Project leader Arūnas Stirkė
Agreement No 1.1.1.2/16/I/001
Research application No. 1.1.1.2/VIAA/4/20/739
Many pharmaceutical companies and the public are demanding new drug-free and immunomodulating technologies for cancer treatment. Fusion of biology and physics should further address biocompatibility guidelines to ensure complete functionality and reliability of microfluidic chips for cancer research. Advances in the field of microfluidics and microfabrication have contributed to the development of dynamic cell culture systems and in vitro models recapitulating the human organs substitutes. The aim of this research is to develop the microfluidic chip with multiple detection modules for evaluation of nsPEF induced gene expression suitable for melanoma gene therapy application.
The project is implemented at the Institute of Solid State Physics of the University of Latvia from 01.01.2021. until 30.06.2023. The total cost of the project is 111 504.90 EUR.
Project progress
30.06.2023.
The ongoing final experiment with the chip is aimed at gathering additional data. Moreover, a successful business trip was made to the University of Rochester Laboratory of Laser Energetics, as documented at https://www.lle.rochester.edu/index.php/omega-laser-facility-2/. During this visit, I conducted THz analysis on hydrogels, which serve as substitutes for the extracellular matrix and facilitate cell adhesion in the chip. Furthermore, the last iteration of the prototype was constructed and evaluated, and a holder for the oxygen sensor fiber and pH sensor components was designed and fabricated.
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| photo No. 1: Prof. A. Jukna (VGTU), Prof. R. Sobolewski (UofR), and Dr. A. Stirke (ISSP UL) discussing about OMEGA laser before going into the facility of High energy density physics | photo No. 1: Prof. V. Petrikyte (LSMU), Prof. R. Sobolewski (UofR), and Dr. A. Stirke (ISSP UL) nearby the THz imagining setup. |
30.04.2023.
The initial iteration of the prototype was constructed and examined, during which enhancements were made. The impact of a microsecond pulsed electric field on cell viability was assessed, leading to the discovery of new issues related to the direct effect of a pulsed electric field on the proteins. When combined with the pulsed electric field, these factors can significantly influence the analysis of the Secreted Embryonic Alkaline Phosphatase reporter system. Consequently, additional experiments were conducted to gain insights into the influence of the pulsed electric field on the physicochemical characteristics of the protein. The manuscript will be finished soon based on this research.
31.01.2023.
During this period the mould fabrication protocol, which were using to cast the critical components of the chip, was changed. It helps as to standardize and increase the producibility of the final chip. Effect of microsecond pulsed electric field on viability of the cells was analyzed. Moreover, experiments of pulsed electric field effects on the activation of the NFAT-SEAP reporter expression system were started. The first version of prototype was fabricated and tested. From the preliminary data we need to improve the resistivity of the contact to the chip electrodes. It will be done soon by improving holder design. Electroporation experiments on a chip is going.
14.09.2022.
During this period the experiment was expanded in various areas. The final design of the chip was finished. Additionally, a new experimental chip was developed and fabricated for pH measurement system calibration. Components and electronics for pH sensor were assembled already. New algorithm to enhance the sensitivity of pH sensor was develop after experiment with pH calibration chip. We are working on it now. A new methodology was developed to glue the oxygen sensor and mini-Luer lock. Simultaneously, a lot of experiments with melanoma cancer cell line is going on. The focus is on the electroporation and adhesion experiments.
12.04.2022.
During a business trip to the partner institution a lab space was prepared for some parallel experiments and for cell growth experiment as well. Prepared semi-finished microfluidic chip parts was assembled for future testing. In parallel, the adhesion experiments of cells on the permeable membrane are going on. Moreover, a new task for development and optimization of new sensors for cell health monitoring was started. The work for new designs suitable for incorporation of oxygen sensors into the developed microfluidic chip was started. We start organizing the 3rd Baltic Biophysics Conference, which will be held on October 6-7th, 2022 in Vilnius. The conference link is here https://bbc.lbfd.lt.
07.01.2022.
Fabrication of microfluidic chip is going well. Gold was thermally evaporated on not heated COC slides to obtain the electrode array for electroporation experiment. Prepared semi-finished microfluidic chip parts was prepared for future assembly. In parallel, the adhesion experiments of cells on the permeable membrane are going on. Early experimental data showed that for good cell adhesion on polycarbonate membrane, used in microfluidic chip as a basement for cells, oxygen plasma treatment of membrane was very important. Counting the adherent cells after 24h of growth on non-treated and 27 minutes plasma and poly-D-lysine treated membrane 20% and more than 94% of seeded CHO-K1 cells were attached respectively. Our last work was accepted for publication in International Journal of Molecular Sciences collection of Feature Papers in Molecular Biophysics which you can find here https://www.mdpi.com/1422-0067/23/1/451/htm
Figure 1. The scheme of experimental flow from already published article.
04.10.2021.
According to pulsed electric field simulation with COMSOL the 3D CAD design was modified. However, 2 mm distance between the electrodes was selected for first test. The CAD designed shadow mask for electrode fabrication was ordered. It will be used for a sputtering of gold on a COC slide to fabricate the electrodes. To maintain the accuracy of inlets and outlets for the handling of liquids in microfluidic chip the laser processing was introduced. Two types of laser processing systems, picosecond, and femtosecond was tested on COC cutting and engrave (Figure 1). Moreover, I was invited represent my scientific career in networking event titled „The Choices of the Modern Scientist“ organized by Lithuanian Agency for Science, Innovation and Technology.
Figure 1. The picture representing results after laser processing of COC slide. A. Engraving using a picosecond laser. B. A hole made by a femtosecond laser. C. The profile of engraved ladder seen at A. insert.
05.07.2021.
Several 3D CAD designs of microfluidic chip was developed. Based on offered design the distribution of pulsed electric field between the electrodes (Figure 1), across the membrane and through the membrane hole was modeled using COMSOL software. It was concluded that the optimal distance between the electrodes should not exceed the 1,5 mm. After the analysis of pulsed electric field distribution across the porous membrane pore it’s seem that the electric field strength multiplies six times. This finding should be kept in mind for future investigation and modeling of impedance spectroscopy or transepithelial electrical resistance measurements. Moreover, the electrode fabrication on selected COC polymer slides was started. Silver was thermally evaporated on heated and not heated COC slides. Adhesion tape test revealed that there is no big difference between heated and not heated samples. To investigate how plasma treatment involves into the adhesion properties of plastic and silver electrodes (thinness around 300nm) the treatment of COC slides with oxygen plasma was used.
Figure 1. The distribution of pulsed electric field corresponding to the distance of electrodes.
31.3.2021.
The literature survey was finished. The collected articles were used for 3D designing and for preparation of lecture about the electrostimulation and measurements of cells in a chips. The 3D Computer-aided design (CAD) program was employed to design the primary and simplified concept of the microfluidic chip with multi-detection properties. 3D concept of the device is very important step before start translating it into a master moulds for the soft-lithography process. Biocompatible materials for melanoma cell line growth were selected for primary concept of the microfluidic chip with multi-detection properties. Reasearcher was trained and certified for a work in a ISSP UL cleanroom.