Sandwich-type ELISA Impedimetric Immunosensor for Early Detection of Prostate-specific Antigen (PSA) in Human Serum

Early detection of cancer is vital for the successful treatment of the disease. Hence, a rapid and sensitive diagnosis is essential before the cancer is spread out to the other body organs. A gold millielectrode (GME) functionalized with a mixed (16-MHA+EG₃SH) self-assembled monolayer (SAM) was used to fabricate a point-of-care immunosensor for the detection of the prostate cancer biomarker: prostate-specific antigen (PSA) in human serum samples. An organic matrix (Amine-PEG₃-biotin/Avidin) was used to reduce non-specific protein adsorption on the electrode surface. A 16-MHA/EG₃SH/Amine-PEG₃-biotin/Avidin GME was exposed to solutions with different sandwich-type immunocomplex (^BtnAb-Ag_PSA-^HRPAb) concentrations and its response was measured using the electrochemical impedance spectroscopy (EIS) technique. This approach was compared with regard to a reference ELISA immunoassay. The obtained results showed that the immunosensor proposed in this work qualifies for its pre-clinical application in the quantification of PSA in serum samples of a representative Mexican population.     

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′)₂ anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.     

Selective optical sensing of biothiols with Ellman's reagent: 5,5′-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles

Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB²⁻) anion as a result of reaction with biothiols was measured at 410 nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57 μM and acceptable linearity ranging from 0.4 to 29.0 μM (r = 0.998).     

Benzimidazole-based chromogenic chemosensor for the recognition of oxalic acid via counter ion displacement assay in semi-aqueous medium

An azo dye-coupled benzimidazole-based receptor 1 was synthesized and investigated as a receptor for metal ions in semi-aqueous medium. The receptor recognizes Cu²⁺ with high selectivity over other metal ions. The resultant complex 1·Cu²⁺ was found to selectively bind oxalic acid via counter ion displacement.     

A nonradioactive electrophoretic mobility shift assay for measurement of pregnane X receptor binding activity to CYP3A4 response element

The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug‐metabolizing enzymes and many other genes. Radiolabeled ³²P‐DNA‐probes had been used in studies of PXR‐DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.     

A rapid,straightforward, and print house compatible mass fabrication method for integrating 3D paper‐based microfluidics

Three‐dimensional (3D) paper‐based microfluidics, which is featured with high performance and speedy determination, promise to carry out multistep sample pretreatment and orderly chemical reaction, which have been used for medical diagnosis, cell culture, environment determination, and so on with broad market prospect. However, there are some drawbacks in the existing fabrication methods for 3D paper‐based microfluidics, such as, cumbersome and time‐consuming device assembly; expensive and difficult process for manufacture; contamination caused by organic reagents from their fabrication process. Here, we present a simple printing–bookbinding method for mass fabricating 3D paper‐based microfluidics. This approach involves two main steps: (i) wax‐printing, (ii) bookbinding. We tested the delivery capability, diffusion rate, homogeneity and demonstrated the applicability of the device to chemical analysis by nitrite colorimetric assays. The described method is rapid (<30 s), cheap, easy to manipulate, and compatible with the flat stitching method that is common in a print house, making itself an ideal scheme for large‐scale production of 3D paper‐based microfluidics.     

Pinus wallichiana-synthesized silver nanoparticles as biomedical agents: in-vitro and in-vivo approach

ABSTRACT

The current study aims to assess the aqueous extract of Pinus wallichiana stem for the synthesis of small spherical-shaped (10–30?nm) silver nanoparticles (AgNPs) and their in-vitro and in-vivo biomedical applications. The biosynthesized AgNPs were nonmutagenic and safe at all test doses as per Ames and acute toxicity assay (20, 40, 60, and 80?mg/kg). The percent writhing inhibitory effect generated by AgNPs was 42.51, 50.84, and 59.06 at test doses of 10, 20, and 30?mg/kg, respectively. The percent decreased in gastrointestinal tract motility observed was 41.34%, 32.69%, and 28.48% at 10, 20, and 30?mg/kg, respectively. They also showed a significant antipyretic effect after 1, 2, and 3?h in comparison to normal saline. The AgNPs of P. wallichiana showed good antibacterial activity against Acinetobacter baumannii (60% with MIC₅₀?=?2.36?mg/ml and MBC?=?5.0?mg/ml). These nanoparticles also possessed good antioxidant activity of 61.77?±?0.828% and 70.25?±?0.56% at 400 and 500?µg/ml, respectively and lack phytoagglutinin potential. Because of their high potency as biomedical agents, these nanoparticles can be a good alternative to the currently available drugs and approaches.     

Bioactivity and molecular docking of synthesized macromolecular ligand and its complex

The development of drug resistant strains of the pathogenic fungi despite the availability of large number of drugs demands the development of new and more potential drug molecules. Dendrimer based drug molecules are comparatively less researched upon and a recent advancement in this field. The present study is concerned with the preparation of macromolecular ligand and its complex using ethylenediamine and methylmethacrylate as starting material. The reaction goes via Michael addition reaction and synthesized dendritic units were used as ligands to obtain metal-ligand complexes. Obtained ligand and its complex were characterized in terms of different techniques such as FTIR, ¹H NMR, ESI-MS, UV–Vis and Elemental analysis. Further, the ligand and its complex were used to determine antifungal activity against C. albicans ATCC 90028 by MICs and Disk diffusion assay. Comet assay and Molecular docking techniques were used to show toxicity effects and ligand–DNA interactions respectively. Ligand and its complex were obtained in very good yield with square planar geometry and having good antifungal potential against C. albicans. It was also found from Molecular docking and Comet assay, that the Copper complex interacts strongly with DNA and shows less toxicity than ligand. The compounds can serve as promising leads for the development of new antifungal agents.     

Determination of 19-nortestosterone residues in aquaculture tissues by enzyme-linked immunosorbent assay and comparison with liquid chromatography and tandem mass spectrometry

19-Nortestosterone (17β-NT) was oximated by carboxymethoxylamine and then coupled with bovine serum albumin (BSA) in a mixed-anhydride reaction in order to produce an antibody. The conjugate rate of 17β-NT and BSA was estimated to be 24 by ultraviolet spectrophotometry. Polyclonal antibody of 17β-NT was acquired from the animal immunized with the conjugate. Through an indirect enzyme-linked immunosorbent assay (ELISA), which demonstrated that the synthesis of immunogen was successful, the titre of antiserum was found to be 6.4?×?10⁵. Based on the purified antibody, a competitive indirect ELISA was developed. ELISA revealed that the limit of detection (LOD) was 0.07?ng?g^?1, the recovery (in edible tissues) was 71–89%, and the working range was 0.05–31.25?ng?g^?1. The preliminary evaluation of assay performance through specificity, sensitivity, precision, and accuracy revealed that this ELISA method could be used in the practical detection of 17β-NT in tissue samples. Moreover, this method was compared with high-performance liquid chromatography tandem mass spectrometry, for which the transition for quantification of 17β-NT was 275.4/109.1.     

生白片的药理实验研究

研究生白片（ＳＢ）对小鼠血虚模型的升白疗效、促进淋巴细胞增殖和保护ＤＮＡ免受损伤的作用。以环磷酰胺制小鼠血虚模型，以生白片加以治疗，观察各研究指标。结果表明生白片具有显著的生白疗效、提高淋巴细胞增殖能力和降低细胞微核率的作用。