Metal-Free Carbon Nitride Nanosheet Supported the Pentacoordinated Silicon Intermediates for Photocatalytic Overall Water Splitting.

Photocatalytic overall water splitting is a promising approach to overcome the environmental and energy crisis. However, developing effective photocatalysts with well activity, light absorption, and photogenerated carrier lifetime is still a challenge. Herein, combining extensive first-principles and nonadiabatic molecular dynamics calculations, we find that microporous carbon-nitride nanosheets with a pyridinic nitrogen, such as C2N and C6N6, possess the pentacoordinated silicon intermediates' bonding environment. The pentacoordinated silicon as intermediates exhibits good photocatalytic performance for the difficult four-electronic oxygen evolution reaction. The overpotential is only 0.55 V for C2N, which is significantly lower than that of the tetracoordinated silicon intermediates (2.00 V). Simultaneously, the decoration of the silicon group not only widens the absorption range of visible light but also maintains the lifetime of photogenerated carriers on the nanosecond scale, which enhances the application efficiency of solar energy. Our work paves a new route for advancing photocatalytic overall water splitting.

Development of an Enzyme-Linked Immunosorbent Assay and Immunoaffinity Column Chromatography for Saikosaponin d Using an Anti-Saikosaponin d Monoclonal Antibody.

This work developed a novel immunochemical approach for the quality control of saikosaponin d using an enzyme-linked immunosorbent assay. Splenocytes from mice immunized with the saikosaponin d-bovine serum albumin conjugate were fused with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma SP2/0 cell line, and a hybridoma secreting monoclonal antibody against saikosaponin d was successfully obtained. The prepared anti-saikosaponin d monoclonal antibody 1E7F3 has a novel characteristic, showing weak reactivity with compounds that are structurally related to saikosaponin d. Using monoclonal antibody 1E7F3, a specific and reliable enzyme-linked immunosorbent assay was developed to detect saikosaponin d. The system shows a full measurement range from 156.25 to 5000.00 ng × mL(-1). Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10.00%. The recovery rates ranged from 92.36% to 101.00%, meeting the requirements for biological samples. There was a good correlation between the enzyme-linked immunosorbent assay and high-performance liquid chromatography analyses of saikosaponin d, and the saikosaponin d levels in formulated Chinese medicines were successfully determined. Furthermore, immunoaffinity column chromatography was established using this anti-saikosaponin d monoclonal antibody, and the elution profile of saikosaponin d was detected by a Bio-Rad QuadTec UV/Vis detector at 203 nm. The results demonstrate that we generated a reliable and more efficient assay system for measuring saikosaponin d and provide a potential approach for purifying and separating saikosaponin d.

Sandwich enzyme-linked immunosorbent assay for naringin.

Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA.

Development of a Fluorescence-Linked Immunosorbent Assay for Baicalin.

Previously, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for baicalin (BAL) and used this assay to investigate the pharmacokinetics of BAL in mice. In this study, an anti-BAL monoclonal antibody (MAb) was purified by the caprylic acid method and then labelled with fluorescein isothiocyanate (FITC). Subsequently, an indirect competitive fluorescence-linked immunosorbent assay (icFLISA) was developed to detect baicalin (BAL) using FITC-labelled anti-BAL MAbs. Characterization of the assay demonstrated an effective BAL measurement range of 6.4 ng/mL to 500 µg/mL (R(2) = 0.997). The relative standard deviations (RSDs) for both intra-assay and inter-assay repeatability and precision were below 10 %. This icFLISA for BAL is simple, rapid and sensitive, with a 390-fold larger linear range and a 2-fold lower limit of detection (LOD) compared with the previously developed icELISA. We observed a strong correlation between the results determined by the icFLISA and icELISA methods. Overall, this study provides a useful method for detecting BAL in medicines, enabling in vivo visualization research.

Development of immunoaffinity chromatography to specifically knockout baicalin from Gegenqinlian Decoction.

Specific knockout technology provides a powerful tool to confirm the role of target compounds in a plant or its derived prescriptions, and this principle is the same as that with knockout genes. In this study, we generated an immunoaffinity column conjugated with an anti-baicalin monoclonal antibody and then loaded Gegenqinlian Decoction extracts, followed by washing with deionized water and an elution solvent. The results of the high-performance liquid chromatography fingerprints and high-performance liquid chromatography with mass spectrometry showed that the immunoaffinity column was able to specifically knockout baicalin, oroxylin A-7-O-glucuronide, wogonoside, wogonin, and baicalein from Gegenqinlian Decoction. A reliable one-step method to specifically knockout baicalin was established with an immunoaffinity column. Gegenqinlian Decoction and its knocked-out fraction induced the expression of superoxide dismutase and were compared in human umbilical vein endothelial cells cultured with a high glucose concentration; the results showed that the Gegenqinlian Decoction and its knocked-out fraction showed no significant difference, which indicated that the baicalin, oroxylin A-7-O-glucuronide, wogonoside, wogonin, and baicalein that were knocked out by the immunoaffinity column might not be key compounds for the induction of Gegenqinlian Decoction superoxide dismutase secretion.

Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody.

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzyme-linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12-2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine.

A Sensitive and Specific Indirect Competitive Enzyme-Linked Immunosorbent Assay for Detection of Paeoniflorin and Its Application in Pharmacokinetic Interactions between Paeoniflorin and Glycyrrhizinic Acid.

This study aimed to develop an indirect competitive enzyme-linked immunosorbent assay based on monoclonal antibodies against paeoniflorin to study the effects of different doses of glycyrrhizinic acid on the pharmacokinetics of paeoniflorin in mice. An anti-paeoniflorin monoclonal antibody was produced from a hybridoma created through a fusion of splenocytes immunized with paeoniflorin-bovine serum albumin and conjugated with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line SP2/0. The resultant antibody was used to develop and validate a rapid, specific and sensitive, indirect competitive enzyme-linked immunosorbent assay for the measurement of paeoniflorin (linear range 4.8-312.5 ng ·â€ŠmL(-1)). The intraday and interday precision values of the indirect competitive ELISA method were well within the recommended range (≤ 10 %), and the recovery rate was, on average, 101.13 %. Pharmacokinetic parameters obtained from mouse blood samples at various intervals following the oral administration of paeoniflorin and glycyrrhizic acid at three doses (1 : 0.3, 1 : 1, 1 : 3, respectively) demonstrated that the highest dose of glycyrrhizic acid inhibits the absorption of paeoniflorin.

Development of ELISA for detection of Rh1 and Rg2 and potential method of immunoaffinity chromatography for separation of epimers.

In this work, hybridomas producing anti-ginsenoside-Rh1 monoclonal antibodies (MAbs) were generated. These MAbs were subsequently used to create indirect competitive enzyme-linked immunosorbent assays (icELISAs). A linear correlation was obtained for G-Rh1 concentrations in the range from 26 to 512ng/mL. The regression equation was y=1.979-0.201Log2(X) with a regression coefficient of 0.9898. Precision and accuracy of the icELISA method were evaluated by the variations between replicates from well to well (intra-assay) and plate to plate (inter-assay). The recovery rates ranged from 93.16% to 108.43%. Testing with the icELISA demonstrated that the MAbs were specific for 20(S)-Rh1 and 20(S)-Rg2 with no cross-reactivity against 20(R)-Rh1 and 20(R)-Rg2. The immunoaffinity chromatography column (IAC) was constructed by covalently coupling monoclonal antibody (MAb) against G-Rh1 to CNBr-activated Sepharose 4B. When 20(R)-type-Rg2 passed through the IAC column, it was adsorbed, but the amount adsorbed was lower than that when 20(S)-type-Rg2 ran through the column. The differences in adsorption between the 20(S) and 20(R) type ginsenosides bring a new approach or method to separate 20(S)-Rg2 and 20(R)-Rg2 by IAC. Our results indicate that the icELISA is a sensitive and efficient approach for the identification of epimers, and the application of IAC using MAbs against small molecules provides a totally new thought and potential method for resolving epimers.