A method is described for the chemiluminescence based determination of the activity of catalase (CAT) using HO-sensitive CdTe quantum dots (QDs). It is based on the finding that the chemiluminescence (CL) of the CdTe/HO system is reduced due to the consumption of HO by the catalytic action of CAT. The Michaelis constant is calculated to be 519 ± 27 mM, showing the potential of the method to accurately measure the K compared to the standard method. The method does not require QDs to be conjugated to biological/organic molecules and therefore is considered to be a rapid and convenient method for determination of CAT in real samples. At an incubation time of 2 s, the LOD was calculated to be 4.5 unit/mL, with a linear range from 6 to 400 unit/mL. The assay is sensitive, simple, and suitable for practical applications. Graphical abstract Schematic representation of chemiluminescence-based catalase U(CAT) assay using the CdSe QD/HO system. The reduction of HO is reflected by the chemiluminescence of the QDs. A mechanism is put forward based on the changes in chemiluminescence intensity of the QDs by the consumption of HO due to the catalytic action of CAT.

Angiogenesis is a hallmark of various pathological conditions and is controlled by a variety of angiogenic factors. Blockade of vascular endothelial growth factor (VEGF) as the most pivotal stimulator of angiogenesis offers a promising therapeutic approach for some diseases, typically cancer. In the present study, a heterodimeric antagonistic VEGF was precisely designed based on structural information of recently‐crystallized VEGF/VEGF receptor‐2 (VEGFR‐2/fetal liver kinase 1/kinase domain region) complex. Directed blocking of kinase domain region occurs via substitution of a VEGF receptor binding site by two peptide segments in one pole, whereas the binding domain of the other pole of VEGF was intact. Candidate peptides for substitution were selected considering to some sequence and structural criteria. A reliable model of modified VEGF was built, refined using molecular dynamics simulation and docked with VEGFR‐2. Docking analysis revealed that binding affinity of mutant VEGF was notably diminished, corroborating our design. Heterodimeric VEGF was expressed, refolded and highly purified by two‐step affinity chromatography. Dimerization of this antagonist was confirmed using some analytical techniques. Spectroscopic studies assured us to obtain the heterodimeric form of VEGF. Some angiogenic in vitro assays such endothelial cell proliferation and tube formation indicated that this antagonist is not only strongly capable of inhibiting angiogenesis (half maximal inhibitory concentration of 33 and 24 ng·mL−1, respectively), but also showed the highest inhibitory effect compared to all other heterodimeric VEGF variants. The high anti‐angiogenic potency of this VEGF antagonist may allow its future use as an anti‐tumor agent. Structured digital abstract WT-VEGF and M-VEGF  bind by comigration in sds page ( View interaction).

Background: Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H 2 O 2-sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme. Results: Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2-35,000 pg mL −1 , with a detection limit (S/N = 3) of 0.5 pg mL −1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R 2 = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%. Conclusions: The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available.

Although the superoxide anion (O2 –·) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.

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