted bright blue‐violet, white and yellow colours under 390 nm, 365 nm and 254 nm UV illumination, respectively. Importantly, multicolour, flexible and transparent films as well as an anticounterfeiting application using this o‐carborane dyad are demonstrated.

Non-adhesive contrast substrate for single-cell trapping and Raman spectroscopic analysis.

Droplet splitting by exploiting tailored surface wettability is emerging as an important pathway to creating ultralow volumes of samples that can have applications in bioassays, tissue engineering, protein chips, and material synthesis. Reduction of droplet volumes enables the encapsulation of single biological cells which allows high throughput screening. In this work, we demonstrate a facile fabrication approach to create a non-adhesive contrast quartz substrate that allows droplet splitting under gravitational force and its utilization to trap single biological cells for Raman spectroscopic studies. The non-adhesive contrast surface is created by low-power continuous-wave laser-assisted removal of the region of interest from a biocompatible non-adhesive silicone oil grafted quartz substrate. For a given laser spot dimension, the hydrophilic zone dimension is controlled via irradiation with varying laser powers. The fabricated non-adhesive contrast surface can split a microliter droplet into pico- and sub-picolitre daughter droplets. By using the substrate, the trapping of a single polystyrene bead is demonstrated and the recording of Raman spectra is carried out. Additionally, the Raman spectra of two biological cells, yeast cells and human mononuclear cells (MNCs), from a daughter droplet are recorded independently and from a mixture of the solutions. This single-cell Raman analysis could find applications in cell identification and type discrimination, biochemical imaging, metabolic and functional characterization, and clinical and toxicity studies.

Electrogenerated chemiluminescence of a Ru(bpy)3 2+/arginine system: a specific and sensitive detection of acetaminophen.

Ru(bpy)3Cl2/TPrA is a prominent and widely used ECL system in analytical science. However, the co-reactant TPrA restricts the variety of applications because of its toxicity, volatility, and high cost. Here, we use arginine (Arg) as an alternative co-reactant for Ru(bpy)32+ by taking advantage of its low cost, non-toxicity, and biocompatibility. The mechanism of the Ru(bpy)32+/Arg system is that the deprotonated Arg can react with Ru(bpy)32+ to release emission. The similarity between the Ru(bpy)32+/Arg, Ru(bpy)32+/TPrA, and Ru(bpy)32+/DBAE systems demonstrates that Arg can be used as an alternative co-reactant for Ru(bpy)32+ ECL. As a proof of concept, we achieve an excellent performance for acetaminophen (Ace) detection based on the specificity of Arg and Ace, with excellent linearity, low detection limits, and good recoveries. This work is promising to expand the scope of the Ru(bpy)32+/Arg system and move forward their applications in bioassays.

Enzyme-based digital bioassay technology - key strategies and future perspectives.

Digital bioassays based on single-molecule enzyme reactions represent a new class of bioanalytical methods that enable the highly sensitive detection of biomolecules in a quantitative manner. Since the first reports of these methods in the 2000s, there has been significant growth in this new bioanalytical strategy. The principal strategy of this method is to compartmentalize target molecules in micron-sized reactors at the single-molecule level and count the number of microreactors showing positive signals originating from the target molecule. A representative application of digital bioassay is the digital enzyme-linked immunosorbent assay (ELISA). Owing to their versatility, various types of digital ELISAs have been actively developed. In addition, some disease markers and viruses possess catalytic activity, and digital bioassays for such enzymes and viruses have, thus, been developed. Currently, with the emergence of new microreactor technologies, the targets of this methodology are expanding from simple enzymes to more complex systems, such as membrane transporters and cell-free gene expression. In addition, multiplex or multiparametric digital bioassays have been developed to assess precisely the heterogeneities in sample molecules/systems that are obscured by ensemble measurements. In this review, we first introduce the basic concepts of digital bioassays and introduce a range of digital bioassays. Finally, we discuss the perspectives of new classes of digital bioassays and emerging fields based on digital bioassay technology.

Upconversion-nanoparticle-functionalized Janus micromotors for efficient detection of uric acid.

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 SiO2. 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.

Design of novel tripyridinophane-based Eu(III) complexes as efficient luminescent labels for bioassay applications.

In this work, the development of highly luminescent europium(III) complexes in water solution is reported, including their syntheses, analyses of their photophysical properties and applications in bioassays. Three Eu(III) complexes are derived from new ligands based on a tripyridinophane platform. There are four distinct sections in the structure of these ligands: an 18-membered polyaminocarboxylic macrocycle to bind efficiently lanthanide ions in aqueous solutions, three chromophoric subunits (4-(phenylethynyl)pyridine moieties) to effectively sensitize the emission of the metal, two peripheral moieties to solubilise the complex in aqueous media (sulfonate, sulfobetaine or glucose groups) and a free NH2 group available for grafting or bioconjugation. In our synthetic procedure, a pivotal macrocyclic platform is obtained with a high yield in the crucial macrocyclization step due to a metal template ion effect (74% yield). In Tris aqueous buffer (pH 7.4), the Eu(III) complexes show a maximum excitation wavelength at 320 nm, a suitable overall quantum yield (14%), a relatively long lifetime (0.80 ms) and a one-photon brightness in the range of 10 000 M-1 cm-1. Importantly, these photophysical properties are retained at dilute concentrations, even in the presence of a very large excess of potentially competing species, such as EDTA or Mg2+ ions. Furthermore, we report the bioconjugation of a Eu(III) complex labelled by an N-hydroxysuccinimide ester reactive group with an antibody (anti-glutathione-S-transferase) and the successful application of the corresponding antibody conjugate in the detection of GST-biotin in a fluoroimmunoassay. These new complexes provide a solution for high sensitivity in Homogeneous Time-Resolved Fluorescence (HTRF®) bioassays.

Highly sensitive photoelectrochemical biosensor based on Au nanoparticles sensitized zinc selenide quantum dots for DNA detection

Herein, a sensitive photoelectrochemical (PEC) biosensor was developed for detection of target DNA based on ZnSe quantum dots (QDs)/Au nanoparticles (NPs) sensitized structure as signal tag and hybrid chain reaction (HCR) as signal amplifier. Initially, the single-stranded target DNA triggered HCR to form a long double-stranded DNA HCR product containing a great number of biotin and AuNPs. Subsequently, the product could be connected with ZnSe QDs modified by avidin through the interaction between avidin and biotin to form AuNPs/ZnSe QDs sensitized structure, in which the photocurrent signal of ZnSe QDs would be significantly enhanced under light irradiation as a result of the surface plasmon resonance of AuNPs. Impressively, the photocurrent intensity obtained by 4 nm AuNPs/ZnSe QDs was about sixfold higher than that of ZnSe QDs alone. As a application in bioassay, the constructed PEC biosensor could detect target DNA in the linear range from 1 fM to 10 nM with a detection limit of 0.33 fM. This strategy opens a new way to construct sensitive PEC biosensor for the detection of biomolecules in the bioanalysis and disease diagnosis.

Short-term changes related to autotetraploidy in essential oil composition of Eucalyptus benthamii Maiden & Cambage and its applications in different bioassays

Some forest trees have been polyploidized to improve their traits and to supply new germplasms for breeding programs. As trees have a long juvenile stage, the early characterization of the chromosome set doubling effects is crucial for previous selection. Thus, we aimed to characterize the chemical variability of essential oils from diploid and autotetraploid germplasms (autotetraploid A and B) of Eucalyptus benthamii, as well as to evaluate their larvicidal and allelopathic effects. Autotetraploid A showed a higher essential oil yield than diploid and autotetraploid B, which did not differ quantitatively. Aromadendrene, viridiflorol and α-pinene were the major compounds in the diploid essential oil. In contrast, compounds were present in autotetraploids, such as 1,8-cineole, limonene, α-terpineol, and α-terpinyl-acetate. Essential oils from the diploid at 50–200 ppm were twice as larvicidal than those from autotetraploids against Aedes aegypti larvae. Considering the phytotoxicity bioassays using Lactuca sativa, essential oils from both ploidy levels affected root growth. Moreover, the essential oils inhibited shoot growth at all concentrations tested (187.5; 375; 750; 1500; and 3000 ppm). Autotetraploid A and B had the same effect on shoot growth as glyphosate. The essential oils had no cytogenotoxic effect on root meristematic cells of L. sativa, whereas phytotoxic potential was identified mainly in shoot growth. This work demonstrated a dramatic change in secondary metabolism (terpene composition) related to an increase in the ploidy level in Eucalyptus germplasms. In addition, we report the novelty of the chemical composition of essential oils among germplasms and their potential use as larvicidal and post-emergence weed control agents.

A low-cost self-dispersing method of droplet array generation enabled by a simple reusable mask for bioanalysis and bioassays.

Discontinuous dewetting is an attractive technique that can produce droplet array of specific volume, geometry and at predefined location on a substrate. Droplet array has great potential in bioanalysis such as high-throughput live cell screening, digital PCR, and drug candidates. Here, we propose a self-dispersing droplet array generation method, which has advantages of low cost, simple operation, and easy large-area production ability. Droplet array of specific volumes was generated on a polymethyl methacrylate (PMMA) substrate using a simple reusable polyimide (PI) adhesive mask. Experiment shows that the generated droplet array can be used to successfully capture single particles which obeys Poisson distribution in a high-throughput manner. Furthermore, a droplet-array sandwiching chip was created based on the self-dispersion method for rapid detection of human serum albumin (HSA) at wide range of 183-11,712μg/mL with low reagent consumption of 2.2 μL, demonstrating its potential applications in convenient high-throughput bioanalysis and bioassays.