In-situ tumor cell culture and active small molecules monitoring based on 3D porous hollow fiber报告人:
陈大竞，杭州师范大学医学部药学院教授，2011年博士毕业于浙江大学生物医学工程专业。2012年赴美国德州大学奥斯汀分校从事博士后研究，2014年于美国常春藤联盟高校达特茅斯学院担任研究助理，2017年通过杭州师范大学卓越人才计划全职引进回国。2017年入选杭州市521全球引才计划、2018年入选浙江省钱江人才计划、2019年入选杭州市级领军人才。主要研究方向为细胞传感器、药物电化学分析及生物医学传感与快速诊断体系等。目前主持国家自然科学基金、浙江省自然科学基金等各类项目。在国内外Chemical Engineering Journal、Analytical Chemistry、Biosensor&Bioelectronics、ACS Sensor、Acta Biomaterialia等期刊发表论文30余篇。获国家科技进步二等奖（8/10）、教育部科技进步一等奖（9/15）。美国加州大学伯克利分校（2010-2011）、意大利圣安娜高等研究院（2008-2009）访问学者。
Objective: Small molecules such as NO and H2O2 play important roles in signal transmission and regulatory of cancer. Real-time monitoring of NO and H2O2 in tumor cells will provide more comprehensive signaling molecular information for the analysis of the cell status and the anti-tumor drug effect. But extremely low in-vitro concentrations and short half-lifes of active small molecules limited the continous monitoring application.
Method: In this experiment, NO and H2O2 sensors were developed based on a three-dimensional polysulfone hollow fiber (PHF) structure. The NO sensor was assembled by layer-by-layer deposition of multi-wall carbon nanotubes (MWCNT) and gold nanoparticles (AuNPs) on the outer surface of the PHF. In H2O2 sensor. horseradish peroxidase (HRP) was immoblized on AuNPs (Fig 1). The electron generated during the electrocatalytic oxidation of NO and H2O2 on AuNPs can be accurately transferred to the electrochemical workstation (CHI660) through CNT meshes. H1299 cells were cultured in the lumen of PHF, isolating from electrochemical detection regions, thus reduced the current affect on cell growth.
Result: The growth state of tumor cells in the lumen of the PHF was characterized by SEM and laser confocal microscopy (Fig 2). The results showed that tumor cells can grow and proliferate normally within the PHF. NO sensing test indicated that with the addition of 2 mM acetylcholine, the current response of the sensor reached 69 nA, while conventional cells test setup only showed a current response of approximately 7 nA (Fig 3). The PHF sensor group was ten-fold higher in the current response than the conventional setup group. Similarly, the H2O2 sensor also showed enhanced sensing performance. The sensor can detect as low as 10nM H2O2 with a sensitivity of 90 nA/μM. The response current showed a good linear relationship in the H2O2 concentration range of 0.01-1 µM (Fig 4). The current signal induced by H2O2 upon drug stimulation with 1 µg/mL PMA, 3.3µM fMLP was 10 to 20-fold higher than conventional setup of cells dispersed in electrolytes.
Conclusion: Our study revealed that PHF is a biocompatible material that can be used for cell growth in its lumen. The sensor showed facile fabrication and greatly improved sensitivity due to its three-dimensional flow-through configuration. Our work enhanced the sensitivity and stability of NO and H2O2 sensor from the perspective of structural design and provided a robust tool in identifying cellular information.