1. Introduction
In the field analysis of cholinesterase inhibitors, one can encounter miniaturized spectrophotometric systems, but reflectance measurements on colored strips are also sometimes encountered [
1]. Based on the principle of attaching a sensor to a color surface, color sensors work by having a white LED light in the vicinity and reflected light that passes through filters on the sensor, determining the resulting color. According to the ratio of light incidents on sensors with different color filters (red, green, or blue), acetylcholinesterase was used as a biorecognition element for the determination of pesticides, medicine, and other toxicologically relevant substances, which inhibits the cholinesterase active site [
2]. The Ellman method was used for the determination of cholinesterase activity [
3]. An enzyme kinetic assay was used to prove the capabilities of the proposed system (
Figure 1).
2. Materials and Methods
This work showed the use of color sensor APDS9960 integrated on the Arduino Nano BLE Sense (Arduino, Italy) microcontroller board with a color-programmable RGB LED (Keyestudio, China). ASA signal white and jet black (Prusa research, Czech Republic) and FFF 3D printer i3 MK3S+ with MMU2S upgrade (Prusa Research, Czech Republic) were used to print the box. Acetylcholinesterase from electric eel was used as a biorecognition element, acetylthiocholine chloride was used as a substrate, and phosphate-buffered saline (pH = 7.4) was established for use in the optimal reaction conditions (All chemicals were obtained from Merck (Germany). The programmed color of the LED was equivalent for the wavelength 412 nm. 5′-dithiobis (2-nitrobenzoic acid, DTNB) was used as the chromogenic reagent for an assay lasting 5 min. Data acquisition was carried out through the Visual Studio code interface.
3. Discussion
The use of a color sensor for photometry has been demonstrate recently [
4]. Also, the diode array color sensor has already been used as a wireless RFID-like color analyzer on surfaces [
5]. To the best of our knowledge, there is not yet a printed paper that maps the coupling of APDS9960 sensor and a RGB LED for photometry. It is the addition of RGB LED that may allow for the response to be monitored in multiple color modes and to switch continuously and verify trends during analysis.
Colorimetric analysis confirmed that the highest difference in signal directly related to cholinesterase activity is in the green channel. It is probably caused by the complementarity of color with the created nitro- 5-thiobenzoic acid, because green color was not used for emission. Decreases in color depth were assessed to be inversely proportional to cholinesterase activity. Validation with inhibitors of cholinesterase is the next step, and changes in RGB LED to LED with emission maximum at approx. 410 nm will be tested for dual-readout photo-/fluorimetry. Tests which use acetylcholinesterase point to the fact that the colorimetric sensor is suitable for the analysis of enzymes, allowing it to serve for diagnosis in clinical biochemistry and toxicology, and it could also be adopted for use in other assays.
Author Contributions
Conceptualization, O.K.; methodology, O.K.; software, O.K.; validation, O.K.; formal analysis, O.K.; investigation, O.K.; resources, O.K. and M.P.; data curation, O.K.; writing—original draft preparation, O.K.; writing—review and editing, O.K. and M.P.; visualization, O.K.; supervision, M.P.; project administration, M.P. and O.K.; funding acquisition, O.K. and M.P. All authors have read and agreed to the published version of the manuscript.
Funding
This research and APC were funded by the Czech Ministry of Education, Youth and Sports [grant number SV/FVZ202103].
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare no conflicts of interest.
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