A simple and visible colorimetric method through Zr⁴⁺–phosphate coordination for the assay of protein tyrosine phosphatase 1B and screening of its inhibitors

Abstract

Inhibitors of protein tyrosine phosphatase 1B (PTP1B) are promising agents for the treatment of type 2 diabetes and obesity, so a colorimetric method has been developed in this work for PTP1B assay and screening of its inhibitors. The method is based on the chelation effect of zirconium (Zr(4+)) ions on the phosphate group, which may induce aggregation of 4-aminophenylphosphate-functionalized gold nanoparticles (APP/AuNPs) and the corresponding color change of the testing solution. Owing to the dephosphorylation of PTP1B, the aggregation of AuNPs will be influenced by PTP1B since there is no coordination reactivity between Zr(4+) ions and 4-aminophenol, the hydrolyzed product of APP catalyzed by the enzyme. Therefore, a simple colorimetric method for the assay of PTP1B activity can be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 650 nm to that at 522 nm vary linearly with the PTP1B activity in the range from 0.005 to 0.18 U mL(-1) with the lowest detection limit of 0.0017 U mL(-1). Moreover, using this proposed method, the inhibition effect of 6-chloro-3-formyl-7-methylchromone, betulinic acid, ursolic acid, and sodium orthovanadate on PTP1B activity can be tested with IC50 values of 10, 13, 9, and 1.1 μM, respectively. Therefore, this new method has great potential not only for the detection of PTP1B activity but also for the screening of the inhibitors.

Bifunctional Magnetic Fe3O4@Cu2O@TiO2 Nanosphere-Mediated Dual-Mode Assay of PTP1B Activity Based on Photocurrent Polarity Switching and Nanozyme-Engineered Biocatalytic Precipitation Strategies

“…Compared with most reported studies, − ,,− the proposed PEC-QCM dual-mode sensing platform exhibits lower detection limits and wider linear ranges (SI Table S1). Especially, the excellent trace assay performance (as low as 10 or 40 fM) implies that the proposed PEC-QCM dual-mode sensing platform has higher assay sensitivity than the colorimetry and fluorescence methods listed in SI Table S1, which may be attributed to the excellent properties of Fe 3 O 4 @Cu 2 O@TiO 2 nanospheres in PEC photocurrent polarity switching and QCM signal amplification processes.…”

“…Reversible phosphorylation of proteins (mediated by kinases and phosphatases), one of the most important posttranslational modifications which mainly occurs in serine, threonine, and tyrosine, plays an important role in cellular processes. , In living cells, disruption of this balance between kinase-mediated phosphorylation and phosphatase-mediated dephosphorylation may lead to critical diseases, including cancers and autoimmune imbalance. − Up till now, studies on phosphatases are still limited compared to the large number of studies on kinases . Traditional methods for protein phosphatase activity assay mainly rely on radioactive elements and are harmful to human health and the environment. , To avoid this potential harm, nonradioactive methods have also been developed, including colorimetry , and fluorescence. − However, the above activity analysis methods depend upon a single-mode readout signal and may be affected by apparatus, operators, and nonstandard analytical procedures …”

“…6 Traditional methods for protein phosphatase activity assay mainly rely on radioactive elements and are harmful to human health and the environment. 7,8 To avoid this potential harm, nonradioactive methods have also been developed, including colorimetry 9,10 and fluorescence. 11−13 However, the above activity analysis methods depend upon a single-mode readout signal and may be affected by apparatus, operators, and nonstandard analytical procedures.…”

High-Frequency Quartz Crystal Microbalance and Dual-Signaling Electrochemical Ratiometric Assays of PTP1B Activity Based on COF@Au@Fc Hybrids

“…It can be seen from Figure S10 that all four inhibitors have inhibitory effects on the activity of PTP1B, and the maximum inhibitory rates are 60.4, 74.1, 92.3, and 76.4%, respectively, based on the QCM method (or 58.9, 72.5, 90.6, and 77.8% based on the EC method, respectively), which is consistent with that reported in the literature. 9,38,39 ■ CONCLUSIONS In summary, by combining the high-frequency (100 MHz) QCM with the dual-signaling EC ratiometric method, the PTP1B-induced dephosphorylation process of the P-peptide is successfully monitored in real time by QCM, and PTP1B activity is assayed sensitively and reliably by the QCM-EC dual-mode sensing platform with an ultralow detection limit.…”

“…Traditionally, methods relying on harmful radioactive elements have been used in the assay of PTP1B activity . In order to avoid the hazards of radioactivity, single-mode methods, including colorimetry (CL), fluorescence (FL), and photoelectrochemistry (PEC), have been reported. It is well known that single detection mode may suffer from insufficient accuracy due to potential interferences such as the presence of confounding elements, variations in operators, instruments, and nonstandardized detection procedures. , Recently, some dual-mode sensing platforms have also been proposed in our group for the PTP1B activity assay, such as PEC-quartz crystal microbalance (QCM) , and PEC-CL .…”