On-line monitoring of a recirculating-flow fluorescent capillary system for exploring the interaction mechanism of carbon dots/metal ions.

The interaction mechanism between carbon dots (CDs) and metal ions is essential for optimizing their design, synthesis, and application. However, it must be accurately distinguished and quantified because of CDs' complex structure, composition, and coexisting various response mechanisms or products. Herein, a recirculating-flow fluorescence capillary analysis (RF-FCA) system was developed to online monitor the fluorescence kinetics of CDs interacting with metal ions. The fluorescence kinetics of purification and dissociation of CDs/metal ion complexes were easy to monitor online by integrating immobilized CDs and RF-FCA. Here, CDs derived from citric acid and ethylenediamine were used as a model system. We found that the fluorescence of CDs is quenched by Cu(II) and Hg(II) only through the formation of a coordination complex, by Cr(VI) only through the inner filtering effect, and by Fe(III) through the above two mechanisms. Then the kinetics of the competitive interaction between metal ions were used to address the difference of binding sites on CDs with metal ions, wherein Hg(II) was bound to other sites of CDs besides the same sites of CDs with Fe(III) and Cu(II). Finally, from the fluorescence kinetics of fluorescent molecules in the CD structure with metal ions, the difference was due to the presence of two fluorescent centers in the carbon core and molecular state in the CDs. Therefore, the RF-FCA system can distinguish and quantify the interaction mechanism between metal ions and CDs effectively and accurately and be a potential detection or performance characterization method.

A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level.

Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/NanoKAZ (NKAZ)-FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.

A Split-Luciferase-Based Cell Fusion Assay for Evaluating the Myogenesis-Promoting Effects of Bioactive Molecules.

A split-luciferase-based cell fusion assay enables high-throughput screening of myogenesis-promoting chemicals in chemical libraries. The assay consists of two C2C12 myoblast-derived cell lines (N- and C-cells), each of which stably expresses either an N- or C-terminal split-firefly luciferase (FLuc) fragment fused to a naturally split DnaE intein (N- and C-probes, respectively). The fusion of N- and C-cells during myogenesis induces bioluminescence (BL) in the cytosol due to a stable reconstitution of the split-FLuc. Thus, the myogenesis-promoting effects of a chemical compound can be determined through the enhanced BL intensity. Here, we describe the preparation of N- and C-cells and determination of the myogenesis-promoting effects of imatinib using a 96-well microplate-based assay.

Exploration and quantification of ascorbate affecting peroxidase-catalyzed chromogenic reactions with a recirculating-flow catalysis detection system.

The use of a recirculating-flow catalysis detection system (RFCD) explored competition and the influence of ascorbic acid (AsA) in peroxidase (POD)-catalyzed reactions. The study identified that AsA is neither the inhibitor of POD nor could directly deplete H2O2; it directly reacts with chromogenic products to form colorless intermediates, which can react with H2O2 to again rapidly re-generate the chromogenic products. If using the reactions (trinder reactions or enzyme-linked reactions) to determine POD activity (EPOD), substrates or analytes, the interference of concomitant AsA should be removed and the conclusions have significance for oxidase/POD catalyzed reactions. In addition, the RFCD system was also used to simultaneously determine EPOD and AsA.

Simultaneous quantification of peroxidase and ascorbic acid in biosamples with an automatic system based on a Fe(iii)/methylthymol blue-carbon dot simulative enzyme.

Peroxidase (POD) and ascorbic acid (AsA) usually coexist in organisms to synergistically protect them from reactive oxygen damage, and their contents undergo dynamic changes under different physiological conditions. What's more, the response of POD-catalytic activity in spectrophotometry has to be corrected using the content of concomitant AsA because we found that there is an extinction reaction between AsA and chromogenic products obtained from POD catalysis. With these implications, by skilfully using the chromogenic and the extinction phenomena in the guaiacol/POD/H2O2 reaction, an automatic analysis system for simultaneous quantification of POD (73-440 U L-1) and AsA (4-60 mg L-1) was successfully established based on flow injection analysis (FIA). Furthermore, under acidic conditions (0.5 mol L-1 of HCl), hydrothermal synthesis (250 °C for 1 h) was used for synthesizing new carbon dots (sPOD-CDs) of methylthymol blue (0.08 g L-1)/FeCl3 (0.8 g L-1), which is a simulative enzyme for POD, and it was first used for catalyzing the guaiacol/H2O2 reaction within the FIA system to replace natural HRP in the extinction reaction. This sPOD-CD solution has no background absorption and its concentration shows excellent correlation with simulative POD-activity. Finally, after optimization, this FIA system was utilized to testify that the reducibility of AsA is due to ascorbate ions and to determine POD and AsA in some plant samples. The standard addition recovery experiment showed that there was no interference from the matrix in real samples (recoveries: 95%-105%), and the obtained POD and AsA results were also consistent with the reference experiments (relative deviation ≤ 2.80%, t-test ≥ 0.07). The proposed FIA system is characterized by high sample-throughput (40 samples per h), better repeatability (relative standard deviation ≤ 1.4%), etc.

A novel immobilization fluorescence capillary analysis method and its applications.

Fluorescence capillary analysis (FCA) realizes trace-level analysis of micro-volume samples; it is easy to operate, extremely low in analytical cost and can significantly lessen environmental pollution from analytical chemistry waste. FCA has the characteristics of green analytical chemistry and has been applied in clinical, biochemical, pharmaceutical, food safety and other fields. FCA basically involves a micro-volume glass capillary, a capillary holder and an ordinary fluorescence detector. The capillary is not only a container for chemical reaction and detection but also functions as a carrier to immobilize enzymes, gene probes or reagents; it can be used repeatedly or can be disposable. In analysis, the capillary which is modified with functional reagents sucks in a measured liquid for the reaction and is then inserted into the holder within the fluorescent detector for measurement. The immobilized FCA method has been successfully used in the determination of reduced coenzyme I, ethanol in liqueur, lactic acid in dairy products, pyruvic acid and glucose in serum, trace-level sulfated bile acid in urine, the ratio of pyruvic/lactic acid in serum, and pyruvic acid in cells as well as in DNA end-labeling and dyeing methods. Further, FCA can also be extended to capillary arrays to complete multipurpose simultaneous determinations and can be combined with mobile phones as fluorescence detectors for use in mobile health analytical technology. FCA will produce considerable social benefits in medicine, pharmacy, fermentation of food, environmental protection and other fields. Therefore, the relevant contents are presented in this tutorial review.

A Detection Method for GLUT4 Exocytosis Based on Spontaneous Split Luciferase Complementation.

Glucose transporter 4 (GLUT4) is an insulin-regulated glucose transporter, which is vital for blood glucose homeostasis. To clarify the physiological roles of GLUT4, quantitative measurement of GLUT4 exocytosis is indispensable. Herein, we show a rapid detection system for GLUT4 on the cell surface using spontaneous split-luciferase reconstitution. Upon insulin-induced GLUT4 exocytosis, GLUT4 was exposed outside, where luciferase is reconstituted and emitted luminescence. Pretreatment with inhibitors reduced the insulin-induced signal elevation. The results indicate that the developed method is applicable to high-throughput analysis on GLUT4 trafficking, which will greatly accelerate comprehensive research on the physiological roles of GLUT4.

A robust split-luciferase-based cell fusion screening for discovering myogenesis-promoting molecules.

Myogenesis-promoting chemicals are an important source of new pharmaceuticals for the treatment of skeletal muscle atrophy that impairs quality of life. This report presents a robust and quantitative bioluminescence-based assay for screening myogenesis-promoting compounds in chemical libraries. The assay system consists of two stable C2C12 myoblast cell lines, each of which expresses either an N-terminal or a C-terminal split luciferase fragment fused to a naturally split DnaE intein as an indicator for cell fusion. Cell fusion during myogenesis induces bioluminescence in the cytosol because of the reconstitution of luciferases. The luminescence intensity quantitatively represents the progress in the cell fusion and therefore indicates the extent of myogenesis. We applied this assay system to a high-throughput screening of myogenesis-promoting compouns in 1191 pharmacologically proven bioactive small molecules, which revealed two chemical compounds as myogenesis-promoting compounds: Imatinib and Doxazosin mesylate. The assay system enabled a robust and quantitative evaluation of the extent of myogenesis through simple luminescence measurements, and is expected to be widely applicable for high-throughput screening of cell fusion-promoting and inhibiting molecules.

Development of a fluorescent capillary biosensor based on self-assembled AuNP/LDH for micro-volume intracellular pyruvate.

Herein, a fluorescent capillary biosensor was developed for quantifying micro-volume intracellular pyruvate (PA), in which AuNPs and lactate dehydrogenase (LDH) were modified on the inner surface of an amination capillary (20 µL) via a self-assembly technique. The PA concentration was quantified by the change in the value of the fluorescence of NADH after sucking a mixed solution of the sample and NADH into the biosensor. This study investigated factors including the degree of protonation of the amino groups on the surface of the capillary, the AuNP concentration and time for self-assembly, the activity concentration and time for the LDH self-assembly, the flow rate and acidity for LDH immobilization, pH, temperature, and reaction time for the NADH/PA/LDH reaction system. Under the optimized conditions, the linear response range of the biosensor towards PA was 2.5-120 µmol L-1, in which the determination limit and detection limit were 2.5 and 0.75 µmol L-1, respectively. The biosensor could be reused more than 41 times when its relative standard deviation (RSD) was controlled at less than 1.5%. At room temperature (approximately 25 °C), the intracellular PA in the erythrocyte of a healthy person was measured using the biosensor, and the PA content was observed to be 241.76 ± 68.05 µmol L-1 (n = 8). The standard addition recovery was 95-106%. Employment of the AuNPs in the PA biosensor not only improved the affinity of the immobilized LDH towards PA and its stability, but also significantly enhanced the service life of the PA biosensor.

Research on a New Micro-Volume Fluorescence Capillary Biosensor Assay for Sequentially Quantifying Pyruvate and Lactate.

It was studied that making conditions of a micro-volume fluorescence capillary biosensor for determining pyruvate (PA) and lactate (LA). The biosensor made under the optimized conditions could be used for sequential quantifications of LA in the range 0.10-1.2 mM and PA in 4-120 µM, and its recovery for PA and LA was in a satisfactory range 97-106% for human serum samples, with detection limits of 0.023 mM for LA (RSD < 1.89%, n = 11) and 0.87 µM for PA (RSD < 1.70%, n = 11). The new assay possessed these advantages that the LDH immobilizing on capillary realized the reuse of expensive enzyme in fluorospectrophotometry, and the consumption of serum samples or chemical reagents decreased to 9 µL in per assay, and the analytes no needed to preseparation, and it also are accurate and reliable. Consequently, the fluorescence capillary biosensor should have a good prospect in assaying PA and LA or LA/PA ratios for clinical medicines or biology field. The optimization conditions and parameters obtained in this study have also a certain guiding significance for the development of biochip based on glass substrate.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

A novel analysis method for lactate dehydrogenase activity in serum samples based on fluorescence capillary analysis.

Based on fluorescence capillary analysis technology, a method for quantitating lactate dehydrogenase (LDH) activity in a micro-volume sample was developed. Sample and reagent consumptions were merely 2 and 16 µL per time, respectively. The optimized test conditions were as follows. The reaction reagent consisted of 0.10 M phosphate buffer (pH 6.5), 0.30 mM NADH and 1.20 mM pyruvate. NADH standard was prepared with a phosphate buffer of pH 8.0, and its linear response was controlled in 0.05 - 0.30 mM. LDH standards containing 2.0 mM PEG could exhibit long-term stability. Under the optimized conditions, a linear response for LDH from 50 to 1200 U L(-1) and a detection limit of 31 U L(-1) were obtained with good precision (RSD: 2.1 - 2.2%, n = 10) and better recovery of 96 - 105%. The method's characteristics was high sensitivity, low consumptions, simple operations, good precision and reliability, lending itself to the miniaturization of fluorophotometer which transformed into a bedside instrument in the hospital.

Shadoo binds lipid membranes and undergoes aggregation and fibrillization.

Lipid membrane can enhance prion protein (PrP) pathological fibrillogenesis. A neuronal paralog of PrP, named Shadoo (Sho), is localized to similar membrane environment as PrP and can also convert to amyloid-like fibrilles. To gain insight into the role of Sho in prion diseases, we studied Sho interactions with cellular membrane models. Sho was found to bind anionic lipid vesicles. Spectroscopic and microscopic data showed that membrane-associated Sho slowly converted into amyloid fibers. Furthermore, binding of Sho to anionic liposomes has a disruptive effect on the integrity of the lipid bilayer leading to the formation of supramolecular lipid-protein complexes. In consequence, the role of Sho in prion diseases might depend on the oligomerization state of Sho but also the nature of these lipoprotein assembles.

Development and preliminary application of a quantitative PCR assay for detecting gtxA-containing Gallibacterium species in chickens.

A quantitative PCR (qPCR) assay using SYBR Green I was developed based on the published sequence of the gtxA gene from Gallibacterium anatis. This method produced reliable specificity, sensitivity, and repeatability. The detection rate of Gallibacterium in 181 clinical samples was 36.5% (66/181) by qPCR, which was superior to the detection rate of Gallibacterium-specific PCR (0/181) and an isolation and identification assay (18.2% or 33/181). No association was found between the prevalence of Gallibacterium and the age of the chickens. Gallibacterium infection was detected in one 4-day-old chicken, showing that infection can occur much earlier than the previously stated fourth week of life. Tissue sample analysis showed that Gallibacterium is mainly located in the trachea and ovaries, based on results from three groups of chicken with different health statuses. Furthermore, a titer analysis suggested that Gallibacterium loads in different organs may correlate with different clinical manifestations of disease. Thus, the qPCR assay developed in the present study is useful for identification and quantitative analysis of gtxA-containing Gallibacterium in various tissue samples from birds and for the assessment of the pathogenic mechanisms of Gallibacterium.