We evaluate enzymatic activity at single protein level.
We can live well when our "cells" are working properly. Within cells are “proteins”, which consist of several types including enzymes. Enzymes play various important roles in “cells” function. For example, if the enzyme that acts as scissors cuts more substrates than usual, or, conversely, stops cutting at all, the cells will not work well. Often times, this change in enzyme activity is known to cause or worsen disease. Thus, we can accurately detect disease by evaluating the enzyme performance or activity at each enzyme level.
Single Enzyme Activity-Based Liquid Biopsy
Diluted blood with multi-colored fluorescent enzyme substrates are added to a microdevice with numerous wells with a diameter of 3 µm. Probability wise, each well will include one or zero enzyme. The enzymes metabolize the fluorescent enzyme substrates, producing fluorescent material in the wells. Fluorescence microscopy provides fluorescent signals with an intensity corresponding to the activity of each enzyme.
Diseases such as cancers are diagnosed based on difference in enzyme activity profiles, such as number of enzymes (based on number of glowing wells) and enzyme activity (fluorescence intensity of each color).
Fluorescent enzyme substrates (fluorescent probes)
We designed and developed our own fluorescent enzyme substrates that fluoresce when metabolized by certain enzyme groups.
To date, more than 100 fluorescent enzyme substrates have been developed, including ALP (Alkaline phosphatases), ENPP (Ectonucleotide pyrophosphatases/phosphodiesterases), MMP (Matrix metalloproteinases), DPP (Dipeptidyl peptidases), Aminopeptidases, etc.
Enzyme subtypes can be distinguished by the differences in the enzyme reactivity to the fluorescent enzyme substrate of each color.
1. High predictive accuracy
Existing diagnostics mostly detect proteins which include DNA, mRNA, and enzymes, but detecting the change levels of such proteins may not accurately capture changes in disease state.
Our technology utilizes "enzyme activity" which is directly related to cellular function and is thought to more accurately reflect the disease state as an indicator.
Diagnoses with high predictive accuracy is made possible by capturing the intensity of activity for each molecule of enzyme.
2. High detection sensitivity
For example, in the conventional ELISA method, 10 million enzymes are required for detection.
Our technology enables enzymes to be detected at single enzyme level with a microdevice, thus allowing for earlier diagnosis.
3. Detectable with less than a drop of blood
Since 1 µL of blood is sufficient for diagnosis, evaluation is possible with a very small amount of sample.
"Multiplexed single-molecule enzyme activity analysis for counting disease-related proteins in biological samples" Shingo Sakamoto, Toru Komatsu, Rikiya Watanabe, Yi Zhang, Taiki Inoue, Mitsuyasu Kawaguchi, Hidehiko Nakagawa, Takaaki Ueno, Takuji Okusaka, Kazufumi Honda, Hiroyuki Noji, and Yasuteru Urano, Science Advances, 2020, 6, eaay0888
This is the result of our CTO Dr. Sakamoto's graduate school work. Using single enzyme activity-based protein profiling (SEAP) technology with fluorescent enzyme substrates and microdevices, he demonstrated that the activity of various phosphate hydrolysis enzymes in blood can be detected at the single molecule level and by subtype. Furthermore, he found that the size of ENPP3 activity clusters in the blood of pancreatic cancer patients was significantly increased compared to healthy subjects, suggesting the applicability of this technique towards disease diagnosis.