Polydopamine@ZIFs with enhanced electrochemiluminescence quenching performance for mycotoxin detection.

Quench-type electrochemiluminescence (ECL) immunosensors are appealing for detecting small molecule contaminants in signal-on mode, for which efficient ECL quenchers are highly desirable. Here, the classical quencher of polydopamine (PDA) was transformed into a unique structure by introducing zeolite imidazole frameworks (ZIFs). Besides the inherent energy transfer quenching effect on ECL, the resulting PDA@ZIFs exhibits a high scavenging property against electrogenerated coreactant-radicals and inhibits the formation of excited luminophore. A quench-type ECL immunosensor for ochratoxin A (OTA) was developed using the PDA@ZIFs as a quencher and the g-C3N4 as a luminophore. The immunosensor showed a good response towards the OTA with a linear range of 10.0 fg/mL-1.0 ng/mL and a detection limit of 4.8 fg/mL. Acceptable recoveries of 85.7 to 109.2 % were achieved for the detection of OTA in spiked foods. This work offers valuable insight for improving the performance of quench-typed ECL biosensors.

Identifying tumor cell-released extracellular vesicles as biomarkers for breast cancer diagnosis by a three-dimensional hydrogel-based electrochemical immunosensor.

Tumor cell-released LC3+ extracellular vesicles (LC3+ EVs) participate in immunosuppression during autophagy and contribute to the occurrence and development of breast cancer. In view of the strong association between the LC3+ EVs and breast cancer, developing an effective strategy for the quantitative detection of LC3+ EVs levels with high sensitivity to identify LC3+ EVs as new biomarkers for accurate diagnosis of breast cancer is crucial, but yet not been reported. Herein, an ultrasensitive electrochemical immunosensor is presented for the quantitative determination of LC3+ EVs using a three-dimensional graphene oxide hydrogel-methylene blue composite as a redox probe, showing a low detection limit and a wide linear range. With this immunosensor, the expression levels of LC3+ EVs in various practical sample groups including different cancer cell lines, the peripheral blood of tumor-bearing mice before and after immunotherapy, and the peripheral blood from breast cancer patients with different subtypes and stages were clearly distinguished. This study demonstrated that LC3+ EVs were superior as biomarkers for the accurate diagnosis of breast cancer compared to traditional biomarkers, particularly for cancer subtype discrimination. This work would provide a new noninvasive detection tool for the early diagnosis and prognosis assessment of breast cancer in clinics.

Ligand-induced Assembly of Copper Nanoclusters with Enhanced Electrochemical Excitation and Radiative Transition for Electrochemiluminescence.

Copper nanoclusters (CuNCs) are emerging electrochemiluminescence (ECL) emitters with unique molecule-like electronic structures, high abundance, and low cost. However, the synthesis of CuNCs with high ECL efficiency and stability in a scalable manner remains challenging. Here, we report a facile gram-scale approach for preparing self-assembled CuNCs (CuNCsAssy ) induced by ligands with exceptionally boosted anodic ECL and stability. Compared to the disordered aggregates that are inactive in ECL, the CuNCsAssy shows a record anodic ECL efficiency for CuNCs (10 %, wavelength-corrected, relative to Ru(bpy)3 Cl2 /tripropylamine). Mechanism studies revealed the unusual dual functions of ligands in simultaneously facilitating electrochemical excitation and radiative transition. Moreover, the assembly addressed the limitation of poor stability of conventional CuNCs. As a proof of concept, an ECL biosensor for alkaline phosphatase detection was successfully constructed with an ultralow limit of detection of 8.1×10-6  U/L.

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.

Construction of Carbon Dots with Wavelength-Tunable Electrochemiluminescence and Enhanced Efficiency.

Tuning the electrochemiluminescence (ECL) wavelength of carbon dots (CDs) with enhanced efficiency is essential for multiplexed biosensing, bioimaging, and energy applications but remains challenging. Herein, we reported a facile route to finely modulate the ECL wavelength of CDs from 425 to 645 nm, the widest range ever reported, along with a more than 5-fold enhancement of ECL efficiency via phosphorous (P) incorporation. The molecular mechanism was explored experimentally and theoretically, which revealed the unusual dual roles of P dopants in the form of P-C and P-O bonding, that is, importing shallow trapping states and promoting an effective intramolecular charge transfer. This work would allow unlocking the key factors of ECL kinetics for heteroatom-doped CDs appearing out of reach and open a new avenue for the rational design of nanocarbon for desirable applications.

Recent advances of functional nucleic acids-based electrochemiluminescent sensing.

Electroluminescence (ECL) has been used in extensive applications ranging from bioanalysis to clinical diagnosis owing to its simple device requirement, low background, high sensitivity, and wide dynamic range. Nucleic acid is a significant theme in ECL bioanalysis. The inherent versatile selective molecular recognition of nucleic acids and their programmable self-assembly make it desirable for the robust construction of nanostructures. Benefiting from their unique structures and physiochemical properties, ECL biosensing based on nucleic acids has experienced rapid growth. This review focuses on recent applications of nucleic acids in ECL sensing systems, particularly concerning the employment of nucleic acids as molecular recognition elements, signal amplification units, and sensing interface schemes. In the end, an outlook of nucleic acid-based ECL biosensing will be provided for future developments and directions. We envision that nucleic acids, which act as an essential component for both bioanalysis and clinical diagnosis, will provide a new thinking model and driving force for developing next-generation sensing systems.

A Dual Functional Self-Enhanced Electrochemiluminescent Nanohybrid for Label-Free MicroRNA Detection.

The development of electrochemiluminescent (ECL) emitters with both intense ECL and excellent film-forming properties is highly desirable for biosensing applications. Herein, a facile one-pot preparation strategy was proposed for the synthesis of a self-enhanced ECL emitter by co-doping Ru(bpy)32+ and (diethylaminomethyl)triethoxysilane (DEAMTES) into an in situ-produced silica nanohybrid (DEAMTES@RuSiO2). DEAMTES@RuSiO2 not only possessed improved ECL properties but also exhibited outstanding film-forming ability, which are both critical for the construction of ECL biosensors. By coupling branched catalytic hairpin assembly with efficient signal amplification peculiarity, a label-free ECL biosensor was further constructed for the convenient and highly sensitive detection of miRNA-21. The as-fabricated ECL biosensor displayed a detection limit of 8.19 fM, much lower than those in previous reports for miRNA-21 and showed superior reliability for detecting miRNA-21-spiked human serum sample, demonstrating its potential for applications in miRNA-associated fundamental research and clinical diagnosis.

Research on the Mechanism of Action of a Citrinin and Anti-Citrinin Antibody Based on Mimotope X27.

Immunoassays are developed based on antigen-antibody interactions. A mimotope is an effective recognition receptor used to study the mechanism of action of antigens and antibodies, and is used for improving the sensitivity of the antibody. In this study, we built a 3D structure of the citrinin (CIT) mimotope X27 and anti-CIT single-chain antibody fragment (ScFv) through a "homologous modeling" strategy. Then, CIT and X27 were respectively docked to anti-CIT ScFv by using the "molecular docking" program. Finally, T28, F29, N30, R31, and Y32 were confirmed as the key binding sites in X27. Furthermore, the result of the phage-ELISA showed that the mutational phage lost the binding activity to the anti-CIT ScFv when the five amino acids were mutated to "alanine", thereby proving the correctness of the molecular docking model. Lastly, a site-directed saturation strategy was adopted for the sites (T28, F29, N30, R31, and Y32). Eighteen different amino acids were introduced to each site on average. The activities of all mutants were identified by indirect competitive ELISA. The sensitivities of mutants T28F, T28I, F29I, F29V, N30T, and N30V were 1.83-, 1.37-, 1.70-, 2.96-, 1.31-, and 2.01-fold higher than that of the wild-type, respectively. In conclusion, the binding model between the CIT and antibody was elaborated for the first time based on the mimotope method, thereby presenting another strategy for improving the sensitivity of citrinin detection in immunoassays.

Facile Preparation of WO3-x Dots with Remarkably Low Toxicity and Uncompromised Activity as Co-reactants for Clinical Diagnosis by Electrochemiluminescence.

The exceptional nature of WO3-x dots has inspired widespread interest, but it is still a significant challenge to synthesize high-quality WO3-x dots without using unstable reactants, expensive equipment, and complex synthetic processes. Herein, the synthesis of ligand-free WO3-x dots is reported that are highly dispersible and rich in oxygen vacancies by a simple but straightforward exfoliation of bulk WS2 and a mild follow-up chemical conversion. Surprisingly, the WO3-x dots emerged as co-reactants for the electrochemiluminescence (ECL) of Ru(bpy)32+ with a comparable ECL efficiency to the well-known Ru(bpy)32+ /tripropylamine (TPrA) system. Moreover, compared to TPrA, whose toxicity remains a critical issue of concern, the WO3-x dots were ca. 300-fold less toxic. The potency of WO3-x dots was further explored in the detection of circulating tumor cells (CTCs) with the most competitive limit of detection so far.

Coupling aptazyme and catalytic hairpin assembly for cascaded dual signal amplified electrochemiluminescence biosensing.

Developing reliable and sensitive detection methods for adenosine triphosphate (ATP) is vital for both clinical diagnosis and food safety. In this work, by coupling aptazyme- and catalytic hairpin assembly (CHA)-based signal amplification and electrochemiluminescence (ECL), an ultrasensitive biosensor for sensing ATP was fabricated using Ru(bpy)32+-doped silica nanoparticles (RuSiO2) as ECL probes and a ferrocene-functionalized hairpin DNA (hairpin-Fc) as quencher. The aptazyme-triggered cleavage of the DNA substrate and the CHA reaction both led to the circular release of trigger DNA, resulting in a significant dual signal amplification, with unprecedented enhancement up to 940-fold. Moreover, the bioconjugation of the DNA substrate with Au@Fe3O4 facilitated the separation and purification of the released trigger DNA, and effectively reduced the background signal. As a result, the as-prepared ECL biosensor exhibited a much lower detection limit of 0.054 pM for ATP, compared to those in previous reports, and showed high reliability for ATP detection in both spiked serum samples and Staphylococcus aureus. This work offers a new perspective for designing nucleic acid-based signal amplification for detecting ATP in bacterial analysis and clinical diagnosis.

Development of Real-Time Immuno-PCR Based on Phage Displayed an Anti-Idiotypic Nanobody for Quantitative Determination of Citrinin in Monascus.

Citrinin (CIT) is a mycotoxin that has been detected in agricultural products, feedstuff, and Monascus products. At present, research has been performed to develop methods for CIT detection, mainly through TLC, HPLC, biosensor, and immunoassay. The immunoassay method is popular with researchers because of its speed, economy, simplicity, and ease of control. However, mycotoxins are inevitably introduced during the determination. Immunoassays require the use of toxins coupled to carrier proteins or enzymes to make competitive antigens. In this study, anti-idiotypic nanobody X27 as CIT mimetic antigen was used as non-toxic surrogate reagents in immunoassay. Therefore, the X27-based real-time immuno-PCR (rtIPCR) method had been established after optimal experiments of annealing temperature and amplification efficiency of real-time PCR, concentration of coating antibody, phage X27, and methyl alcohol. The IC50 value of the established method in the present study is 9.86 ± 2.52 ng/mL, which is nearly equivalent to the traditional phage ELISA method. However, the linear range is of 0.1-1000 ng/mL, which has been broadened 10-fold compared to the phage ELISA method. Besides, the X27-based rtIPCR method has no cross-reactivity to the common mycotoxins, like aflatoxin B1 (AFB1), deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). The method has also been applied to the determination of CIT in rice flour and flour samples, and the recovery was found to be in the range of 90.0-104.6% and 75.8-110.0% respectively. There was no significant difference in the results between the rtIPCR and UPLC-MS. The anti-idiotypic nanobody as a non-toxic surrogate of CIT makes rtIPCR a promising method for actual CIT analysis in Monascus products.

Deleting the citrinin biosynthesis-related gene, ctnE, to greatly reduce citrinin production in Monascus aurantiacus Li AS3.4384.

For thousands of years, fermentation products of the filamentous fungi Monascus spp. have been used extensively in the food and pharmaceutical industries. However, their development is limited because of the health threats from the mycotoxin citrinin, known to be produced by these fungi. Citrinin is recognized as a hepato-nephrotoxin which possesses potential genotoxicity, tumorigenicity, carcinogenicity, embryotoxicity, and teratogenicity. Studies have shown that citrinin biosynthesis is intimately related to pksCT, orf1, ctnA, orf3, ctnB and ctnG. The ctnE gene, which is located 3.3kb upstream of ctnA, encodes a protein that showed significant similarity to the dehydrogenase. In this study, the role of ctnE in citrinin biosynthesis was investigated by means of gene knockout technology. The ctnE disruptant significantly reduced citrinin production by 96%, which suggested that ctnE is important in citrinin biosynthesis. Moreover, the mutant produced 40% more pigments than the wild-type. This work contributes to the study of the citrinin biosynthesis pathway in Monascus, and the methodology described in this article can fundamentally lower the risk of citrinin contamination in Monascus aurantiacus Li AS3.4384 which has important significance for food safety.