ABSTRACT
Objective A label-free electrochemical immunosensor was developed for the detection of nuclear matrix protein-22 (NMP22) as a biomarker of bladder cancer. Methods The study was based on the establishment and validation of the methodology. Urine samples were collected from 20 patients with bladder cancer and 20 controls in the affiliated Hongqi hospital of Mudanjiang medical university from September in 2017 to July in 2019 to validate the developed method. A screen-printed electrode (SPE) was modified with a film of a composite made from the reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) immobilized Zn-based-Metal-organic frameworks deposited with Au nanoparticles (rGO-TEPA@Au-ZIF8). Primary antibody against NMP22 was immobilized on the Au nanoparticles on the surface of the modified SPE, which then was blocked with bovine serum albumin to elimiate nonspecific binding sites. The process of the construction of the proposed sensorwas characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Differential pulse voltammetry was used to evaluate the linear range, recovery, precision, selectivity and stability. The data were analyzed by Mann-Whitney U test. Results Under optimal conditions, the immunosensor exhibited a linear range of 0.01-1000 ng/mlwith a detection limit of 3.33 pg/ml (S/N=3) and a standard recovery of 97.65％-107.05％. The levels of NMP22 in urine samples from patients with bladder cancer [66.03 (4.34, 91.74)]ng/ml determined by the proposed sensor were significantly higher than those of controls 0.54(0.06, 8.84) ng/ml(P=0.001). Conclusion The immunosensor can achieve sensitive, rapid and acucurate detection of NMP22, and has potential application prospects in monitoring tumor markers.
ABSTRACT
Objective@#A label-free electrochemical immunosensor was developed for the detection of nuclear matrix protein-22 (NMP22) as a biomarker of bladder cancer.@*Methods@#The study was based on the establishment and validation of the methodology. Urine samples were collected from 20 patients with bladder cancer and 20 controls in the affiliated Hongqi hospital of Mudanjiang medical university from September in 2017 to July in 2019 to validate the developed method. A screen-printed electrode (SPE) was modified with a film of a composite made from the reduced graphene oxide-tetraethylene pentamine (rGO-TEPA) immobilized Zn-based-Metal-organic frameworks deposited with Au nanoparticles (rGO-TEPA@Au-ZIF8). Primary antibody against NMP22 was immobilized on the Au nanoparticles on the surface of the modified SPE, which then was blocked with bovine serum albumin to elimiate nonspecific binding sites. The process of the construction of the proposed sensorwas characterized by cyclic voltammetry and electrochemical impedance spectroscopy. Differential pulse voltammetry was used to evaluate the linear range, recovery, precision, selectivity and stability. The data were analyzed by Mann-Whitney U test.@*Results@#Under optimal conditions, the immunosensor exhibited a linear range of 0.01-1000 ng/mlwith a detection limit of 3.33 pg/ml (S/N=3) and a standard recovery of 97.65%-107.05%. The levels of NMP22 in urine samples from patients with bladder cancer [66.03 (4.34, 91.74)]ng/ml determined by the proposed sensor were significantly higher than those of controls 0.54(0.06, 8.84) ng/ml(P=0.001).@*Conclusion@#The immunosensor can achieve sensitive, rapid and acucurate detection of NMP22, and has potential application prospects in monitoring tumor markers.
ABSTRACT
Objective To study the effect of T-2 toxin on proliferation and cell cycle of rat chondrocytes,in order to provide a new idea in molecular mechanism of T-2 toxin-induced chondrocyte damage.Methods Primary chondrocytes of neonatal Wistar rats were isolated and stained by toluidine blue staining and type Ⅱ collagen immunofluorescence staining.The effects of different concentrations of T-2 toxin [0 (control),1,5,10,20,50,100 μg/L)] on proliferation of chondrocytes for 24 h were detected by cell counting kit-8 (CCK-8) method,and control,1 (low dose),5 (medium dose),and 10 μg/L (high dose) T-2 toxin were selected for subsequent experiment;cell cycle changes were detected by flow cytometry;Real-time PCR and Western blotting were used to detect the effects of T-2 toxin on mRNA and protein expressions of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in chondrocytes.Results With increase of T-2 toxin concentration (control,1,5,10,20,50,100 μg/L),the cell survival rates [(100.00 ± 0.00)％,(93.12 ± 1.66)％,(77.12 ± 1.11)％,(59.44 ± 4.09)％,(46.64 ± 3.86)％,(38.15 ± 3.37)％,(33.79 ± 0.99)％] were decreased,and the differences were statistically significant (F =139.21,P ＜0.05).The percentages of quiescent phase/pre-DNA synthesis phase (G0/G1 phase) ceils in 1,5,10 μg/L T-2 toxin groups [(22.03 ± 0.42)％,(30.54 ± 2.61)％,(36.01 ± 1.51)％] were significantly higher than that in control group [(13.79 ± 1.65)％,P ＜ 0.05];the percentages of DNA synthesis phase (S phase) cells [(60.27 ± 3.53)％,(53.88 ±4.38)％,(49.55 ± 2.49)％] were significantly lower than that in control group [(76.72 ± 4.24)％,P ＜ 0.05].The differences of mRNA levels of PCNA and Cyclin D1 between groups were statistically significant (F =46.80,17.97,P ＜ 0.05),and 5,10 μg/L T-2 toxin groups (0.77 ± 0.13,0.79 ± 0.08,0.60 ± 0.07,0.56 ± 0.05) were lower than the control group (0.99 ± 0.02,1.01 ± 0.01,P ＜ 0.05).The expressions of PCNA protein in 5,10 μg/L T-2 toxin groups (0.69 ± 0.03,0.49 ± 0.03) were lower than that in control group (0.92 ± 0.05,P ＜ 0.05);the expressions of Cyclin D1 protein in 1,5,10 μg/L T-2 toxin groups (0.80 ± 0.06,0.60 ± 0.07,0.33 ± 0.13) were lower than that in control group (0.95 ± 0.07,P ＜ 0.05).Conclusion T-2 toxin can inhibit the proliferation of chondrocytes,which may be worked through influencing the expression of cell cycle protein,causing cell cycle arrest,thereby inhibiting DNA synthesis.