Brightness Analysis per Moving Particle: In Situ Analysis of Alkaline Phosphatase in Living Cells

Abstract

In situ quantitative analysis of enzymes such as phosphatase is important to understand a number of involved biological processes ranging from various metabolisms to signal transduction and cellular regulation. In this paper, a novel in situ measurement strategy was proposed to detect alkaline phosphatase (ALP) activity in different locations within single living cells. The principle is based on the measurement of the resonance light scattering brightness ratio (SBR) per moving nanoparticle that forms in an ALP-related chemical reaction. In the method, a novel resonance light scattering correlation spectroscopy (RLSCS) system was developed using two lasers for illumination or two detection channels. Using the gold nanoparticles (AuNPs) as probes, the Au@Ag nanoparticles (Au@Ag NPs) formed due to the ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AAP) and the subsequent reduction–deposition reaction of Ag ions that occurred on the AuNPs. The SBR value per moving particle was determined based on the obtained RLS intensity traces and RLSCS curves. The SBR value was found to be not influenced by the intracellular viscosity and size that was confirmed in the experiments. The linear relation between the SBR and ALP activity was established and applied to detect ALP activity and evaluate the inhibition of different drugs. Finally, the method was successfully used to in situ measure ALP activity within living cells. The method overcomes the shortcoming of conventional methods that lack quantitative analysis and are susceptible to intracellular viscosity.

Alkaline Phosphatase-Decorated Au Nanoparticles Immobilized on Bi2S3 Particles Combined with Ag2S Quantum Dots as a Photoelectrochemical Sensing Platform for Alkaline Phosphatase Activity and Fluoride Ions

“…Therefore, ALP is widely recognized as a critical diagnostic and treatment predictor. 6 It is imperative to develop a convenient and sensitive method to measure the ALP activity. Furthermore, enzyme activity can be influenced by specific substances, opening the possibility of screening enzyme inhibitors.…”

“…For example, alkaline phosphatase (ALP) catalyzes hydrolysis and phosphor-transfer reactions of proteins, nucleic acids, and small-molecules orbiomolecules both inside and outside the organism. , Deviations in ALP levels are linked to various disorders, including bone disease, hepatitis, diabetes, prostate cancer, and hepatocellular carcinoma. Therefore, ALP is widely recognized as a critical diagnostic and treatment predictor . It is imperative to develop a convenient and sensitive method to measure the ALP activity.…”

Resonance Light-Scattering Correlation Spectroscopy and Its Application in Analytical Chemistry for Life Science

“…Recently, quantitative analysis on proteins or enzymes within a single living cell was also established using the ratiometric diffusion time or brightness per particle as measurement parameters. 35,36 Until now, the confocal RLSCS technique has showed broad applications in homogeneous systems and living cells. 1,37−39 However, the illumination area in confocal RLSCS is limited within several hundred nanometers.…”

“…Subsequently, confocal RLSCS methods were developed to characterize nanoparticles in solution, such as the rotational and translational diffusion coefficients, concentration, aspect ratio, size, size distribution, etc. − Further, homogeneous detection strategies were developed for DNA detection, immunoassay, , enzymatic activity and apoptosis, study on self-thermophoresis of a nanomotor, formation of protein corona in the NPs, etc. Recently, quantitative analysis on proteins or enzymes within a single living cell was also established using the ratiometric diffusion time or brightness per particle as measurement parameters. , Until now, the confocal RLSCS technique has showed broad applications in homogeneous systems and living cells. ,− …”

Supramolecular Host‐Guest Interaction‐Driven Electrochemical Recognition for Pyrophosphate and Alkaline Phosphatase Analysis

“…Alkaline phosphatase (ALP), which is a kind of membranebound enzyme, can catalyze regularly the de-and transphosphorylation of phosphate-containing biomolecules, [1][2][3] which is involved usually for intracellular regulation and signal transduction in the process of cell apoptosis and growth. [4,5] Some recent studies show abnormal ALP expression has associated with various diseases (prostate cancer, liver disease, bone disease, etc. ), [6,7] so ALP is one of the most broadly utilized biomarkers/indicators in ALP-related clinical diagnosis and disease treatment.…”