A sandwich-type electrochemical immunosensor based on Au-rGO composite for CA15-3 tumor marker detection.

A sensitive detection of carbohydrate antigen 15-3 (CA15-3) levels may allow for early diagnosis and monitoring the treatment of breast cancer, but this can only be made in routine clinical practice if low-cost immunosensors are available. In this work, we developed a sandwich-type electrochemical immunosensor capable of rapid detection of CA15-3 with an ultra-low limit of detection (LOD) of 0.08 fg mL-1 within a wide linear concentration range from 0.1 fg mL-1 to 1 µg mL-1. The immunosensor had a matrix of a layer-by-layer film of Au nanoparticles and reduced graphene oxide (Au-rGO) co-electrodeposited on screen-printed carbon electrodes (SPCE). The high sensitivity was achieved by using secondary antibodies (Ab2) labeled with horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) as signal amplifiers, and hydroquinone (HQ) was used as an electron mediator. The immunosensor was selective for CA15-3 in human serum and artificial saliva samples, robust, and stable to permit storage at 4 °C for more than 30 days. With its high performance, the immunosensor may be incorporated into future point-of-care (POC) devices to determine CA15-3 in distinct biological fluids, including in blood and saliva samples.

End-labeling-based electrochemical strategy for detection of adenine methylation in nucleic acid by differential pulse voltammetry.

A promising electrochemical strategy for assay of N6-methyladenosine (m6A)/N6-methyladenine (6mA) in RNA/DNA is proposed. The key of this strategy is the end-labeling of nucleic acid, which makes it possible to detect methylation level in unknown sequence. Firstly, the end of m6A-RNA or 6mA-DNA was labeled with sulfhydryl group through T4 polynucleotide kinase (T4 PNK) and then directly assembled on a gold nanoparticle-modified glassy carbon electrode (AuNPs/GCE). Secondly, methylation sites in RNA/DNA were specifically recognized by anti-m6A-antibody, and then, horseradish peroxidase-labeled goat anti-rabbit IgG (HRP-IgG) was further conjugated on the antibody. Thirdly, HRP-IgG catalyzed the hydroquinone oxidation reaction to generate amplified current signal which correlates with the amount of m6A/6mA in nucleic acid. This method showed a wide linear range from 0.0001 to 10 nM for m6A-RNA, 0.001 to 100 nM for 6mA-dsDNA, and 0.0001 to 10 nM for 6mA-ssDNA. The method was successfully applied to detection of m6A/6mA in RNA/DNA from HeLa cells and E. coli cells and validation of the decrease of m6A-RNA in HeLa cells after treatment with FTO protein.

Fully integrated sampler and dilutor in an electrochemical paper-based device for glucose sensing.

An electroanalytical platform capable to take and dilute the sample has been designed in order to fully integrate the different steps of the analytical process in only one device. The concept is based on the addition of glass-fiber pads for sampling and diluting to an electrochemical cell combining a paper-based working electrode with low-cost connector headers as counter and reference electrodes. In order to demonstrate the feasibility of this all-in-one platform for biosensing applications, an enzymatic sensor for glucose determination (requiring a potential as low as -0.1 V vs. gold-plated wire by using ferrocyanide as mediator) was developed. Real food samples, such as cola beverages and orange juice, have been analyzed with the bioelectroanalytical lab-on-paper platform. As a proof-of-concept, and trying to go further in the integration of steps, sucrose was successfully detected by depositing invertase in the sampling strip. This enzyme hydrolyzes sucrose into fructose and glucose, which was determined using the enzymatic biosensor. This approach opens the pathway for the development of devices applying the lab-on-paper concept, saving costs and time, and making possible to perform decentralized analysis with high accuracy.

Rationally engineered high-performance BiVO4/Ag3VO4/SnS2 photoelectrodes for ultrasensitive immunosensing of CYFRA21-1 based on HRP-tyramine-triggered insoluble precipitates.

A photoelectrochemical (PEC) biosensor capable of detecting cytokeratin 19 fragment 21-1 (CYFRA21-1) was optimized by taking advantage of the powerful conjugate repeats of horseradish peroxidase and tyramine (HRP-tyramine)-triggered enzymatic biocatalytic precipitation (BCP) on high-performance BiVO4/Ag3VO4/SnS2 photoelectrodes. Compared with the ubiquitous BCP strategy, we identified a design supporting conjugate repeats generated by HRP and tyramine-triggered immeasurable insoluble precipitates in the presence of hydrogen peroxide and 4-chloro-1-phenol (4-CN), and the steric hindrance improved sensitivity. Moreover, by virtue of BiVO4, Ag3VO4, SnS2 excellent level matching structure and chemical stability, a heterojunction (BiVO4/Ag3VO4/SnS2) with high light absorption efficiency has been successfully prepared. The novel heterostructure system of BiVO4/Ag3VO4/SnS2 with high detection current and low background signal exhibited high-performance PEC determination. Generally, the hitherto untapped biosensor resource realized the sensitive detection of CYFRA21-1 with a wide linear range from 50 fg/mL to 200 ng/mL, and a detection limit of 15 fg/mL, which illustrated the potential for biotechnological applications.

A versatile and ultrasensitive molecularly imprinted electrochemiluminescence sensor with HRP-encapsulated liposome labeled by light-triggered click reaction for pesticide residues.

A novel approach for trace detection of fipronil with a molecularly imprinted electrochemiluminescence sensor (MIECLS) is proposed. The sensitivity is significantly improved via signal amplification of the enzymatic reaction of horseradish peroxidase (HRP) released from encapsulated liposomes which linked onto the template molecules after rebinding. The molecularly imprinted polymer membrane was prepared through the electropolymerization of monomers with fipronil as a template. After the elution of the template molecules, the analyte fipronil was reabsorbed into the cavities. HRP-encapsulated liposomes were linked to the target molecules by light-triggered click reaction. The higher the concentration of the target was, the more HRP-encapsulated liposomes were present on the molecularly imprinted polymer (MIP) sensor. Then, HRP was liberated from liposomes, and the catalytic degradation of hydrogen peroxide (H2O2) by HRP occurs, which changed the electrochemiluminescence intensity of luminol significantly. The change of the ∆ECL intensity was linearly proportional to the logarithm of the fipronil concentration ranging from 1.00 × 10-14 to 1.00 × 10-9 mol/L, and the detection limit was 7.77 × 10-16 mol/L. The recoveries obtained ranged from 95.7 to 105.8% with RSD < 5%. The sensitivity of the detection was significantly improved, and the analysis process was simplified in that the incubation step required in the conventional method was avoided. The sensor proposed provides a feasible platform for ultra-trace amount determination.

Carboxylic acid-tethered polyaniline as a generic immobilization matrix for electrochemical bioassays.

Polyaniline (PANI) was functionalized by thiol-ene click chemistry to obtain carboxylic acid-tethered polyaniline (PCOOH). The versatility of PCOOH as an immobilization matrix was demonstrated by constructing four different biosensors for detection of metabolites and cancer biomarker. Immobilization efficiency of PCOOH was investigated by surface plasmon resonance and fluorescence microscopic analysis which revealed dense immobilization of biomolecules on PCOOH as compared to conventional PANI. A sandwich electrochemical biosensor was constructed using PCOOH for detection of liver cancer biomarker, α-fetoprotein (AFP). The sensor displayed sensitivity of 15.24 µA (ng mL-1)-1 cm-2, with good specificity, reproducibility (RSD 3.4%), wide linear range (0.25-40 ng mL-1) at - 0.1 V (vs. Ag/AgCl), and a low detection limit of 2 pg mL-1. The sensor was validated by estimating AFP in human blood serum samples where the AFP concentrations obtained are consistent with the values estimated using ELISA. Furthermore, utilization of PCOOH for construction of enzymatic biosensor was demonstrated by covalent immobilization of glucose oxidase, uricase, and horseradish peroxidase (HRP) for detection of glucose, uric acid, and H2O2, respectively. The biosensors displayed reasonable sensitivity (50, 148, 127 µA mM-1 cm-2), and linear ranges (0.1-5, 0.1-6, 0.1-7 mM) with a detection limit of 10, 1, and 8 µM for glucose, uric acid, and H2O2, respectively. The present study demonstrates the capability of PCOOH to support and enable oxidation of H2O2 generated by oxidase enzymes as well as HRP enzyme catalyzed reduction of H2O2. Thus, PCOOH offers a great promise as an immobilization matrix for development of high-performance biosensors to quantify a variety of other disease biomarkers. Carboxylic acid-tethered polyaniline synthesized by thiol-ene click chemistry was used as matrix to construct four different electrochemical biosensors for detection of cancer biomarker α-fetoprotein, glucose, uric acid, and H2O2.

Hierarchically Porous Biocatalytic MOF Microreactor as a Versatile Platform towards Enhanced Multienzyme and Cofactor-Dependent Biocatalysis.

Metal-organic frameworks (MOFs) have recently emerged as excellent hosting matrices for enzyme immobilization, offering superior physical and chemical protection for biocatalytic reactions. However, for multienzyme and cofactor-dependent biocatalysis, the subtle orchestration of enzymes and cofactors is largely disrupted upon immobilizing in the rigid crystalline MOF network, which leads to a much reduced biocatalytic efficiency. Herein, we constructed hierarchically porous MOFs by controlled structural etching to enhance multienzyme and cofactor-dependent enzyme biocatalysis. The expanded size of the pores can provide sufficient space for accommodated enzymes to reorientate and spread within MOFs in their lower surface energy state as well as to decrease the inherent barriers to accelerate the diffusion rate of reactants and intermediates. Moreover, the developed hierarchically porous MOFs demonstrated outstanding tolerance to inhospitable surroundings and recyclability.

Direct bioelectrocatalysis by redox enzymes immobilized in electrostatically condensed oppositely charged polyelectrolyte electrode coatings.

The immobilization of enzymes on an electrode surface is critical in preserving enzyme activity and providing a sufficient electron transfer pathway for bioelectrocatalysis. Here, we present a novel single-step, cross-linker free immobilization for direct bioelectrocatalysis using an ionic strength induced phase inversion of oppositely charged polyelectrolytes. Cationic poly-guanidinyl-propyl-methacrylate (pGPMA, PG) and anionic inorganic polyphosphate, sodium hexametaphosphate (P6) were used to make an electrostatically condensed phase (PGP6). A mixture of PGP6 and laccase (LAC) from Tramates versicolor or HRP (HRP) from Armoracia rusticana were deposited on the electrode surface and were submerged in DI water to form white porous electrode coatings. Each electrode showed a current generation corresponding to the respective substrates via direct bioelectrocatalysis.

A dual-enzyme, micro-band array biosensor based on the electrodeposition of carbon nanotubes embedded in chitosan and nanostructured Au-foams on microfabricated gold band electrodes.

We report the development of a dual-enzyme electrochemical biosensor based on microfabricated gold band array electrodes which were first modified by gold foam (Au-foam) in order to dramatically increase the active surface area. The resulting nanostructured Au-foam deposits then served as a highly porous 3D matrix for the electrodeposition of a nanocomposite film consisting of multi walled carbon nanotubes embedded in a chitosan matrix (CS:MWCNT) designed to provide a conducting, biocompatible and chemically versatile surface suitable for the attachment of a wide range of chemically or biologically active agents. Finally, a dual enzyme mixture of glucose oxidase (GOx) and horseradish peroxidase (HRP) was immobilised onto the CS:MWCNT nanocomposite film surface. It is shown that the resulting sensing platform developed demonstrates excellent analytical performance in terms of glucose detection with a sensitivity of 261.8 µA mM-1 cm-2 and a reproducibility standard deviation (RSD) of 3.30% as determined over 7 measurements. Furthermore, long term stability studies showed that the electrodes exhibited an effectively unchanged response to glucose detection after some 45 days. The example of glucose detection presented here illustrates the fact that the particular combination of nanostructured materials employed represents a very flexible platform for the attachment of enzymes or indeed any other bioactive agent and as such may form the basis of the fabrication of a wide range of biosensors. Finally, since the platform used is based on lithographically-deposited gold electrodes on silicon, we note that it is also very suitable for further miniaturisation, mass production and packaging- all of which would serve to reduce production costs.

A highly sensitive immunoassay of pesticide and veterinary drug residues in food by tandem conjugation of bi-functional mesoporous silica nanospheres.

A novel type of enzyme-antibody conjugation using mesoporous silicon nanospheres (MSN) was developed, which amplified the labeling signal and highly increased the sensitivity of enzyme-linked immunosorbent assay (ELISA) for the determination of pesticide and veterinary drug residues in food. First, conjugates were prepared through layer-by-layer immobilization of an enzyme and an antibody on an MSN scaffold. Then the MSN scaffold was employed for labeling and signal amplification to develop a sensitive colorimetric immunoassay through the catalytic oxidation reaction of 5,50-tetramethylbenzidine (TMB). When this MSN-based ELISA was applied to detect chloramphenicol, avermectin, tetracycline and streptomycin in food samples, it provided linear ranges of 0.025 ng ml-1-25 ng ml-1, 0.05 ng ml-1-10 ng ml-1, 0.025 ng ml-1-10 ng ml-1 and 0.05 ng ml-1-25 ng ml-1, respectively, with low detection limits down to 0.011 ng mL-1, 0.134 ng mL-1, 0.015 ng ml-1 and 0.106 ng ml-1, respectively. For avermectin, it provided a 16.7-fold decrease of the limit of detection in contrast to that of standard ELISA without the loss of method specificity and accuracy. This novel immunoassay was hypersensitive, simple and easy-to-use, which made it high potential in applying for the accurate analysis of harmful substances in food.

Detection of the SARS-CoV-2 humanized antibody with paper-based ELISA.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng µL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

A triple-channel sensing array for protein discrimination based on multi-photoresponsive g-C3N4.

Graphitic carbon nitride (g-C3N4) as an outstanding photoresponsive nanomaterial has been widely used in biosensing. Other than the conventional single channel sensing mode, a triple-channel sensing array was developed for high discrimination of proteins based on the photoresponsive g-C3N4. Besides the photoluminescence and Rayleigh light scattering features of g-C3N4, we exploit the new photosensitive colorimetry of g-C3N4 as the third channel optical input. The triple-channel optical behavior of g-C3N4 can be synchronously changed after interaction with the protein, resulting in the distinct response patterns related to each specific protein. Such a triple-channel sensing array is demonstrated for highly discriminative and precise identification of nine proteins (hemoglobin, trypsin, lysozyme, cytochrome c, horseradish peroxidase, transferrin, human serum albumin, pepsin, and myoglobin) at 1 µM concentration levels with 100% accuracy. It also can discriminate proteins being present at different concentration and protein mixtures with different content ratios. The practicability of this sensor array is validated by high accuracy identification of nine proteins in human urine samples. This indicates that the array has a great potential in terms of analyzing biological fluids. Graphic abstract .

In situ encapsulation of horseradish peroxidase in zeolitic imidazolate framework-8 enables catalyzing luminol reaction under near-neutral conditions for sensitive chemiluminescence determination of cholesterol.

HRP@ZIF-8 nanocomposite was prepared by in situ encapsulation of horseradish peroxidase (HRP) in the frame of zeolitic imidazolate framework-8 (ZIF-8) with a simple one-pot method. The HRP@ZIF-8 nanocomposite displays outstanding thermal stability and efficiently catalyzes the chemiluminescence (CL) reaction of luminol with hydrogen peroxide (H2O2) under near-neutral pH condition (pH 7-8). This CL system has a good response to H2O2 with a linear range of 0.1-100.0 µmol L-1. The limit of detection (LOD) is 0.06 µmol L-1 H2O2. By marriage with cholesterol oxidase, cholesterol is determined with a linear range from 0.1 to 100.0 µmol L-1 and a LOD of 0.04 µmol L-1. The relative standard deviations (RSD) are 1.7% and 2.5%, respectively, in 11 repeated measurements of 50.0 µmol L-1 solutions of H2O2 and cholesterol, indicating excellent precision of the method. The method shows good selectivity and has been applied to the determination of total cholesterol in real serum samples. No significant difference has been observed between the results obtained by this method and the cholesterol oxidase-peroxidase coupling method. Graphical abstract Schematic presentation of in situ one-pot synthesis of horseradish peroxidase@zeolitic imidazolate framework-8 (HRP@ZIF-8) nanocomposite and chemiluminescence determination of cholesterol with HRP@ZIF-8 catalyzing luminol-H2O2 system.

Oriented boronate affinity-imprinted inverse opal hydrogel for glycoprotein assay via colorimetry.

A biomimetic antibody is described for colorimetric determination of glycoprotein, and horseradish peroxidase (HRP) is used as model analyte. Use is made of oriented surface imprinted inverse opal hydrogel particles functionalized with phenylboronic acid. The inverse opal hydrogel particles were negatively replicated from silica colloidal crystal beads (SCCBs), so that they were endowed with larger specific surface area than the bulk structure. Benefit from that, there were abundant surface molecularly imprinting sites in the hydrogel particles. Because the imprinting sites match the structure of the template molecules, they can recognize HRP with high selectivity and sensitivity. The recognized glycoprotein was bonded with the phenylboronic acid within the sites. The bonded HRP was determined by colorimetry of 3, 3', 5, 5'-tetramethylbenzidine (TMB) single-component solution at 450 nm, and it shows a 16.03 imprinting factor under optimized conditions. Alpha-fetoprotein (AFP) was also investigated and demostrated the value of this strategy in practical applications. The results show that the absorbance increases linearly in the 1-50 ng mL-1 AFP concentration range and has a 1.32 ng mL-1 detection limit. The assay of human serum was realized by the standard addition method. This strategy is promising to open new horizons for glycoprotein assay. Graphical abstract Schematic representation of the preparation of oriented boronate affinity-imprinted inverse opal hydrogel particles for glycoprotein assay.

A sandwich electrochemiluminescent assay for determination of concanavalin A with triple signal amplification based on MoS2NF@MWCNTs modified electrode and Zn-MOF encapsulated luminol.

An ultrasensitive sandwich electrochemiluminescence (ECL) biosensor was designed for determination of concanavalin A (ConA) through the specific carbohydrate-ConA interactions. Three-dimensional porous metal-organic framework (Zn-MOF) was synthesized, which loaded a large amount of luminescent reagents as luminol by encapsulating into its pores to form Zn-MOF@luminol complex. Interestingly, Zn-MOF also acted as the coreactant accelerator in the luminol-H2O2 ECL system. This Zn-MOF@luminol complex was used as the signal probe to achieve a super strong and stable ECL signal. In addition, three-dimensional hierarchical molybdenum disulfide nanoflower and multiwalled carbon nanotubes complex (MoS2NF@MWCNTs) with peroxidase-mimicking enzyme property were used as a substrate to modify the glassy carbon electrode to further enhance the ECL signal of luminol by promoting decomposition of H2O2 into reactive oxygen species (ROSs). In addition to the horseradish peroxidase (HRP) catalysis effect on the luminol ECL signal, a triple amplified ConA sandwich ECL sensor with high sensitivity sensor was constructed. The linear range for ConA detection was from 0.5 pg/mL to 100 ng/mL with a detection limit of 0.3 pg/mL (S/N = 3). The recovery test for ConA in human serum samples was performed with satisfactory results. Graphical abstract.

Colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction for determination of bovine pregnancy-associated glycoproteins.

DNA aptamers that bind to bovine pregnancy-associated glycoproteins (bPAGs) were selected by the systematic evolution of ligands by exponential enrichment (SELEX) procedure coupled to surface plasmon resonance (SPR) and high-throughput sequencing (HTS) technology. After seven rounds of selection using carboxylated magnetic beads (MB) coated with bovine pregnancy-associated glycoproteins 9 (bPAG9) and bovine serum albumin (BSA) as target and counter targets, respectively, two aptamers designated as A1 and A24 showed high affinities to bPAG9 (Kd = 1.04 and 2.5 nM). Moreover, the specificity was determined by testing the non-targets bPAG4, bPAG6, bPAG16, BSA, and ovalbumin (OVA). Results showed that two aptamers demonstrated broad group specificity to bPAG family. Subsequently, a colorimetric sandwich enzyme-linked aptamer assay was developed for ultrasensitive detection of bPAG9 based on hybridization chain reaction (HCR) amplification strategy. The method exhibited a broad determination from 0.134 to 134 ng/mL with a detection limit of 0.037 ng/mL. The method has been successfully applied to determine bPAGs in real samples. The results demonstrate that the developed aptamers could be used as promising molecular probes for the development of pregnancy diagnostic tools. Graphical abstract In this study, we first selected aptamers against bovine pregnancy-associated glycoproteins (bPAGs) as molecular recognition elements and then developed a colorimetric enzyme-linked aptamer assay utilizing hybridization chain reaction to detect bPAGs in the serum.The GA can't be deleted, the modified GA can not upload. So themodified GA and figures will be send to CorrAdmin3@spi-global.com.

A ratiometric fluorescent assay for evaluation of alkaline phosphatase activity based on ionic liquid-functionalized carbon dots.

A ratiometric fluorescent assay is fabricated for the evaluation of alkaline phosphatase (ALP) activity. This assay is composed of ionic liquid-functionalized carbon dots (IL-CDs) with blue fluorescence signal at 470 nm and 2,3-diaminophenazine (DAP) with yellow fluorescence signal at 570 nm. IL-CDs were synthesized via electrochemical method by using ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate) and ultrapure water as precursors. DAP is produced by the oxidation reaction between o-phenylenediamine and H2O2 under the catalysis of horseradish peroxidase. H2O2 is reduced by ascorbic acid which is the hydrolysis product of ascorbic acid 2-phosphate under the catalysis of ALP, finally reducing the amount of DAP. The activity of ALP is evaluated through the ratiometric fluorescent signal between IL-CDs and DAP via Förster resonance energy transfer. Under optimal experimental conditions, this ratiometric fluorescent assay has a response that covers the 0.04 to 3.2 U L-1 (12 to 960 pM) ALP activity. This assay possesses ultralow detection limit of 0.012 U L-1 (3.6 pM) for ALP and high selectivity for ALP among several enzymes. The method was used to measure ALP activity in human serum samples with satisfying results. Graphical abstract Schematic presentation of IL-CDs-based ratiometric fluorescent assay for ALP activity evaluation via FRET strategy between IL-CDs and DAP. This ratiometric fluorescent assay possessed low detection limit of ALP activity (0.012 U L-1) and high selectivity among several enzymes.

Synergy between nanozymes and natural enzymes on the hybrid MoS2 nanosheets/graphite microfiber for enhanced voltammetric determination of hydrogen peroxide.

A biosensor for hydrogen peroxide (H2O2) has been developed based on the use of MoS2 nanosheets and graphite that are assembled to form a microfiber hybrid structure. The MoS2 nanosheets are synthesized in situ on a graphite microfiber. The chemical composition and surface morphology of the microfiber hybrid structure has been characterized. The microfiber is shown to display peroxidase-mimicking activity. In the next step, horseradish peroxidase, methylene blue, and chitosan are co-immobilized on the microfiber electrode. The use of MoS2 nanosheets warrants high electrochemical activity of immobilized enzyme on the electrode surface. The modified microfiber electrode, best operated at a voltage of - 0.3 V (vs. Ag/AgCl), can be used to sense H2O2 with a linear response in the 0.1 to 90 µM concentration range and with a determination limit of 30 nM (at S/N = 3). The good response is attributed to the synergistic enhancement of the synthetic nanozymes (few-layered MoS2 nanosheets) and immobilized natural horseradish peroxidase (HRP). Grapical abstract.

Rolling circle amplification based colorimetric determination of Staphylococcus aureus.

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.µL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.

Nanobody-based electrochemical competitive immunosensor for the detection of AFB1 through AFB1-HCR as signal amplifier.

A novel nanobody (Nb)-based voltammetric immunosensor coupled with horseradish peroxidase concatemer-modified hybridization chain reaction (HRP-HCR) signal amplifying system is described to realize the rapid and ultrasensitive detection of AFB1. To design such an immunoassay, anti-AFB1 Nbs with smaller molecular size were coated densely onto the surface of Au nanoparticle-tungsten disulfide-multi-walled carbon nanotubes (AuNPs/WS2/MWCNTs) functional nanocomposites as an effective molecular recognition element, whereas AFB1-streptavidin (AFB1-SA) conjugates were ingeniously bound with biotinylated HCR dsDNA nanostructures as the competitor, amplifier, and signal report element. In the presence of AFB1 targets, a competitive immunoreaction was performed between the analyte and AFB1-SA-labeled HCR (AFB1-HCR) platform. Upon the addition of SA-modified polyHRP (SA-polyHRP), AFB1-HCR nanostructures containing abundant biotins were allowed to cross-link to a quantity of HRP by streptavidin-biotin chemistry for signal amplification and signal conversion. Under optimal conditions, the immunosensor displayed a good linear correlation toward AFB1 ranging from 0.5 to 10 ng mL-1 with a sensitivity of 2.7 µA • (mL ng-1) and an ultralow limit of detection (LOD) of 68 fg mL-1. The specificity test showed that the AFB1 immunosensor had no obvious cross-reaction with OTA, DON, ZEN, and FB1. The signal of this sensor decreased by 10.18% in 4 weeks indicating satisfactory stability, and its intra- and inter-laboratory reproducibility was 3.42~10.35% and 4.03%~12.11%, respectively. This biosensing system will open up new opportunities for the detection of AFB1 in food safety and environmental analysis and extend a wide range of applications in the analysis of other small molecules. Graphical abstract.