Enzyme- and label-free cascade isothermal amplification aptasensor for the ultrasensitive detection of ochratoxin A.

BACKGROUND: Ultrasensitive detection is crucial for the early warning and intervention of risk factors, ultimately benefiting the environment and human health. Low levels of ochratoxin A (OTA) present a hidden yet significant threat, and rapid detection via high-performing biosensors is therefore essential. RESULTS: A cascade isothermal amplification aptasensor (CIA-aptasensor) was designed for OTA detection. On the surface of a magnetic bead probe, the OTA level was converted into positively correlated trigger cDNA through its competitive binding with OTA-Apt. The released trigger cDNA activated catalytic hairpin assembly followed by coupling with a hybridization chain reaction to achieve CIA. After adding graphene oxide and SYBR Green I, the background interference was eliminated to specifically obtain OTA-related fluorescence. The ultrasensitive limit of detection was 0.22 pg mL-1, an improvement of 1368-fold over conventional enzyme-linked aptamer sorbent assay by the same OTA-Apt, demonstrating satisfactory reliability and practicability. Thus, the CIA-aptasensor provides an enzyme- and label-free simplified homogeneous system with minimal background interference using isothermal conditions. SIGNIFICANCE: This study provides a polymerase chain reaction-like approach for enhancing the sensitivity and performance of a biosensor, which could be extended for the application of CIA and label-free signaling strategy to other risk factors.

Hierarchical magneto-colorimetric labels for immediate lateral flow immunoassay of chlorothalonil residues.

Quantitative detection of pesticide residues in food and environmental samples using an improved lateral flow immunoassay (LFIA) is of considerable importance for real-time analysis. This paper proposes a highly sensitive LFIA platform based on a hierarchical magneto-colorimetric compact. This compact serves as both the target magnetic enrichment substrate and a photosensitive label. Initially, a large porous dendritic silica template is prepared and doped with superparamagnetic ferric oxide nanoparticles (Fe3O4 NPs) and colloidal gold nanoparticles (AuNPs) at high densities within its vertical channels. The sequential assembly of central-radial channels allow for the three-dimensional integration of these two components, enabling independent control of their discrete functions without mutual interference. Following alkyl organosilicon encapsulation and silica sealing, the composite spheres are then applied in LFIA to detect chlorothalonil residues. Fe3O4 NPs enhance the binding efficiency to target analytes, while AuNPs amplify the signal, leveraging their high loading densities and robust optical properties. The developed LFIA platform exhibited a detection limit of 0.34 ng/mL for chlorothalonil and a linear range of 0.0085-824 ng/mL. The recoveries varied between 85.1 % and 103.1 %, and the relative standard deviations were 1.25%-8.84 %. This LFIA approach demonstrates high sensitivity, specificity, reproducibility and flexible detection modes, making it highly suitable for the on-site monitoring of pesticide residues.

Boronic functionalized aptamer macroarrays with dual-recognition and isothermal amplification of lipopolysaccharide detection.

A highly sensitive dual-recognition fluorescence amplification method is presented for lipopolysaccharide (LPS) detection based on boronic functionalized aptamer macroarrays with dual-recognition and isothermal amplification. The surface of the polystyrene microplate was firstly carboxylated, and then, 3-aminophenylboronic acid was conjugated to the carboxyl groups through EDC/NHS reaction, creating boronic acid groups as the capture moiety for LPS. A recognition DNA aptamer labeled with the fluorescent dye 6-FAM, which exhibits specificity towards LPS, was selected as the signal reporting moiety. By introducing primers and Klenow enzyme, the fluorescent-labeled aptamers are released from the microplate bottom, and double-stranded structures were formed via isothermal amplification. The addition of SYBR Green I, which strongly fluoresces upon binding to the double-stranded structures, enables signal amplification and detection. This detection method exhibits a linear range of 1-10,000 ng/mL and has a detection limit as low as 401.93 pg/mL. This analytical approach shows high selectivity and sensitivity and may serve as a universal platform in lipopolysaccharide detection.

3D-Bioprinted Hepar-on-a-Chip Implanted in Graphene-Based Plasmonic Sensors.

Precise three-dimensional (3D) bioprinting designs enable the fabrication of unique structures for 3D-cell culture models. There is still an absence of real-time detection tools to effectively track in situ 3D-cell performance, hindering a comprehensive understanding of disease progression and drug efficacy assessment. While numerous bioinks have been developed, few are equipped with internal sensors capable of accurate detection. This study addresses these challenges by constructing a 3D-bioprinted hepar-on-a-chip embedded with graphene quantum dot-capped gold nanoparticle-based plasmonic sensors, featuring strong surface-enhanced Raman scattering (SERS) enhancement, biostability, and signal consistency. Such an integrated hepar-on-a-chip demonstrates excellent capability in the secretion of liver function-related proteins and the expression of drug metabolism and transport-related genes. Furthermore, the on-site detection of cell-secreted biomarker glutathione transferase α (GST-α) was successfully achieved using the plasmonic probe, with a dynamic linear detection range of 20-500 ng/mL, showcasing high anti-interference and specificity for GST-α. Ultimately, this integrated hepar-on-a-chip system offers a high-quality platform for monitoring liver injury.

Hepar-on-a-sensor-platform with hybridization chain reaction amplification strategy to intuitively monitor the hepatoxicity of natural compounds.

The irrational use of natural compounds in the treatment of diseases can lead to serious side effects, especially hepatoxicity, and its toxic effects are usually cumulative and imperceptible. Therefore, an accurate sensing platform is urgently needed to monitor the hepatotoxicity of natural compounds. Here, we deposited a thermo-responsive alginate-RGD/Pluronic hydrogel to construct an in vitro three-dimensional(3D) hepar-platform, and a thorough validation was adopted to evaluate the bioprinted hepatic constructs. The engineered hepar-platform was then employed to access its biological response toward Emodin (EM) and Triptolide (TP), two typical hepatotoxic natural compounds. Subsequently, we integrated it with a robust fluorescent sensor based on hybridization chain reaction amplification strategy (HCR) to monitor the early hepatotoxic biomarker - glutathione-S-transferase-alpha (GST-α) secreted by this 3D constructs. Our study was the first attempt to construct an accurate hepar-on-a-sensor platform that could effectively detect GST-α for monitoring the hepatoxic effects of natural compounds. The limit of detection of the platform was 0.3 ng ml-1 and the accuracy of this platform was verified by enzyme linked immunosorbent assay. Furthermore, the variation of GST-α induced by EM and TP was consistent with hepatotoxicity studies, thus providing an important application value for evaluating the hepatotoxicity of natural compounds. STATEMENT OF SIGNIFICANCE: 1. We deposited a thermo-responsive alginate-RGD/Pluronic hydrogel to construct an in vitro three-dimensional(3D) hepar-platform, and elucidated the essential reasons why hybrid bioinks more suitable for 3D extrusion from biomaterials itself. Also, a thorough validation associated with a series of important proteins and genes involved in liver cell metabolism was adopted to evaluate the bioprinted hepatic constructs accurately 2. Glutathione-S-transferase-alpha is a soluble trace biomarker for acute hepatotoxic injury, the hepatotoxic effects of natural compounds on the secretion of GST-α has not been reported to date. We integrated our 3D hepar-platform with recognition molecules-aptamers and HCR amplification strategy to monitor the variation of GST-α, aiming at developing a robust and stable fluorescent biosensing platform to monitor the hepatoxicity of natural compounds.

Supramolecular self-assembled AIE molecules are used in the search for target proteins in norcantharidin.

Norcantharidin (NCTD), a demethylated derivative of cantharidin, is an anticancer active component in traditional Chinese medicine. At present, the main methods for finding its target proteins are pharmacological methods and biophysical screening, which cannot achieve the purpose of efficient and accurate screening. Here we established a new analytical method for specific fishing and assisted imaging for norcantharidin target proteins. For the AIE supramolecule probe, the benzophenone azide (BPA) fluorescent nanoparticles with strong AIE properties were encapsulated in biocompatible DSPE-PEG that covalently coupled with NCTD (named BPA@NCTD NPs). The target proteins of NCTD can be captured by BPA@NCTD NPs, and then be detected to investigate the potential signaling pathways. The screened differential proteins were analysed through the protein and signaling pathway database, and multiple signaling pathways were obtained and verified. The mechanism of norcantharidin in inhibiting the migration and invasion of A549 cells through the P53 signaling pathway was confirmed by Western blot experiments. Our research showed that AIE supramolecule probe BPA@NCTD NPs has the dual functions of specific screening of A549 cells target proteins and biological imaging, which not only offers a good anti-fluorescence quenching ability for the dynamic imaging process of NCTD, but also provides a novel and efficient specific method for efficient analysis of target proteins and signal pathways.

A theranostic microneedle array patch for integrated glycemia sensing and self-regulated release of insulin.

Diabetes can cause various complications and affect the normal functioning of the human body. A theranostic and diagnostic platform for real-time glycemia sensing and simultaneous self-regulated release of insulin is desired to improve diabetic patients' life quality. Here, we describe a theranostic microneedle array patch, which enables the achievement of visualization quantification of glycemia and simultaneously self-regulated release of insulin. The microneedle patch (MNDF) was fabricated by crosslinking of 3-aminophenylboronic acid (ABA)-modified sodium alginate and chondroitin sulfate. The hierarchical structure consisted of a tip part containing mineralized insulin particles and glucose oxidase (GOD) for insulin release, and a base surface embodying 3,3',5,5'-tetramethylbenzidine (TMB) and (horseradish peroxidase) HRP for real-time glycemia sensing. In the presence of glucose, GOD converts glucose into H+ and H2O2, driving gradual dissolution of the calcium layer of insulin particles, resulting in long-acting release of insulin. By the bio-catalytic action of HRP, the generated H2O2 brings about a visible color change allowing the glucose level at the base surface to be read out. We believe that the theranostic microneedle array patch can act as a promising alternative for future clinical applications.

A silver@gold nanoparticle tetrahedron biosensor for multiple pesticides detection based on surface-enhanced Raman scattering.

The detection of multiple pesticides in food and environment is of great importance for human health and safety. In this study, the DNA backbone structure and Ag@Au nanoparticles (NPs) to construct a nano-tetrahedron with the help of the surface-enhanced Raman scattering (SERS) effect by controlling the formation of SERS hotspots and subsequently realized the simultaneous detection of multiple pesticides. The DNA aptamers corresponding to the three pesticides of profenofos, acetamiprid and carbendazim were embedded into the three edges of the DNA tetrahedral skeleton, and the tetrahedral corners were connected to modify the Ag@Au NPs with different Raman signaling molecules. When aptamers recognize the related pesticides, the DNA backbone is deformed. Then Ag@Au NPs approach to each other with SERS hotspots formed and the intensity of the Raman signal increased, realizing the detection of the pesticide content. The biosensor constructed from the SERS substrate with higher sensitivity and lower detection limit (profenofos: 0.0021 ng mL-1; acetamiprid: 0.0046 ng mL-1; carbendazim: 0.0061 ng mL-1). The practicability of this proposed method was verified by adding the recovery rate detection and the accuracy of the method was examined by the analysis of the HPLC-MS method. The proposed SERS biosensor could distinguish and detect three pesticides in food and environmental samples with high sensitivity and low detection limit that can be used in practical applications.

Simultaneous and ultrasensitive detection of three pesticides using a surface-enhanced Raman scattering-based lateral flow assay test strip.

Chlorothalonil (CHL), imidacloprid (IMI) and oxyfluorfen (OXY) are commonly used in combination to increase crop yield. However, these three pesticides are toxic to aquatic organisms and do not easily degrade. In this study, a surface-enhanced Raman scattering-based lateral flow assay (SERS-LFA) test strip was prepared by combining antibodies with SERS nanotags, and then competitive immune binding was used to detect the three pesticides simultaneously. Moreover, the two-way binding effect of ssDNA-streptavidin bound to Ag4-NTP@AuNPs and Ag4-NTP@AuNPs with antibodies was used to further amplify the detection signal. Under the optimal conditions, the SERS-LFA test strips exhibited high sensitivity, a low detection limit, short detection time, high specificity and low cost. Furthermore, the detection range was within the values prescribed by international detection standards. By measuring the intensity of the SERS signal on the test line of the paper strip, accurate quantitative analysis was achieved. The practical application of the proposed system was demonstrated by simultaneous detection of CHL, IMI and OXY in environmental and food samples with satisfactory results.

Oxidase-mimicking activity of ultrathin MnO2 nanosheets in a colorimetric assay of chlorothalonil in food samples.

Chlorothalonil is a class of 2B carcinogen which is widely used in the prevention and treatment of fungal diseases in food samples. Its residual problem has been increasingly concerned by society. In this paper, a fast and simple colorimetric assay based on Manganese dioxide nanosheets (MnO2 NSs)-oxidize 3,3',5,5'-tetramethylbenzidine (TMB) platform was used to detect residual pesticide chlorothalonil in food samples. Under optimal conditions, the half maximal inhibitory concentration and the limit of detection of chlorothalonil were 3.27 and 0.024 ng/mL. There were no obvious cross-reactivity between chlorothalonil and interference substances. The recoveries shown the satisfactory results. The results of colorimetric assay for the authentic samples were largely consistent with gas chromatography. Therefore, the proposed method would be convenient and satisfactory analytical methods for the monitoring of chlorothalonil. Furthermore, the MnO2 - TMB system was used to produce test strips for quick and convenient visual detection of chlorothalonil with good performance.

Ratiometric Fluorescent Quantum Dot-Based Biosensor for Chlorothalonil Detection via an Inner-Filter Effect.

A new sensitive sensor for detecting chlorothalonil (CHL) based on the inner-filter effect (IFE) between gold nanoparticles (AuNPs) and ratiometric fluorescent quantum dots (RF-QDs) was developed. Here, RF-QDs were designed by two different color CdTe QDs. Based on the IFE, the AuNPs can quench the fluorescence of the RF-QDs. Because of the electrostatic attraction between protamine (PRO) and the AuNPs, the PRO can restore fluorescence effectively. Papain (PAP) can easily hydrolyze PRO and causes the quench of fluorescence quenching. The addition of CHL can inhibit PAP activity and restore the fluorescent signal. Through the characterization of the structural changes of PAP, the inhibition and mechanism of CHL on PAP activity were studied. The ability of CHL to inhibit PAP activity was evaluated by measuring the fluorescence of the RF-QDs. Under the optimal conditions, this sensing platform shows a response to CHL in the range of 0.34-2320 ng/mL and a detection limit of 0.0017 ng/mL. Based on the CHL inhibition of PAP activity, the RF-QDs showed good selectivity for CHL. The practical application of the proposed system was demonstrated by detecting CHL in food and environmental samples with satisfying results.

Development of immunoassays for detecting oxyfluorfen residue in agricultural and environmental samples.

An enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassays (CLEIA) were developed to detect oxyfluorfen in agricultural and environmental samples. The hapten of oxyfluorfen was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) to produce immunogen and coating antigen. One cell line (1A7D6F5) that stably secretes anti-oxyfluorfen monoclonal antibody (mAb) is obtained by cell fusion. Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC20) of ELISA are 0.065 mg L-1 and 0.0048 mg L-1, while those of CLEIA are 0.021 mg L-1 and 0.0016 mg L-1, respectively. The immunoassays show no obvious cross-reactivities with the analogues of oxyfluorfen except for benzofluorfen and bifenox. The recoveries of oxyfluorfen in the spiked samples of soil, grape, peach, apple and pear are in the range of 74.1-107.2% with a RSD of 2.7-9.7% for ELISA, and 77.2-106.4% with a RSD of 2.4-7.9% for CLEIA. The results of immunoassays for the authentic samples are significantly correlated with those detected by gas chromatography (GC).

Dual-labeled time-resolved fluoroimmunoassay for simultaneous detection of clothianidin and diniconazole in agricultural samples.

Europium (Eu(3+)) and samarium (Sm(3+)) were used as fluorescent labels to develop a highly sensitive dual-labeled time-resolved fluoroimmunoassay (TRFIA) for detect clothianidin and diniconazole in food samples. Under the optimized assay conditions, 50% inhibition concentration (IC50) and the limit of detection (LOD, IC10) of clothianidin were 5.08 and 0.021 µg/L, and 13.14 and 0.029 µg/L for diniconazole. The cross-reactivities (CRs) were negligible except dinotefuran (9.4%) and uniconazole (4.28%). The recoveries of clothianidin and diniconazole ranged from 79.3% to 108.7% in food samples. The results of TRFIA for the authentic samples were validated by gas chromatography (GC) analyses, and a satisfactory correlations were obtained. These results indicated that the method was an alternative tool for simultaneous detection of clothianidin and diniconazole in food samples.

Simultaneous detection of imidacloprid and parathion by the dual-labeled time-resolved fluoroimmunoassay.

A highly sensitive direct dual-labeled time-resolved fluoroimmunoassay (TRFIA) to detect parathion and imidacloprid simultaneously in food and environmental matrices was developed. Europium (Eu(3+)) and samarium (Sm(3+)) were used as fluorescent labels by coupling separately with L1-Ab and A1P1-Ab. Under optimal assay conditions, the half-maximal inhibition concentration (IC50) and limit of detection (LOD, IC10) were 10.87 and 0.025 µg/L for parathion and 7.08 and 0.028 µg/L for imidacloprid, respectively. The cross-reactivities (CR) were negligible except for methyl-parathion (42.4 %) and imidaclothiz (103.4 %). The average recoveries of imidacloprid ranged from 78.9 to 104.2 % in water, soil, rice, tomato, and Chinese cabbage with a relative standard deviation (RSD) of 2.4 to 11.6 %, and those of parathion were from 81.5 to 110.9 % with the RSD of 3.2 to 10.5 %. The results of TRFIA for the authentic samples were validated by comparison with gas chromatography (GC) analyses, and satisfactory correlations (parathion: R (2) = 0.9918; imidacloprid: R (2) = 0.9908) were obtained. The results indicate that the dual-labeled TRFIA is convenient and reliable to detect parathion and imidacloprid simultaneously in food and environmental matrices.

Sensitive time-resolved fluoroimmunoassay for quantitative determination of clothianidin in agricultural samples.

Europium (Eu(3+))-labeled antibody was used as a fluorescent label to develop a highly sensitive time-resolved fluoroimmunoassay (TRFIA) for determination of clothianidin residues in agricultural samples. Toward this goal, the Eu(3+)-labeled polyclonal antibody and goat anti-rabbit antibody were prepared for developing and evaluating direct competitive TRFIA (dc-TRFIA) and indirect competitive TRFIA (ic-TRFIA). Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) of clothianidin were 9.20 and 0.0909 µg/L for the dc-TRFIA and 2.07 and 0.0220 µg/L for the ic-TRFIA, respectively. The ic-TRFIA has no obvious cross-reactivity with the analogues of clothianidin except for dinotefuran. The average recoveries of clothianidin from spiked water, soil, cabbage, and rice samples were estimated to range from 74.1 to 115.9 %, with relative standard deviations of 3.3 to 11.7 %. The results of TRFIA for the blind samples were largely consistent with gas chromatography (R (2) = 0.9902). The optimized ic-TRFIA might become a sensitive and satisfactory analytical method for the quantitative monitoring of clothianidin residues in agricultural samples.

Development of immunoassays for detecting clothianidin residue in agricultural products.

Two enzyme-linked immunosorbent assays (ELISAs) based on polyclonal antibodies (PcAbs) for clothianidin are described: colorimetric detection format (ELISA) and pattern of chemiluminescent assay (CLEIA). Clothianidin hapten was synthesized and conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to produce immunogen and coating antigen. Anticlothianidin PcAbs were obtained from immunized New Zealand white rabbits. Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC20) of clothianidin were 0.046 and 0.0028 mg/L for the ELISA and 0.015 and 0.0014 mg/L for the CLEIA, respectively. There were no obvious cross-reactivities of the antibodies with its analogues except for dinotefuran. Recoveries of 76.4-116.4% for the immunoassays were achieved from spiked samples. The results of immunoassays for the spiked and authentic samples were largely consistent with gas chromatography. Therefore, the proposed immunoassays would be convenient and satisfactory analytical methods for the monitoring of clothianidin in agricultural products.