A fast estimation platform of anticancer drugs for personal precision medicine by using double reaction hydrogel let dielectrophoretic separation technique
- Publication Date: 2021-02-09
| Execution Methods | Cancer is one of the most serious genetic diseases in humans and can lead to the death of most patients. Melanoma is an uncontrolled accumulation of malignant melanocytes and is one of the most dangerous and deadly diseases of skin tumors. Current medical interventions, such as radiation therapy, chemotherapy, or immunotherapy, are insufficient to effectively treat malignant metastatic melanoma in the lungs. Gene sequencing is performed on patients before administration to find out whether the patient has a mutation in a specific gene. However, due to the complexity of cancer, it is often the case that the known cancer gene does not have a mutation, or the location of the mutation does not correspond to the drug. At this time, how to administer the medicine can only rely on the experience of the doctor. In order to solve this problem, the existing technology is to extract the patient's blood, isolate the cancer cells, enlarge the culture, and conduct drug testing before administration to select the best drug. This can avoid wasting medical resources and reduce patient suffering, but there are still the following problems. (1) The experiment takes a long time and drags down the disease. (2) It needs to consume a lot of consumables and is expensive. (3) Manpower consuming. (4) It cannot be measured in time and cannot be monitored in real-time under real medication. The key point for the above reasons is that the current technology for measuring cell survival after the medication is insufficient. The minimum number of cells must be more than 2,000 to detect significant results, and it is expensive and cannot be reused. In order to solve the above problems, we designed a solution. The bead microfluidic system addresses the above problems. 1. This project develops a dual-reaction hydrogel and microbead DEP sorting chip for rapid evaluation of the efficacy of cancer drugs. 2. This project mainly focuses on temperature-sensitive and acid-base types, and develops a dual-reactive, highly sensitive, and highly biocompatible reversible hydrogel for the 3D culture of melanoma cells. 3. This project develops a bead microchannel chip through microfabrication and embeds the cultured melanoma cells in a dual-reactive hydrogel material for 3D culture. 4. Develop microfluidic sorting chips and integrate three-dimensional electrodes. Using the different metabolism of cells in the liquid beads, the changes in the overall conductivity of the liquid beads are used as indicators, and the dielectrophoresis force is used to separate the liquid beads with different cell activities. select. 5. Investigate the effect of different dielectrophoresis parameters (frequency, voltage, bead flow rate, etc.) on drug potency sorting, and optimize the microfluidic sorting chip. |
| Performance Evaluation | 1. Dual-reactive hydrogel: Utilize temperature-sensitive and acid-base properties to develop a dual-reactive reversible hydrogel with high sensitivity and high biocompatibility. 2. Droplet generation chip: Using different phase fluids (water glue/oil), the melanoma cells and anticancer drugs are embedded in the droplets by means of squeezing. Generates many tiny droplets (d = 100 ± 5 μm) and limits the diffusion of metabolites from cells, reducing reagents, concentrating products, and amplifying detection. 3. Dielectrophoretic sorting wafer: Using the alternating electric field to generate dielectrophoretic force, the liquid beads with different conductivity are subjected to standard-free sorting. In the case of the same collection efficiency, the increase of the conductivity of the droplets will reduce the minimum applied voltage. The conductivity range is 28-482 μS/cm, and the relationship coefficient is 6.5 (μS/cm)/V, R2 = 0.97. 4. Dielectrophoretic sorting wafer: The dielectrophoretic force generated by the electric field of 200 Vpp and 10 kHz was applied, and the collection rate of hydrogel beads embedded with more than 3 cells reached 93.5% (n = 103). 5. Dielectrophoretic sorting wafer: The DEP force generated by the electric field of 200 Vpp and 10 kHz was applied, and the collection rate of the hydrogel beads containing anticancer drugs and melanoma cells was reduced to 10.6% (n = 132 ). |
| Conclusion & Suggestion | First of all, I would like to thank the ENABLE project for its subsidy. Through this ENABLE project subsidy, this cross-disciplinary team can be established and cooperated. Precision medicine is the current effort of researchers all over the world to detect individual differences and give optimal medical procedures to the trend of future medical development. For cancer treatment, before chemotherapy (chemotherapy), patients can use gene sequencing to find out whether the patient has a specific gene mutation, so as to increase the efficacy of chemotherapy drugs. But even so, the cure rate of cancer treatment is still not very high. Taking melanoma as an example, its occurrence is mainly due to the mutation of the BRAF (V600E) gene, but about half of the patients are insensitive to BRAF inhibitors, and the treatment effect is very poor. micro. Therefore, in addition to gene sequencing, the efficacy of drugs on individual patients is also an important part of precision medicine. Through this cooperation, our team has designed and developed a highly sensitive dual-reaction hydrogel, combined with microfluidic chip and dielectrophoresis technology, with the efficacy of melanoma anti-cancer drugs as the target, and developed a calibration-free rapid drug efficacy evaluation platform. . And this new type of hydrogel is highly sensitive to temperature (thermal sensitive hydrogel, liquid below 30°C; colloid above 30°C), and the liquid bead is composed of a temperature-sensitive hydrogel and an acid-alkaline hydrogel (pH-sensitive hydrogel). The hydrogel contains a hydrophilic group and a hydrophobic group, and the two types of functional groups will compete with each other. In a low-temperature environment, the hydrophilic group makes the hydrogel connected by hydrogen bonds to expand in water, forming a uniform solution, which is a liquid hydrogel; on the contrary, when the temperature is higher than 30 °C, the hydrogel will change from liquid to gel. The acid-base hydrogel will swell and de-swell with the change of pH value, and it has good results in the basic physical property test and biocompatibility test. Such hydrogels could be used for drug delivery or as biosensors in future applications. On the other hand, the droplet microfluidic generation chip can stably and effectively embed the target (cells and drugs) in a droplet with a diameter of 100 ± 5 μm, so that the metabolites of the cells are confined in the droplet, and then Change the conductivity of the droplet. The droplet microfluidic sorting chip integrates three-dimensional electrodes and microfluidic channels, and sorts droplets of different conductivity by means of dielectrophoresis, so as to understand the metabolism of melanoma cells under the influence of anticancer drugs. The decrease in activity was used to evaluate the efficacy of anticancer drugs. In the conductivity range of 28 −482 μS/cm, the correlation coefficient between the minimum voltage applied to the sorting wafer and the conductivity is 6.5(μS/cm)/V. The dielectrophoretic force generated by the application of 200 Vpp and 10 kHz electric field, the collection rate of hydrogel beads embedded with more than 3 cells can reach 93.5% (n = 103); Cancer drug and melanoma cells with hydrogel beads, the collection rate dropped to 10.6% (n = 132). This result shows that the efficacy of the drug inhibits the metabolism of melanoma cells, and even kills the cells, resulting in a very low change in conductivity, which cannot be collected by the sorting wafer. Compared with the traditional drug screening method, this system uses the sorting rate as an indicator of the efficacy of anticancer drugs, which can reduce the consumption of a lot of time, reagents, and samples. In the future, this dielectrophoretic microbead sorting chip combined with a highly sensitive dual-reaction hydrogel has great potential for development towards an automated drug screening and efficacy analysis platform. |
| Appendix | Appendix 1 |