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  • Adsorption between TC-stabilized AuNPs and the phosphate group: application of the PTP1B activity assay.

Adsorption between TC-stabilized AuNPs and the phosphate group: application of the PTP1B activity assay.

The Analyst (2015-11-03)
Jun Lv, Xiaonan Wang, Yuanyuan Zhang, Defeng Li, Juan Zhang, Lizhou Sun
ABSTRACT

Based on the adsorption between tetracycline (TC) and phosphate groups, a general colorimetric method is explored in this work by using TC-stabilized gold nanoparticles (TC/AuNPs) and 4-aminophenyl phosphate-functionalized Fe3O4 magnetic nanoparticles (APP/MNPs). Taking protein tyrosine phosphatase 1B (PTP1B) as an example, 4-aminophenyl phosphate (APP) can be hydrolyzed into 4-aminophenol (AP) by PTP1B, resulting in the disappearance of the phosphate group on the outer layer of MNPs and the loss of corresponding adsorptive ability. Upon addition of TC/AuNP solution, TC/AuNPs will remain in the supernatant solution after magnetic separation and a high absorbance value can be observed. So PTP1B activity is related to the concentrations of TC/AuNPs in the supernatant solution. In this work, the enzyme activity can be determined at levels as low as 0.0885 U mL(-1) and over a linear detection range as wide as 0.1 U mL(-1) to 0.9 U mL(-1). Moreover, using the proposed method, the inhibition effect of betulinic acid (BA) and sodium orthovanadate (Na3VO4) on PTP1B activity can be tested with IC50 values of 30 μM and 4 μM, respectively. Therefore, a universal platform for the accurate colorimetric analysis of kinase and phosphatase activities can be established through the adsorption between TC and phosphate groups.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium orthovanadate, ≥90% (titration)
Sigma-Aldrich
Citrate Concentrated Solution, BioReagent, suitable for coagulation assays, 4 % (w/v)
Sigma-Aldrich
Protein Tyrosine Phosphatase 1B human, recombinant, expressed in E. coli
Sigma-Aldrich
Betulinic acid, technical grade, 90%
Sigma-Aldrich
Betulinic acid, ≥98% (HPLC)
Sigma-Aldrich
Sodium orthovanadate, 99.98% trace metals basis
Sigma-Aldrich
1,2-Dichloroethane, anhydrous, 99.8%
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Citrate Concentrated Solution, BioUltra, for molecular biology, 1 M in H2O
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1,2-Dichloroethane, ACS reagent, ≥99.0%
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N-Hydroxysuccinimide, 98%
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Sodium tetraborate, 99.998% trace metals basis
Sigma-Aldrich
Sodium tetraborate, 99%