Bimetallic Single-Atom Nanozyme-Based Electrochemical-Photothermal Dual-Function Portable Immunoassay with Smartphone Imaging.

Rapid and accurate detection of human epidermal growth factor receptor 2 (HER2) is crucial for the early diagnosis and prognosis of breast cancer. In this study, we reported an iron-manganese ion N-doped carbon single-atom catalyst (FeMn-NCetch/SAC) bimetallic peroxidase mimetic enzyme with abundant active sites etched by H2O2 and further demonstrated unique advantages of single-atom bimetallic nanozymes in generating hydroxyl radicals by density functional theory (DFT) calculations. As a proof of concept, a portable device-dependent electrochemical-photothermal bifunctional immunoassay detection platform was designed to achieve reliable detection of HER2. In the enzyme-linked reaction, H2O2 was generated by substrate catalysis via secondary antibody-labeled glucose oxidase (GOx), while FeMn-NCetch/SAC nanozymes catalyzed the decomposition of H2O2 to form OH*, which catalyzed the conversion of 3,3',5,5'-tetramethylbenzidine (TMB) to ox-TMB. The ox-TMB generation was converted from the colorimetric signals to electrical and photothermal signals by applied potential and laser irradiation, which could be employed for the quantitative detection of HER2. With the help of this bifunctional detection technology, HER2 was accurately detected in two ways: photothermally, with a linear scope of 0.01 to 2.0 ng mL-1 and a limit of detection (LOD) of 7.5 pg mL-1, and electrochemically, with a linear scope of 0.01 to 10 ng mL-1 at an LOD of 3.9 pg mL-1. By successfully avoiding environmental impacts, the bifunctional-based immunosensing strategy offers strong support for accurate clinical detection.

Modulable 3D-printed plantibody-laden platform enabling microscale affinity extraction and ratiometric front-face fluorescence detection of microcystin-LR in marine waters.

A 3D-printed stereolithographic platform for selective biorecognition is designed to enable convective microscale affinity extraction of microcystin-LR (MC-LR) followed by direct solid-phase optosensing exploiting ratiometric front-face fluorescence spectroscopy. For this purpose, a recombinant monoclonal plantibody (recAb) is covalently attached to a 3D-printed structure for sorptive immunoextraction, whereupon the free and unbound primary amino moieties of the recAb are derivatized with a fluorescent probe. The fluorophore-recAb-MC-LR laden device is then accommodated in the cuvette holder of a conventional fluorometer without any instrumental modification for the recording of the solid-phase fluorescence emission. Using Rodbard's four-parameter sigmoidal function, the 3D-printed bioselective platform features a limit of detection (LOD) of 28 ng L-1 using a sample volume of 500 mL, device-to-device reproducibility down to 12%, and relative recoveries ranging from 91 to 100% in marine waters. Printed prototypes are affordable, just 0.4 € per print and ≤ 10 € per device containing recAb. One of the main assets of the miniaturized immunoextraction device is that it performs comparably well in terms of analytical figures of merit with costly mass spectrometric-based analytical methodologies, such as HPLC-MS/MS. The device is readily applicable to high-matrix samples, such as seawater, as opposed to previous biosensing platforms, just applied to freshwater systems.

Ag/MoO3-Pd-mediated gasochromic reaction: An efficient dual-mode photoelectrochemical and photothermal immunoassay.

Herein, we presented a dual-readout gasochromic immunosensing platform for accurate and sensitive detection of carcinoembryonic antigen (CEA) based on Ag-doped/Pd nanoparticles loaded MoO3 nanorods (Ag/MoO3-Pd). Initially, the presence of analyte CEA would prompt the formation of sandwich-type immunoreaction, accompanied by the introduction of Pt NPs labeled on detection antibody. Upon the addition of NH3BH3, the product hydrogen (H2) will interact with Ag/MoO3-Pd as a bridge between the sensing interface and the biological assembly platform. Both photocurrent and temperature signals can serve as readouts due to the significantly increased PEC performance and enhanced photothermal conversion capability of H-Ag/MoO3-Pd (the product of Ag/MoO3-Pd react with H2) compared to Ag/MoO3-Pd. In addition, the DFT results show that the band gap of Ag/MoO3-Pd becomes narrower after the reaction with H2, thus improving the utilization of light, which theoretically explains the internal mechanism of gas sensing reaction. Under optimal conditions, the designed immunosensing platform showed good sensitivity for CEA detection with the limit of detection (LOD) of 26 pg mL-1 (photoelectrochemical mode) and 98 pg mL-1 (photothermal mode). This work not only presents the possible reaction mechanism of Ag/MoO3-Pd and H2, but also creatively applicate it in photothermal biosensors that give a new path for devising dual-readout immunosensor.

Smartphone-Based Electrochemical Immunoassay for Point-of-Care Detection of SARS-CoV-2 Nucleocapsid Protein.

Large-scale, rapid, and inexpensive serological diagnoses of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) are of great interest in reducing virus transmission at the population level; however, their development is greatly plagued by the lack of available point-of-care methods, leading to low detection efficiency. Herein, an ultrasensitive smartphone-based electrochemical immunoassay is reported for rapid (less than 5 min), low-cost, easy-to-implement detection of the SARS-CoV-2 nucleocapsid protein (SARS-CoV-2 N protein). Specifically, the electrochemical immunoassay was fabricated on a screen-printed carbon electrode coated with electrodeposited gold nanoparticles, followed by incubation of anti-N antibody (Ab) and bovine serum albumin as the working electrode. Accompanied by the antigen-antibody reaction between the SARS-CoV-2 N protein and the Ab, the electron transfer between the electroactive species [Fe(CN)6]3-/4- and the electrode surface is disturbed, resulting in reduced square-wave voltammetry currents at 0.075 V versus the Ag/AgCl reference electrode. The proposed immunoassay provided a good linear range with SARS-CoV-2 N protein concentrations within the scope of 0.01-1000 ng/mL (R2 = 0.9992) and the limit of detection down to 2.6 pg/mL. Moreover, the detection data are wirelessly transmitted to the interface of the smartphone, and the corresponding SARS-CoV-2 N protein concentration value is calculated and displayed. Therefore, the proposed portable detection mode offers great potential for self-differential diagnosis of residents, which will greatly facilitate the effective control and large-scale screening of virus transmission in resource-limited areas.

CRISPR-Cas12a-Derived Photoelectrochemical Biosensor for Point-Of-Care Diagnosis of Nucleic Acid.

This work presented a point-of-care (POC) photoelectrochemical (PEC) biosensing for the detection of human papillomavirus-16 (HPV-16) on a portable electrochemical detection system by using CRISPR-Cas12a trans-cleaving the G-quadruplex for the biorecognition/amplification and a hollow In2O3-In2S3-modified screen-printed electrode (In2O3-In2S3/SPE) as the photoactive material. G-quadruplexes were capable of biocatalytic precipitation (H2O2-mediated 4-chloro-1-naphthol oxidation) on the In2O3-In2S3/SPE surface, resulting in a weakened photocurrent, but suffered from trans-cleavage when the CRISPR-Cas12a system specifically recognized the analyte. The photocurrent results could be directly observed with the card-sized electrochemical device via a smartphone, which displayed a high-value photocurrent for these positive samples, while a low-value photocurrent for the target-free samples. Such a system exhibited satisfying photocurrent responses toward HPV-16 within a wide working range from 5.0 to 5000 pM and allowed for detection of HPV-16 at a concentration as low as 1.2 pM. The proposed assay provided a smartphone signal readout to enable the rapid screening PEC determination of HPV-16 concentration without sophisticated instruments, thus meeting the requirements of remote areas and resource-limited settings. We envision that combining an efficient biometric PEC sensing platform with a wireless card-sized electrochemical device will enable high-throughput POC diagnostic analysis.

3D printed spinning cup-shaped device for immunoaffinity solid-phase extraction of diclofenac in wastewaters.

This article reports current research efforts towards designing bespoke microscale extraction approaches exploiting the versatility of 3D printing for fast prototyping of novel geometries of sorptive devices. This is demonstrated via the so-called 3D printed spinning cup-based platform for immunoextraction of emerging contaminants using diclofenac as a model analyte. A new format of rotating cylindrical scaffold (containing a semispherical upper cavity) with enhanced coverage of biorecognition elements, and providing elevated enhancement factors with no need of eluate processing as compared with other microextraction stirring units is proposed. Two distinct synthetic routes capitalized upon modification of the acrylate surface of stereolithographic 3D printed parts with hexamethylenediamine or branched polyethyleneimine chemistries were assayed for covalent binding of monoclonal diclofenac antibody.Under the optimized experimental conditions, a LOD of 108 ng L-1 diclofenac, dynamic linear range of 0.4-1,500 µg L-1, and enrichment factors > 83 (for near-exhaustive extraction) were obtained using liquid chromatography coupled with UV-Vis detection. The feasibility of the antibody-laden device for handling of complex samples was demonstrated with the analysis of raw influent wastewaters with relative recoveries ranging from 102 to 109%. By exploiting stereolithographic 3D printing, up to 36 midget devices were fabricated in a single run with an estimated cost of mere 0.68 euros per 3D print and up to 16 €/device after the incorporation of the monoclonal antibody.

Recent Progress in Rapid Determination of Mycotoxins Based on Emerging Biorecognition Molecules: A Review.

Mycotoxins are secondary metabolites produced by fungal species, which pose significant risk to humans and livestock. The mycotoxins which are produced from Aspergillus, Penicillium, and Fusarium are considered most important and therefore regulated in food- and feedstuffs. Analyses are predominantly performed by official laboratory methods in centralized labs by expert technicians. There is an urgent demand for new low-cost, easy-to-use, and portable analytical devices for rapid on-site determination. Most significant advances were realized in the field bioanalytical techniques based on molecular recognition. This review aims to discuss recent progress in the generation of native biomolecules and new bioinspired materials towards mycotoxins for the development of reliable bioreceptor-based analytical methods. After brief presentation of basic knowledge regarding characteristics of most important mycotoxins, the generation, benefits, and limitations of present and emerging biorecognition molecules, such as polyclonal (pAb), monoclonal (mAb), recombinant antibodies (rAb), aptamers, short peptides, and molecularly imprinted polymers (MIPs), are discussed. Hereinafter, the use of binders in different areas of application, including sample preparation, microplate- and tube-based assays, lateral flow devices, and biosensors, is highlighted. Special focus, on a global scale, is placed on commercial availability of single receptor molecules, test-kits, and biosensor platforms using multiplexed bead-based suspension assays and planar biochip arrays. Future outlook is given with special emphasis on new challenges, such as increasing use of rAb based on synthetic and naïve antibody libraries to renounce animal immunization, multiple-analyte test-kits and high-throughput multiplexing, and determination of masked mycotoxins, including stereoisomeric degradation products.

Design and Analytical Performances of a Diclofenac Biosensor for Water Resources Monitoring.

Because the broadly consumed pain killer diclofenac (DCF) is a recognized pollutant, monitoring of its concentration is routinely performed in surface waters. As a valuable alternative to chromatographic and immunochemical assays, we developed a piezoelectric immunosensor to quantify DCF, first in buffer (PBS) and then in river water samples. A sensing layer comprising DCF was built up on the surface of silica-coated quartz sensors using a robust coupling chemistry. Binding of a highly affine monoclonal anti-DCF antibody was monitored in real time by quartz crystal microbalance with dissipation (QCM-D) measurements from which were determined a dissociation constant KD of 0.24 nM and an acoustic antibody surface coverage of 1120 ng/cm2 at saturation. On the other hand, an optical antibody surface coverage of 260 ng/cm2 was determined by combined nanoplasmonic sensing measurement, giving a hydration percentage of 75% for the antibody monolayer. DCF assay was further set up following a competitive format for which binding of antibody to the sensing layer is inhibited by DCF in solution. The piezoelectric sensor response expressed as frequency shift ΔF was inversely related to the concentration of DCF with a dynamic range of 15-46 nM and a limit of detection (LoD) of 9.5 nM (2.8 µg/L) in PBS. This piezoelectric immunosensor was eventually applied to the assay of DCF in surface water samples taken at three different locations in the Seine and Marne rivers. The calculated concentration of DCF in these samples was in good agreement with official data published by the French center of water analysis eaufrance.

Reliable Sensing Platform for Plasmonic Enzyme-Linked Immunosorbent Assays Based on Automatic Flow-Based Methodology.

Plasmonic enzyme-linked immunosorbent assays (ELISA) using the localized surface plasmon resonance (LSPR) of metal nanoparticles has emerged as an appealing alternative to conventional ELISA counterparts for ultrasensitive naked-eye detection of biomolecules and small contaminants. However, batchwise plasmonic ELISA involving end-point detection lacks ruggedness inasmuch as the generation or etching of NP is greatly dependent on every experimental parameter of the analytical workflow. To tackle the above shortcomings, this paper reports on an automatic flow methodology as a reliable detection scheme of hydrogen peroxide related enzymatic bioassays for ultrasensitive detection of small molecules. Here, a competitive ELISA is combined with the in-line generation of plasmonic gold nanoparticles (AuNPs) followed by the real-time monitoring of the NP nucleation and growth rates and size distribution using a USB miniaturized photometer. Glucose oxidase was labeled to the secondary antibody and yielded hydrogen peroxide that acted as the measurand and the reducing agent of the Au(III)/citrate system in the flow network. High-throughput plasmonic assays were feasible by assembling a hybrid flow system composed of two microsyringe pumps, a perfluoroalkoxy alkane reaction coil, and a 26-port multiposition valve and operated under computer-controllable flow conditions. The ultratrace determination of diclofenac in high matrix samples, e.g., seawater, without any prior sample treatment was selected as a proof-of-concept application of the flow-based platform for determination of emerging contaminants via plasmonic ELISA. The detection limit (0.001 µg L-1) was 1 order of magnitude lower than that endorsed by the first EU Watch List for diclofenac as a potentially emerging contaminant in seawater and also than that of a conventional colorimetric ELISA, which in turn is inappropriate for determination of diclofenac in seawater at the levels endorsed by the EU regulation. The proposed automatic fluidic approach is characterized by the reproducible timing in AuNPs nucleation and growth along with the unsupervised LSPR absorbance detection of AuNPs with a dynamic range for diclofenac spanning from 0.01 to 10 µg L-1. Repeatability and intermediate precision (given as normalized signal readouts) in seawater were <4% and <14%, respectively, as compared to RSDs as high as 30% as obtained with the batchwise plasmonic ELISA counterpart.

H2-Based Electrochemical Biosensor with Pd Nanowires@ZIF-67 Molecular Sieve Bilayered Sensing Interface for Immunoassay.

With the introduction of gas-based contactless electrochemical biosensors lies the prospects of separating the sensing interface from the bioassembly platform, enhancing stability, and exploring signal transduction mechanism, all intimately linking to development of immunoassay. Herein, we report on a H2-based electrochemical biosensor whose signals derived from the chemical signal transduction between a H2 and Pd nanowires@ZIF-67 (ZIF: Zeolitic Imidazolate Frameworks) bilayered sensing interface for immunoassay. Dendritic Pt nanoparticles (DPNs) conjugated on the detection antibody were introduced on the interface of a magnetic microsphere according to an immune sandwich assembly between the antigen and antibody. H2 as a bridge originates from DPNs catalyzing NH3BH3 and links biological signals to electrical signals by reacting with Pd nanowires. Nevertheless, the response of Pd nanowires being extremely effected by O2 in air due to the competitive adsorption on the surface of Pd nanostructures as well as the reaction between chemisorbed O (Pd-O) and adsorbed dihydrogen lead to a decrease in H absorption into PdHx and poor sensing responses under low target concentration. Porous ZIF-67 (window aperture 0.331 nm) as a molecular sieve self-assembling on the surface of the Pd nanowires film could easily permeate H2 (kinetic diameter of 0.289 nm), while interferential O2 (kinetic diameter of 0.346 nm) has difficultly passing through the ZIF-67 layer to contact Pd nanowires and achieves a response of a lower concentration target as well as faster response rate. Under optimal conditions, H2-based electrochemical biosensors exhibit great response toward target alpha-fetoprotein (AFP) within a dynamic working range of 0.1-50 ng mL-1 at a detection limit of 0.04 ng mL-1. Our strategy provides a reusable sensing interface, high specificity, and acceptable accuracy for the immunoassay. In addition, it also expands a promising platform for application as a molecular sieve in electrochemical biosensors.

Palindromic Molecular Beacon Based Z-Scheme BiOCl-Au-CdS Photoelectrochemical Biodetection.

This work presented an innovative and rationally engineered palindromic molecular beacon (PMB) based "Z-scheme" photoelectrochemical (PEC) biosensing protocol for the selective screening of kanamycin (Kana) through DNA hybridization-induced conformational conversion. Interestingly, the ingeniously designed PMB integrated the multifunctional elements including recognition region, primer-like palindromic fragment, and polymerization-nicking template. The cosensitized structures consisted of CdS quantum dot functionalized hairpin DNA2 (QD-HP2) and region-selectively deposited gold nanoparticles onto {001} facets of bismuth oxychloride (BiOCl-Au). Compared with BiOCl-Au alone, the attachment of CdS QDs onto BiOCl-Au (i.e., BiOCl-Au-CdS QDs) exhibited evidently enhanced photocurrent intensity thanks to the synergistic effect of Z-scheme BiOCl-Au-CdS QDs. After incubation with target Kana, Kana-aptamer binding could induce the exposure of PMB region for hairpin DNA1 (HP1). The exposed palindromic tails hybridized with each other (like a molecular machine) to consume the substrates (dNTPs) and fuels (enzyme) for the releasing of numerous nick fragments (Nick). The as-generated nick fragments could specifically hybridize with the complementary region of QD-HP2, thus resulting in decreasing photocurrent because of the increasing spatial distance for electron transfer between two-type photosensitizers. Under optimum conditions, the PMB-based sensing system exhibited satisfying photocurrent responses toward target Kana within the working range from 50 to 5000 fM at a low detection limit of 29 fM. Impressively, the concept of a palindromic fragment-mediated primer-free biosensing strategy offers a new avenue for advanced development of efficient and convenient biodetection systems.

Microcystin-LR Enrichment from Freshwater by a Recombinant Plant-derived Antibody Using Sol-Gel-Glass Immunoextraction.

Eutrophication of water bodies can promote cyanobacterial (blue-green algae) blooms, which has become a source of increasing concern for both recreational and drinking water use. Many bacterial species can produce toxins that pose threats to wildlife, domestic animals and humans. Microcystin-leucine-arginine (MC-LR) is the most frequent and most toxic microcystin congener. For the first time, lab-scale investigations were performed to test the application of a recombinant plant-derived anti-MC-LR antibody immobilized on an immunoaffinity support material to selectively extract the toxin from spiked freshwater samples. As a comparison, its hybridoma-derived counterpart (murine monoclonal antibody) was evaluated. The antibody-doped material was prepared via an optimized sol-gel process; its stability and binding efficiency of MC-LR in spiked freshwater samples were thoroughly tested using the ELISA and orthogonal LC-MS methods. For removal, two column-based procedures with sequential or continuous cyclic sample addition and a suspension mode (moving adsorbent) were tested. Noteworthy the results obtained with a crude antibody fraction were fully compatible with the highly purified preparation. This study paves the way for further investigation being focused on novel applications of plant-derived anti-MC-LR antibodies in bioremediation to selectively deplete the toxin from freshwater: a green and promising technology without secondary pollution.

Capacitive sensor for detection of benzo(a)pyrene in water.

An affinity sensor based on capacitive transduction was developed to detect benzo(a)pyrene (BaP) in river water. Two types of recognition elements, the synthetic receptor analogues molecularly imprinted polymers (MIPs) and natural monoclonal antibody (mAb) were tested for this type of biosensor. Different polymerization strategies to obtain MIPs were compared. One approach comprised a preliminary batch synthesis of beads that were subsequently coupled covalently to an electrode surface. Another approach consisted of the in-situ synthesis of MIPs directly onto the electrode surface using electropolymerization. The latter approach provided the best results. To choose the optimal recognition element mAb and MIP-modified electrodes different sets were evaluated with regards to their sensitivity, selectivity, linear range and re-usability. The mAb-modified electrodes were considerably more sensitive toward BaP (ng L-1 range vs µg L-1 range for the MIP-modified one), and showed a broader linear working range than the MIP-modified electrodes. The latter revealed more suitable for group-selective determination of PAHs. The developed capacitive sensor was tested for the detection of BaP in naturally-contaminated water samples collected in different places of Ghent, Belgium. The results obtained with the sensor were coherent and reproducible, and were in a good agreement with the confirmation technique, HPLC-FlD.

Detecting Nonvolatile Life- and Nonlife-Derived Organics in a Carbonaceous Chondrite Analogue with a New Multiplex Immunoassay and Its Relevance for Planetary Exploration.

Potential martian molecular targets include those supplied by meteoritic carbonaceous chondrites such as amino acids and polycyclic aromatic hydrocarbons and true biomarkers stemming from any hypothetical martian biota (organic architectures that can be directly related to once living organisms). Heat extraction and pyrolysis-based methods currently used in planetary exploration are highly aggressive and very often modify the target molecules making their identification a cumbersome task. We have developed and validated a mild, nondestructive, multiplex inhibitory microarray immunoassay and demonstrated its implementation in the SOLID (Signs of Life Detector) instrument for simultaneous detection of several nonvolatile life- and nonlife-derived organic molecules relevant in planetary exploration and environmental monitoring. By utilizing a set of highly specific antibodies that recognize D- or L- aromatic amino acids (Phe, Tyr, Trp), benzo[a]pyrene (B[a]P), pentachlorophenol, and sulfone-containing aromatic compounds, respectively, the assay was validated in the SOLID instrument for the analysis of carbon-rich samples used as analogues of the organic material in carbonaceous chondrites or even Mars samples. Most of the antibodies enabled sensitivities at the 1-10 ppb level and some even at the ppt level. The multiplex immunoassay allowed the detection of B[a]P as well as aromatic sulfones in a water/methanol extract of an Early Cretaceous lignite sample (c.a., 140 Ma) representing type IV kerogen. No L- or D-aromatic amino acids were detected, reflecting the advanced diagenetic stage and the fossil nature of the sample. The results demonstrate the ability of the liquid extraction by ultrasonication and the versatility of the multiplex inhibitory immunoassays in the SOLID instrument to discriminate between organic matter derived from life and nonlife processes, an essential step toward life detection outside Earth.

Cloning and plant-based production of antibody MC10E7 for a lateral flow immunoassay to detect [4-arginine]microcystin in freshwater.

Antibody MC10E7 is one of a small number of monoclonal antibodies that bind specifically to [Arg4]-microcystins, and it can be used to survey natural water sources and food samples for algal toxin contamination. However, the development of sensitive immunoassays in different test formats, particularly user-friendly tests for on-site analysis, requires a sensitive but also cost-effective antibody. The original version of MC10E7 was derived from a murine hybridoma, but we determined the sequence of the variable regions using the peptide mass-assisted cloning strategy and expressed a scFv (single-chain variable fragment) format of this antibody in yeast and a chimeric full-size version in leaves of Nicotiana tabacum and Nicotiana benthamiana to facilitate inexpensive and scalable production. The specific antigen-binding activity of the purified antibody was verified by surface plasmon resonance spectroscopy and ELISA, confirming the same binding specificity as its hybridoma-derived counterpart. The plant-derived antibody was used to design a lateral flow immunoassay (dipstick) for the sensitive detection of [Arg4]-microcystins at concentrations of 100-300 ng/L in freshwater samples collected at different sites. Plant-based production will likely reduce the cost of the antibody, currently the most expensive component of the dipstick immunoassay, and will allow the development of further antibody-based analytical devices and water purification adsorbents for the efficient removal of toxic contaminants.

Tight Molecular Recognition of Benzo[a]pyrene by a High-Affinity Antibody.

Benzo[a]pyrene, which is produced during the incomplete combustion of organic material, is an abundant noxious pollutant because of its carcinogenic metabolic degradation products. The high-affinity (KD ≈3 nm) monoclonal antibody 22F12 allows facile bioanalytical quantification of benzo[a]pyrene even in complex matrices. We report the functional and X-ray crystallographic analysis of 22F12 in complex with 3-hydroxybenzo[a]pyrene after cloning of the V-genes and production as a recombinant Fab fragment. The polycyclic aromatic hydrocarbon is bound in a deep pocket between the light and heavy chains, surrounded mainly by aromatic and aliphatic amino acid side chains. Interestingly, the hapten-antibody interface is less densely packed than expected and reveals polar, H-bond-like interactions with the polycyclic aromatic π-electron system, which may allow the antibody to maintain a large, predominantly hydrophobic binding site in an aqueous environment while providing sufficient complementarity to its ligand.

Signal-On Photoelectrochemical Immunoassay for Aflatoxin B1 Based on Enzymatic Product-Etching MnO2 Nanosheets for Dissociation of Carbon Dots.

Aflatoxin B1 (AFB1) monitoring has attracted extensive attention because food safety is a worldwide public health problem. Herein, we design a novel simultaneously visual and photoelectrochemical (PEC) immunosensing system for rapid sensitive detection of AFB1 in foodstuff. The immunoreaction was carried out on anti-AFB1 antibody-modified magnetic beads by using glucose oxidase (GOx)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags with a competitive-type immunoassay format, while the visual and PEC evaluation was performed via carbon quantum dots (CQDs)-functionalized MnO2 nanosheets. Accompanying the formation of immunocomplexes, the carried GOx initially oxidized the substrate (glucose) for the generation of H2O2, which reduced/etched MnO2 nanosheets into Mn2+ ions, thereby resulting in the dissociation of CQDs from the electrode. Within the applied potentials, the photocurrent of MnO2-CQDs-modified electrode decreased with the increasing H2O2 level in the detection cell. Meanwhile, a visual detection could be performed according to the change in the color of MnO2-CQDs-coated electrode. To elaborate, this system was aggregated into a high-throughput microfluidic device to construct a semiautomatic detection cell. Under optimal conditions, the photocurrent increased with the increasing target AFB1 within a dynamic working range from 0.01 to 20 ng mL-1 with a limit of detection (LOD) of 2.1 pg mL-1 (ppt). The developed immunoassay exhibited good reproducibility and acceptable accuracy. In addition, the method accuracy relative to AFB1 ELISA kit was evaluated for analyzing naturally contaminated or spiked peanut samples, giving the well-matched results between two methods. Although our strategy was focused on the detection of target AFB1, it is easily extended to screen other small molecules or mycotoxins, thereby representing a versatile immunosensing scheme.

Microarray-Based Immunoassay for Parallel Quantification of Multiple Mycotoxins in Oat.

Because multianalyte methods are highly desirable in order to keep analysis time and costs low, the biosensor development increasingly focuses on parallel analysis of several mycotoxins. Here, we describe an indirect competitive immunoassay on regenerable, reusable glass microchips for the parallel determination of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisin B1 in oat extracts, using a fully automated flow-through device with chemiluminescence readout.

Silver Nanolabels-Assisted Ion-Exchange Reaction with CdTe Quantum Dots Mediated Exciton Trapping for Signal-On Photoelectrochemical Immunoassay of Mycotoxins.

Mycotoxins, highly toxic secondary metabolites produced by many invading species of filamentous fungi, contaminate different agricultural commodities under favorable temperature and humidity conditions. Herein, we successfully devised a novel signal-on photoelectrochemical immunosensing platform for the quantitative monitoring of mycotoxins (aflatoxin B1, AFB1, used as a model) in foodstuffs on the basis of silver nanolabels-assisted ion-exchange reaction with CdTe quantum dots (QDs) mediated hole-trapping. Initially, a competitive-type immunoreaction was carried out on a high-binding microplate by using silver nanoparticle (AgNP)-labeled AFB1-bovine serum albumin (AFB1-BSA) conjugates as the tags. Then, the carried AgNPs with AFB1-BSA were dissolved by acid to release numerous silver ions, which could induce ion-exchange reaction with the CdTe QDs immobilized on the electrode, thus resulting in formation of surface exciton trapping. Relative to pure CdTe QDs, the formed exciton trapping decreased the photocurrent of the modified electrode. In contrast, the detectable photocurrent increased with the increase of target AFB1 in a dynamic working range from 10 pg mL(-1) to 15 ng mL(-1) at a low limit of detection (LOD) of 3.0 pg mL(-1) under optimal conditions. In addition, the as-prepared photoelectrochemical immunosensing platform also displayed high specificity, good reproducibility, and acceptable method accuracy for detecting naturally contaminated/spiked blank peanut samples with consistent results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.

Competitive Upconversion-Linked Immunosorbent Assay for the Sensitive Detection of Diclofenac.

Photon-upconverting nanoparticles (UCNPs) emit light of shorter wavelength under near-infrared excitation and thus avoid optical background interference. We have exploited this unique photophysical feature to establish a sensitive competitive immunoassay for the detection of the pharmaceutical micropollutant diclofenac (DCF) in water. The so-called upconversion-linked immunosorbent assay (ULISA) was critically dependent on the design of the upconversion luminescent detection label. Silica-coated UCNPs (50 nm in diameter) exposing carboxyl groups on the surface were conjugated to a secondary anti-IgG antibody. We investigated the structure and monodispersity of the nanoconjugates in detail. Using a highly affine anti-DCF primary antibody, the optimized ULISA reached a detection limit of 0.05 ng DCF per mL. This performance came close to a conventional enzyme-linked immunosorbent assay (ELISA) without the need for an enzyme-mediated signal amplification step. The ULISA was further employed for analyzing drinking and surface water samples. The results were consistent with a conventional ELISA as well as liquid chromatography-mass spectrometry (LC-MS).