Chengfei Zhao

Sensitive electrochemical immunoassay of metallothionein-3 based on K3[Fe(CN)6] as a redox-active signal and C-dots/Nafion film for antibody immobilization

The fabrication of a facile, sensitive, and versatile immunosensor for the quantification of metallothionein-3 (MT-3) is proposed in this work. The K3[Fe(CN)6]-chitosan-glutaraldehyde (K-CS-GA) conjugate prepared from K3[Fe(CN)6], chitosan and glutaraldehyde was employed as the redox-active signal source. Carbon nanodots (C-dots) were coupled with Nafion to form the nanocomposite architecture layer to carry antibodies (Abs). C-dots enhanced the electrochemical response of the proposed immunosensor to improve the detection sensitivity. The fabrication steps of the immunosensor were characterized using differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Antigen determination was achieved via the decreased current response of the K3[Fe(CN)6] caused by the insulated coupled antigen. The detected signals were proportional to the logarithm of the concentrations of MT-3 ranging from 5 pg mL(-1) to 20 ng mL(-1) with a detection limit of 2.5 pg mL(-1) in PBS. The proposed immunosensor showed high sensitivity, good selectivity and reproducibility. Furthermore, detection results using real serum samples showed the immunosensors potential applications in clinical diagnostics.

A chronocoulometric LNA sensor for amplified detection of K-ras mutation based on site-specific DNA cleavage of restriction endonuclease

An amplified chronocoulometric Locked nucleic acid (LNA) sensor (CLS) for selective electrochemical detection of K-ras mutation was developed based on site-specific DNA cleavage of restriction endonuclease EcoRI. Thiolated-hairpin LNA probe with palindrome structure of stem was immobilized on the gold nanoparticles modified gold electrode (NG/AuE). It can be cleaved by EcoRI in the absence of K-ras mutation-type DNA (complementary with the loop part of hairpin probe), but cannot be cleaved in the presence of mutation-type DNA. The difference before and after enzymatic cleavage was then monitored by chronocoulometric biosensor. Electrochemical signals are generated by chronocoulometric interrogation of Hexaammineruthenium (III) chloride (RuHex) that quantitatively binds to surface-confined hairpin LNA probe via electrostatic interactions. The results suggested this CLS had a good specificity to distinguish the K-ras mutation-type, wild-type and non-complementary sequence. There was a good linear relationship between the charge and the logarithmic function of K-ras mutation-type DNA concentration. The detection limit had been estimated as 0.5 fM. It is possible to qualitatively and quantitatively detect K-ras point mutation in pancreatic cancer.

Simple and effective label-free electrochemical immunoassay for carbohydrate antigen 19-9 based on polythionine-Au composites as enhanced sensing signals for detecting different clinical samples

Carbohydrate antigen 19-9 (CA19-9) is an important biomarker for the early diagnosis and clinical monitoring of pancreatic cancer. Reliable, simple, and accurate methods for the detection of CA19-9 are still urgently needed. In this study, polythionine-Au composites (AuNPs@ PThi) were designed and prepared through one-pot reaction using HAuCl4 as the co-oxidant and raw material in thionine solution containing FeCl3 as the oxidant. AuNPs@PThi-immobilized glassy carbon electrode was used as a sensitive redox probe for electrochemical interface. AuNPs@PThi not only favored the amplification of electrochemical signals but also facilitated excellent environmental friendliness for bioassay. Maximizing the electrochemical properties of AuNPs@PThi, an effective label-free electrochemical immunoassay for the ultrasensitive and reliable detection of CA19-9 was developed. Under optimal conditions, the linear range of the proposed immunosensor was estimated to range from 6.5 to 520 U/mL, with a detection limit of 0.26 U/mL at a signal-to-noise ratio of 3. The prepared immunosensor for CA19-9 detection showed high sensitivity, stability, and reproducibility. Furthermore, the fabricated immunosensor based on AuNPs@PThi can effectively detect and distinguish clinical serum samples of pancreatic cancer and normal control with accuracy and convenience.

Adropin deficiency worsens HFD-induced metabolic defects

The limited efficacy of current treatment methods and increased type 2 diabetes mellitus (T2DM) incidence constitute an incentive for investigating how metabolic homeostasis is maintained, to improve treatment efficacy and identify novel treatment methods. We analyzed a three-generation family of Chinese origin with the common feature of T2DM attacks and fatty pancreas (FP), alongside 19 unrelated patients with FP and 58 cases with T2DM for genetic variations in Enho, serum adropin, and relative Treg amounts. Functional studies with adropin knockout (AdrKO) in C57BL/6J mice were also performed. It showed serum adropin levels were significantly lower in FP and T2DM patients than in healthy subjects; relative Treg amounts were also significantly decreased in FP and T2DM patients, and positively associated with adropin (r=0.7220, P=0.0001). Sequencing revealed that the patients shared a Cys56Trp mutation in Enho. In vivo, adropin-deficiency was associated with increased severity of glucose homeostasis impairment and fat metabolism disorder. AdrKO mice exhibited reduced endothelial nitric oxide synthase (eNOS) phosphorylation (Ser1177), impaired glycosphingolipid biosynthesis, adipocytes infiltrating, and loss of Treg, and developed FP and T2DM. Adropin-deficiency contributed to loss of Treg and the development of FP disease and T2DM.

CALCB splice region pathogenic variants leading to plasma cell neurotropic enrichment in type 1 autoimmune pancreatitis

Recently, we have demonstrated that PRSS1 mutations cause ectopic trypsinogen activation and thereby result in type 1 autoimmune pancreatitis (AIP). However, the molecules involved in inducing obliterative vasculitis and perineural inflammation in the pancreas are not well-described. The present study applied whole-exome sequencing (WES) to determine the underlying etiology and revealed novel missense splice region variants, CALCB c.88T>C (p.Ser30Pro) and IR [1]-mutants, in 2 of the 3 families and 2 of 26 unrelated patients with type 1 AIP. In vitro, both of the mutants displayed decreased βCGRP, ERK1/2 phosphorylation, and co-localized with endoplasmic reticulum and Golgi apparatus. The novel pathogenic variant identified in this case should contribute to our understanding of the expanding spectrum of AIP.