scholarly journals Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples

2020 ◽  
Vol 6 (11) ◽  
pp. eaay0888 ◽  
Author(s):  
Shingo Sakamoto ◽  
Toru Komatsu ◽  
Rikiya Watanabe ◽  
Yi Zhang ◽  
Taiki Inoue ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Single Molecule ◽  
Biological Samples ◽  
Protein Profiling ◽  
Single Molecule Level ◽  
Multiple Substrates ◽  
Hydrolyzing Enzymes ◽  
Enzyme Molecules ◽  
Related Proteins ◽  
Single Enzyme

We established an ultrasensitive method for identifying multiple enzymes in biological samples by using a multiplexed microdevice-based single-molecule enzymatic assay. We used a paradigm in which we “count” the number of enzyme molecules by profiling their single enzyme activity characteristics toward multiple substrates. In this proof-of-concept study of the single enzyme activity–based protein profiling (SEAP), we were able to detect the activities of various phosphoric ester–hydrolyzing enzymes such as alkaline phosphatases, tyrosine phosphatases, and ectonucleotide pyrophosphatases in blood samples at the single-molecule level and in a subtype-discriminating manner, demonstrating its potential usefulness for the diagnosis of diseases based on ultrasensitive detection of enzymes.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

scholarly journals Single-molecule enzymology: What can it tell us?

2003 ◽  
Vol 25 (4) ◽  
pp. 24-27 ◽  
Author(s):  
Clive R. Bagshaw
Keyword(s):  
Enzyme Activity ◽  
Single Molecule ◽  
Enzyme Molecule ◽  
Basic Principles ◽  
Single Enzyme ◽  
Single Molecule Enzymology

In the last decade, several methods have been developed to measure enzyme activity at the level of a single enzyme molecule. This represents a considerable technical feat, but what does it allow us to learn about enzymes? Here, the basic principles are reviewed to show that new forms of heterogeneity in activity may be revealed and evidence gained for rare states that would otherwise be swamped in bulk assays.

scholarly journals Mechanical Modulation of Enzyme Activity by Rationally Designed DNA Tweezers: From the Ensemble to the Single-Molecule Level

2015 ◽  
Vol 108 (2) ◽  
pp. 186a-187a
Author(s):  
Soma Dhakal ◽  
Minghui Liu ◽  
Matthew R. Adendorff ◽  
Mark Bathe ◽  
Hao Yan ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Single Molecule ◽  
Single Molecule Level ◽  
Mechanical Modulation

Photoinduced electron transfer (PET)-probes for the study of enzyme activity at the ensemble and single molecule level

2007 ◽  
Author(s):  
Sigrun Henkenjohann ◽  
Sebastian van de Linde ◽  
Sören Doose ◽  
Philip Tinnefeld ◽  
Markus Sauer
Keyword(s):  
Electron Transfer ◽  
Enzyme Activity ◽  
Single Molecule ◽  
Photoinduced Electron Transfer ◽  
Single Molecule Level ◽  
Pet Probes

Optical Fiber Microarrays for Chemical and Biological Measurements

2008 ◽  
Vol 1133 ◽  
Author(s):  
David R Walt ◽  
Christopher LaFratta ◽  
Michael Webb ◽  
Zhaohui Li ◽  
Hans-Heiner Gorris ◽  
...  
Keyword(s):  
Single Molecule ◽  
Biological Fluids ◽  
High Sensitivity ◽  
Kinetic Properties ◽  
Fluorescent Product ◽  
Coherent Imaging ◽  
Fiber Arrays ◽  
Stochastic Nature ◽  
Enzyme Molecules ◽  
Single Enzyme

Abstract We have used coherent imaging fiber arrays as a platform for preparing chemical sensors and biosensors. Sensors can be made with spatially-discrete sensing sites for multi-analyte determinations. Micrometer sized sensors have been fabricated by etching the cores of an optical imaging fiber to create microwells and loading them with microspheres. These arrays possess both high sensitivity and reproducibility and can be used for making thousands of measurements simultaneously such as for genetic analysis or for the analysis of complex biological fluids. Both optical and optoelectrochemical arrays have been used for multiplexed sensing. In another scheme, the arrays can be used for single molecule detection. In this format, individual molecules, such as enzymes, can be trapped in the microwells by sealing each microwell with a silicone gasket. The enzyme molecules catalyze the formation of a fluorescent product that can be detected readily. The kinetic properties of hundreds to thousands of single enzyme molecules can be monitored simultaneously using this format. By observing the stochastic nature of the single molecule responses, new mechanistic insights into the fundamental nature of the enzymes can be obtained.

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