Fluorescent Light-up Probe with Aggregation-Induced Emission Characteristics for Alkaline Phosphatase Sensing and Activity Study

Abstract

Fluorogens with aggregation-induced emission (AIE) characteristics have attracted intensified research interest in biosensing applications, and those with specific targeting ability are especially desirable. In this work, we designed and synthesized an AIE fluorescent probe by functionalizing a tetraphenylethylene (TPE) fluorogen with two phosphate groups (TPE-phos) for the detection of alkaline phosphatase (ALP) and its enzymatic activity based on the specific interaction between the probe and ALP. The probe is virtually nonfluorescent in aqueous media due to good water solubility. In the presence of ALP, the phosphate groups are cleaved through enzymatic hydrolysis, yielding a highly fluorescent product as a result of activated AIE process. This light-up probe shows excellent selectivity toward ALP among a group of proteins. The detection limit is found to be 11.4 pM or 0.2 U L(-1) in Tris buffer solution with a linear quantification range of 3-526 U L(-1). The assay is also successfully performed in diluted serum with a linear range up to 175 U L(-1), demonstrating its potential application in clinical analysis of ALP levels in real samples. Furthermore, by conducting kinetic analysis of the enzyme using TPE-phos as the substrate, the kinetic parameter kcat/KM is determined to be 5.1×10(5) M(-1) s(-1), indicating a high efficiency of the substrate.

Fluorescence light-up AIE probe for monitoring cellular alkaline phosphatase activity and detecting osteogenic differentiation

“…31,34 For ALP, papain (Pap), trypsin (Try) and lysozyme (Lys), the concentration was 40 mU mL À1 ; for bovine serum albumin (BSA) and a-amylase (Amy), the concentration was 20 nM. After incubating for 60 min, the fluorescence intensities were measured using a LS55 luminescence spectrometer.…”

“…For most AIE probes, a high signal-to-background ratio was realized. Though the ALP turnon probes based on the phosphorylated TPE derivatives were reported previously, 31,32 their applications in detecting the ALP activity in living cells, especially for stem cells, are still limited and remain to be explored.…”

A fluorescent probe for alkaline phosphatase via excited state intramolecular proton transfer

“…A c c e p t e d M a n u s c r i p t 15 in Figure 5H, the blue-green fluorescence is almost indiscernible. The results indicate that the intracellular fluorescence changes in HeLa cells are indeed triggered by ALP.…”

“…A number of methods for detecting the activity/level of ALP have been reported in the past years, such as colorimetric, electrochemical, surface enhanced resonance Raman scattering (SERRS) methods and fluorescence technique [8][9][10][11][12][13][14][15][16].…”

AIE Based Coumarin Chromophore - Evaluation and Correlation Between Solvatochromism and Solvent Polarity Parameters

“…AIE materials are successfully put into application in the construction of OLEDs [1][2][3][4][5], bioimaging systems [6][7][8] and chemical sensors [9,10]. Most of the fluorescent organic molecules show fluorescence in dilute solutions but it suffer from the aggregation-caused quenching (ACQ) effect due to strong π-π stacking interactions in extended π-conjugated systems and dipoledipole interactions in D-π-A systems.…”