Riboflavin deficiency reduces bone mineral density in rats by compromising osteoblast function.

Insufficient riboflavin intake has been associated with poor bone health. This study aimed to investigate the effect of riboflavin deficiency on bone health in vivo and in vitro. Riboflavin deficiency was successfully developed in rats and osteoblasts. The results indicated that bone mineral density, serum bone alkaline phosphatase, bone phosphorus, and bone calcium were significantly decreased while serum ionized calcium and osteocalcin were significantly increased in the riboflavin-deficient rats. Riboflavin deficiency also induced the reduction of Runx2, Osterix, and BMP-2/Smad1/5/9 cascade in the femur. These results were further verified in cellular experiments. Our findings demonstrated that alkaline phosphatase activities and calcified nodules were significantly decreased while intracellular osteocalcin and pro-collagen I c-terminal propeptide were significantly increased in the riboflavin-deficient osteoblasts. Additionally, the protein expression of Osterix, Runx2, and BMP-2/Smad1/5/9 cascade were significantly decreased while the protein expression of p-p38 MAPK were significantly increased in the riboflavin-deficient cells compared to the control cells. Blockage of p38 MAPK signaling pathway with SB203580 reversed these effects in riboflavin-deficient osteoblastic cells. Our data suggest that riboflavin deficiency causes osteoblast malfunction and retards bone matrix mineralization via p38 MAPK/BMP-2/Smad1/5/9 signaling pathway.

Nanobody-based magnetic chemiluminescence immunoassay for one-pot detection of ochratoxin A.

Ochratoxin A (OTA) contamination seriously threatens food safety and human health and requires sensitive and rapid tools for monitoring. In this study, a convenient enzyme-linked immunosorbent assay based on Avi-labeled nanobody Nb-2G/streptavidin-alkaline phosphatase and magnetic beads (MBS-ELISA) was established for the sensitive detection of OTA, which could be used for one-pot detection without immobilization. After optimization, the 50% inhibitory concentration (IC50) and the lowest limit of detection value of the MBS-ELISA was 1.17 ng/mL and 0.07 ng/mL and the linear range was 248.8 pg/mL-5.28 ng/mL, respectively, which accords with state criteria for food safety. The developed one-step MBS-ELISA was almost 20-times more sensitive than the classic BA-ELISA and could generate results within 15 min, which was significantly less than the classic BA-ELISA at approximately 3 h. The MBS-ELISA indicated good recovery (86.4-114.3%) in spiked sorghum, buckwheat, and mung bean. Thus, MBS-ELISA represents a very promising strategy for the simple, rapid, and accurate detection of OTA and other toxic and hazardous contaminants.

Aptamer-based photonic crystals enable ultra-trace detection of staphylococcal enterotoxin B without labels.

Staphylococcal enterotoxin B (SEB) is one of the most common serotypes in staphylococcal food-poisoning cases. A rapid, sensitive, and simple method for SEB detection is crucial for public health. A photonic crystal (PC) sensing material for label-free detection of ultra-trace SEB was proposed in this study. Gold nanoparticle-doped silica microspheres were stacked to form an opal PC through self-assembly, and SEB aptamers, as the recognition element, were modified onto the PC. When the target protein of SEB came in contact with the PC sensing material, the reflection peak intensity of PCs decreased accordingly. The detection range was 1 × 10-6 to 1 ng mL-1, and the detection limit was 0.103 × 10-6 ng mL-1. Furthermore, the PC sensing material had great specificity and accuracy, which can be used for real sample monitoring. This PC sensing material achieved ultra-sensitive detection, which did not involve complicated preparation processes and reporter labelling.

A novel biosensor based on antibody controlled isothermal strand displacement amplification (ACISDA) system.

The overuse of antibiotics has aroused widespread concern in recent decades. Their residues in food and environment may pose potential risks to human health. Therefore, highly sensitive and rapid detection methods of antibiotics are urgently needed. Inspired by allosteric transcription factors (aTFs), we proposed a novel strategy for small molecules detection based on antibody controlled isothermal chain displacement amplification (ACISDA). A combination of nicking endonuclease, Klenow Fragment polymerase, specific antibody and a pair of antigen-labeled DNA regulate the synthesis of a G-quadruplex by isothermal chain displacement amplification. The presence of a target induces the antibody dissociation from the antigen-labeled DNA, which induces the synthesis of a G-quadruplex, and a fluorescent signal is produced by thioflavine T (ThT) binding to G-quadruplex. To test this notion, norfloxacin-conjugated DNA (named Primer-NOR) was prepared and ACISDA system was established combining with anti-norfloxacin antibody. This system could detect norfloxacin in a linear range of 0.1 ∼ 500 ng/mL with detection limit of 0.04 ng/mL, and this system could be applied to the detection of norfloxacin in real samples with good performance. Meanwhile, this system could also realize washing-free, immobilization-free and "ready-to-use", and could be used for other small molecules quickly by replacing the antigen-labeled DNA and specific antibody.

A gold nanoparticle-based lateral flow immunoassay for atrazine point-of-care detection using a handhold scanning device as reader.

A method is described to achieve accurate quantitative detection of atrazine (ATZ) in maize by using lateral flow strips based on gold nanoparticles (GNPs) and a handheld scanning reader. GNPs of 15 nm in diameter were applied as label, and a lateral flow immune assay strip was prepared. The linear range was 5.01-95.86 ng mL-1 with a detection limit of 4.92 ng mL-1 in phosphate buffer, 4 times better than the readout by the naked eye. ATZ-spiked corn samples were also analysed. The accuracy of results of spiked samples was confirmed by ELISA and liquid chromatography-tandem mass spectrometry (HPLC), which proved the reliability of the proposed method. A handhold device with an optical scanning system was designed for on-site quantitative detection. Combined with the pretreatment, the assay could be completed in less than 20 min.

Bifunctional ligand-mediated amplification of polydiacetylene response to biorecognition of diethylstilbestrol for on-site smartphone detection.

Polydiacetylene (PDA) is very suited for sensitively detecting large biomolecules, and its unique chromatic properties enable visual read-out. However, application to the selective detection of small molecules remains challenging. Here, bifunctional ligands are studied to amplify the color change of PDA for biorecognition of small molecules for the smartphone-based detection of diethylstilbestrol (DES). PDA is decorated with streptavidin (PDA-SA, blue), and biotin-modified DES (bio-DES) is prepared as a bifunctional ligand to couple with PDA-SA and DES antibody. Since multiple bio-DES can bind to a single SA, then multiple SAs on PDA lead to an increased surface coverage of the vesicle. In samples without DES, PDA-SA-bio-DES-DES antibody complexes will form, leading to a color transition (blue to red); this color transition is greatly amplified by antibody-induced aggregation of the complexes. When DES is present, aggregation is inhibited due to competition for the antibody and PDA-SA-bio-DES retains its blue color. A linear relationship (0.4-1250 ng mL-1) is found between the colorimetric response and the logarithmic DES concentration, with adequate selectivity, accuracy (82.24-118.64%), and precision (below 8.24%). Finally, a paper-based DES PDA biosensor is developed with visual and smartphone-based detection limits of 10 ng mL-1 and 0.85 ng mL-1 in water, respectively.

Rapid and ultrasensitive detection of DNA and microRNA-21 using a zirconium porphyrin metal-organic framework-based switch fluorescence biosensor.

Sensitive and accurate detection of nucleic acid biomarkers is critical for early cancer diagnosis, disease monitoring, and clinical treatment. In this study, we developed a switch fluorescence biosensor for simple and high-efficient detection of nucleic acid biomarkers using 6-carboxyfluorescein (FAM)-modified single-stranded DNA (ssDNA) probes (FAM-P1/P2), and zirconium porphyrin metal-organic framework nanoparticles (ZrMOF) acted as fluorescence quencher. FAM-P1/P2 probes were adsorbed on ZrMOF surface because of π-π stacking, hydrogen bonding, and electrostatic interactions. Fluorescence quenching event occurred by fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) processes, thereby achieving the "off" fluorescence status. Once the specific binding was formed between the fluorescence probes and the targets, the rigid double-stranded DNA (dsDNA) structures were released from ZrMOF surface, resulting in the recovery of fluorescence and the "on" status. Because of the superior adsorption ability of ZrMOF toward ssDNA than dsDNA, the switch of fluorescence signals from "off" to "on" allowed rapid and ultrasensitive detection of ssDNA (T1) and microRNA-21 (miR-21) within 30 min. The limit of detection (signal-to-noise ratio = 3) for T1 and miR-21 were 2 fM and 11 aM, respectively. Moreover, the proposed strategy was very simple as it worked by the facile adsorption-quenching-recovery mechanism without difficult and complicated immobilization processes. Also, this biosensor showed an excellent analytical performance in the detection of miR-21 in human serum samples. Therefore, this biosensor might be considered a potential tool for the detection of DNA and miRNA biomarkers in clinical samples.

Development and perspectives of rapid detection technology in food and environment.

Food safety become a hot issue currently with globalization of food trade and food supply chains. Chemical pollution, microbial contamination and adulteration in food have attracted more attention worldwide. Contamination with antibiotics, estrogens and heavy metals in water environment and soil environment have also turn into an enormous threat to food safety. Traditional small-scale, long-term detection technologies have been unable to meet the current needs. In the monitoring process, rapid, convenient, accurate analysis and detection technologies have become the future development trend. We critically synthesizing the current knowledge of various rapid detection technology, and briefly touched upon the problem which still exist in research process. The review showed that the application of novel materials promotes the development of rapid detection technology, high-throughput and portability would be popular study directions in the future. Of course, the ultimate aim of the research is how to industrialization these technologies and apply to the market.

Raman spectroscopy-based adversarial network combined with SVM for detection of foodborne pathogenic bacteria.

Raman spectroscopy combined with artificial intelligence algorithms have been widely explored and focused on in recent years for food safety testing. It is still a challenge to overcome the cumbersome culture process of bacteria and the need for a large number of samples, which hinder qualitative analysis, to obtain a high classification accuracy. In this paper, we propose a method based on Raman spectroscopy combined with generative adversarial network and multiclass support vector machine to classify foodborne pathogenic bacteria. 30,000 iterations of generative adversarial network are trained for three strains of bacteria, generative model G generates data similar to the actual samples, discriminant model D verifies the accuracy of the generated data, and 19 feature variables are obtained by selecting the feature bands according to the Raman spectroscopy pattern. Better classification results are obtained by optimising the parameters of the multi-class support vector machine, etc. Our detection and classification method not only solves the problem of needing a large number of samples as training set, but also improves the accuracy of the classification model. Therefore, this GAN-SVM classification model provides a new idea for the detection of bacteria based on Raman spectroscopy technology combined with artificial intelligence algorithms.

A highly sensitive and dual-readout immunoassay for norfloxacin in milk based on QDs-FM@ALP-SA and click chemistry.

A dual-readout immunoassay based on QDs-FM@ALP-SA and click chemistry was developed for quick and sensitive detection of norfloxacin (NOR), which is an important fluoroquinolone antibiotic. In the system, the NOR-biotin conjugate (NOR-Biotin) was synthesized by click chemistry for signal transformation, and alkaline phosphatase-labeled streptavidin (ALP-SA) was attached to quantum dot fluorescence microspheres (QDs-FM) by an activated ester method to form QDs-FM@ALP-SA for signal amplification. Here, QDs-FM was a dual-functional carrier: it was used not only as a chemiluminescence signal amplification carrier but also as a fluorescent signal due to its fluorescence character. The NOR antibody was coated on a 96-well chemiluminescence microtiter plate, and NOR-Biotin was bound to the antibody specifically. Then, QDs-FM@ALP-SA was combined with NOR-Biotin to develop a direct competition chemiluminescence/fluorescence immunoassay (dc-CLIA/FIA). The IC50 values were 0.345 and 1.206 ng/mL for dc-CLIA/FIA, respectively. The linear range was 0.013-12.48 ng/mL and 0.042-39.86 ng/mL, respectively. The recovery from the standard fortified blank milk samples was in the range of 86.44%-101.3%. Therefore, this method could be a useful tool for routine screening of NOR residues in milk.

A highly sensitive method for simultaneous detection of hAAG and UDG activity based on multifunctional dsDNA probes mediated exponential rolling circle amplification.

DNA glycosylase is an indispensable DNA damage repair enzyme which can recognize and excise the damaged bases in the DNA base excision-repair pathway. The dysregulation of DNA glycosylase activity will give rise to the dysfunction of base excision-repair and lead to abnormalities and diseases. The simultaneous detection of multiple DNA glycosylases can help to fully understand the normal physiological functions of cells, and determine whether the cells are abnormal in pre-disease. Regrettably, the synchronous detection of functionally similar DNA glycosylases is a great challenge. Herein, we developed a multifunctional dsDNA probe mediated exponential rolling circle amplification (E-RCA) method for the simultaneously sensitive detection of human alkyladenine DNA glycosylase (hAAG) and uracil-DNA glycosylase (UDG). The multifunctional dsDNA probe contains the hypoxanthine sites and the uracil sites which can be recognized by hAAG and UDG respectively to generate apyrimidinic (AP) sites in the dsDNA probe. Then the AP sites will be recognized and cut by endonuclease Ⅳ (Endo IV) to release corresponding single-stranded primer probes. Subsequently, two padlock DNA templates are added to initiate E-RCA to generate multitudinous G-quadruplexes and/or double-stranded dumbbell lock structures, which can combine N-methyl mesoporphyrin IX (NMM) and SYBR Green Ⅰ (SGI) for the generation of respective fluorescent signals. The detection limits are obtained as low as 0.0002 U mL-1 and 0.00001 U mL-1 for hAAG and UDG, respectively. Notably, this method can realize the simultaneous detection of two DNA glycosylases without the use of specially labeled probes. Finally, this method is successfully applied to detect hAAG and UDG activities in the lysates of HeLa cells and Endo1617 cells at single-cell level, and to detect the inhibitors of DNA glycosylases.

Detection of 4 quinolone antibiotics by chemiluminescence based on a novel Nor-Biotin bifunctional ligand and SA-ALP technology.

A simple and effective direct competitive chemiluminescence immunoassay for the detection of 4 kinds of quinolone antibiotics in milk was established using Nor-Biotin (biotin-modified norfloxacin [NOR]) bifunctional ligand and alkaline phosphatase-conjugated streptavidin signal amplification technology. The polyclonal antibody was obtained after the immunization of New Zealand White rabbits using norfloxacin-derived antigen. "Click chemistry" was used for the rapid and facile synthesis of the Nor-Biotin bifunctional ligand. After the optimization of the incubation time and reaction buffer, the direct competitive chemiluminescence assay method was developed and used for sensitive detection of 4 kinds of quinolone drugs (NOR, pefloxacin, ciprofloxacin, and danofloxacin). The IC50 of the 4 kinds of quinolone drugs ranged from 7.35 to 24.27 ng/mL, and the lowest detection limits ranged from 0.05 to 0.16 ng/mL, which were below their maximum residue levels, approved by the EU for treatment of food-producing animals. To demonstrate the applicability of the assay, artificially contaminated milk samples with the 4 quinolone drugs were analyzed. The mean recovery rates of the drugs ranged from 86.31% to 112.11%.

State-of-the-art progress of switch fluorescence biosensors based on metal-organic frameworks and nucleic acids.

Metal-organic frameworks (MOFs) have captured substantial attention of an increasing number of scientists working in sensing analysis fields, due to their large surface area, high porosity, and tunable structure. Recently, MOFs as attractive fluorescence quenchers have been extensively investigated. Given their high quenching efficiency toward the fluorescence intensity of dyes-labeled specific biological recognition molecules, such as nucleic acids, MOFs have been widely developed to switch fluorescence biosensors with low background fluorescence signal. These strategies not only lead to specificity, simplicity, and low cost of biosensors, but also possess advantages such as ultrasensitive, rapid, and multiple detection of switch fluorescence methods. At present, researches of the analysis of switch fluorescence biosensors based on MOFs and nucleic acids mainly focus on sensing of different types of in vitro and intracellular analytes, indicating their increasing potential. In this review, we briefly introduce the principle of switch fluorescence biosensor and the mechanism of fluorescence quenching of MOFs, and mainly discuss and summarize the state-of-the-art advances of MOFs and nucleic acids-based switch fluorescence biosensors over the years 2013 to 2020. Most of them have been proposed to the in vitro detection of different types of analytes, showing their wide scope and applicability, such as deoxyribonucleic acid (DNAs), ribonucleic acid (RNAs), proteins, enzymes, antibiotics, and heavy metal ions. Besides, some of them have also been applied to the bioimaging of intracellular analytes, emerging their potential for biomedical applications, for example, cellular adenosine triphosphate (ATP) and subcellular glutathione (GSH). Finally, the remaining challenges in this sensing field and prospects for future research trends are addressed. Graphical abstract.

The mechanism and prevention of mitochondrial injury after exercise.

With the development of society, physical activity has come to be an effective means by which people pursue good health to improve the quality of life. However, with the increase of intensity and the passage of time, exercise injury has become a hazard that can no longer be ignored. It is imperative to find effective ways to inhibit or reduce the negative effects of exercise. Mitochondria are important organelles involved in exercise and play an important role in exercise injury and prevention. Studies have found that exercise preconditioning and increased mitochondrial nutrition can effectively decrease mitochondrial damage after exercise. Against this background, some of the newest developments in this important field are reviewed here. The results discussed indicate that exercise preconditioning and supplement mitochondrial nutrition need to be increased to prevent exercise-related injuries.

Simultaneous detection of diethylstilbestrol and estradiol residues with a single immunochromatographic assay strip.

An immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneously rapid and sensitive detection of diethylstilbestrol (DES) and estradiol (E2) in milk and tissue samples. For this purpose, two monoclonal antibodies raised against those two estrogens were conjugated to gold nanoparticles and were applied to the conjugate pads of the test strip. The competitors of the DES-BSA/E2-BSA conjugates were immobilized onto a nitrocellulose membrane at two detection zones to form T1 and T2, respectively. The immunochromatographic assay had a visual detection limit of DES at 30 ng/g in milk powder, 25 ng/g in liquid milk, and 25 ng/g in shrimp tissue, respectively, and the results can be judged within 7-10 min. The visual detection limit of E2 was 75 ng/g in milk powder, 65 ng/g in liquid milk, and 60 ng/g in shrimp tissue, respectively, and the results can be judged within 3-4 min. It had advantages in easy operation without requiring sophisticated equipment and specialized skills. By testing thirty milk and shrimp tissue samples from the local market, the method was compared with the HPLC-MS / MS method, and there was no statistical difference between the two methods. Furthermore, the immunochromatographic assay had good specificity, simple procedure, and low cost. This protocol was well suited for the food safety monitoring and early warning.

The orphan nuclear receptor Nur77 plays a vital role in BPA-induced PC12 cell apoptosis.

Bisphenol A (BPA) is a typical environmental endocrine disruptor that can migrate into organisms through skin contact, breathing, diet and various other approaches. The reproductive toxicity and neurotoxicity of BPA has been confirmed by several toxicological studies. However, the neurotoxicity of BPA is still controversial. In the present study, we used PC12 cells as a model to investigate the mechanism of BPA-induced neuronal apoptosis. BPA exposure reduced cell viability, altered cell morphology and aggravated intracellular Lactate dehydrogenase (LDH) release, intracellular Ca2+ concentration, Reactive oxygen species (ROS) levels, apoptosis and the reduction in the mitochondrial transmembrane potential (ΔΨm). Moreover, the results of the Western blot (WB) and Real-time quantitative polymerase chain reaction (RT-qPCR) assays indicated that the expression levels of Nur77 in the BPA group were down-regulated and accompanied by the downregulation of the NF-κb/Bcl-2 proteins and the upregulation of cleaved-caspase 3, which is a marker of apoptosis. However, these changes were significantly reversed with the upregulation of the Nur77 protein by introducing plasmids carrying the nur77 gene. These results indicated that BPA-induced apoptosis was closely related to Nur77-mediated inhibition of the NF-κb/Bcl-2 pathway.

Development of a fast and ultrasensitive black phosphorus-based colorimetric/photothermal dual-readout immunochromatography for determination of norfloxacin in tap water and river water.

A rapid and ultrasensitive method for colourimetric/photothermal dual-readout detection was developed using an 808 nm NIR laser and a thermal imaging app on mobile phone. Norfloxacin was used as a model contaminant to demonstrate this universal rapid detection method. It is innovatively, to use the advanced two-dimensional material black phosphorus as a colourimetric/photothermal reagent for the first time. The samples were added to the strip, and the analytes were selectively captured on the conjugate pad by monoclonal antibody-modified magnetic/upconversion nanocomposites. The samples flowed through the strips by capillary action until reaching the control line, where immune complex formation occurred due to the presence of secondary antibody. The added black phosphorus could be captured by the the antigens which were directly exposed to the test line and a brown band could be observed by naked eye. Upon illumination by NIR light for 1 min, the real-time temperature is obtained for quantitative analysis through the thermal imaging performed by mobile phone camera. This method can achieve the detection of norfloxacin in water samples within 20 min, and the detection limits of colorimetric and photothermal readout can reach 45 pg mL-1. Compared with conventional strips, this method provided an increased sensitivity by about two orders of magnitude, with a integrated portable laser and a mobile phone. It is a valuable method for rapid detection and can be applied to other environmental contaminants as well.

A Multichannel Flexible Optoelectronic Fiber Device for Distributed Implantable Neurological Stimulation and Monitoring.

Optical fibers made of polymeric materials possess high flexibility that can potentially integrate with flexible electronic devices to realize complex functions in biology and neurology. Here, a multichannel flexible device based on four individually addressable optical fibers transfer-printed with flexible electronic components and controlled by a wireless circuit is developed. The resulting device offers excellent mechanics that is compatible with soft and curvilinear tissues, and excellent diversity through switching different light sources. The combined configuration of optical fibers and flexible electronics allows optical stimulation in selective wavelengths guided by the optical fibers, while conducting distributed, high-throughput biopotential sensing using the flexible microelectrode arrays. The device has been demonstrated in vivo with rats through optical stimulation and simultaneously monitoring of spontaneous/evoked spike signals and local field potentials using 32 microelectrodes in four brain regions. Biocompatibility of the device has been characterized by behavior and immunohistochemistry studies, demonstrating potential applications of the device in long-term animal studies. The techniques to integrate flexible electronics with optical fibers may inspire the development of more flexible optoelectronic devices for sophisticated applications in biomedicine and biology.

Selection of an artificial paraquat-specific binding protein from a ribosome display library based on a lipocalin scaffold.

In this study, an anticalin that could specifically bind paraquat (PQ), a quaternary nitrogen herbicide, as a new set of engineered receptor protein with antibody-like properties was generated to detect PQ concentration. To this end, a native and random library was constructed and engineered to allow in vitro transcription and translation using an Escherichia coli lysate system. Meanwhile, a PQ derivative that carries an active aliphatic carboxylate group at the end of an aliphatic spacer arm was synthesized. Then, this compound was coupled covalently to the carrier protein bovine serum albumin/ovalbumin and amino-functionalized paramagnetic beads. Alternating selection in solution and immobilization in microtiter wells were used to pan mRNA-ribosome-antibody complexes. After several rounds of ribosome display, three variants were selected from a random library of the bilin-binding protein. The variants that could bind complex PQ with high affinity and exhibit IC50 values as low as 14.039 ± 0.970 ng/mL were identified. Moreover, the limits of detection reached 0.083 ± 0.011 ng/mL. Our data suggest that the generation of anticalins may provide a promising alternative to recombinant antibody fragments to create a stable receptor protein against hapten with bioanalytical relevance.