Lab-on-a-chip (LOC) or microfluidic technology has advanced its way in number of studies and applications with its useful advantages such as small sample consumption, high throughput screening, fast analysis, and portability. Recently, pathogen detection system based on Lab-on-a-Chip has gained much attention due to its suitability on the condition at low concentration sample which pathogen cells are contained. Although extensive research was conducted to develop novel pathogen detection microdevice with analysis system, such as colorimetric detection or electrical signal detection, off-chip preparation is still inevitable. This off-chip sample pretreatment is still a challenge to realize micro total analysis system, which integrates all of the components needed for pathogen detection. In our study, we focused on the sample volume capacity on the microdevice, while maintaining high sensitivity to capture and release the target cell from the sample.
In our study, we have demonstrated effective large volume processing sample pretreatment microdevice that can perform highly sensitive, rapid and simple pathogen capture for the further detection. To overcome the limitation of the previously established 2D flat, channel shaped sample pretreatment methods, we integrated 3D sol-gel matrix into the microdevice to increase the surface area for the antibody immobilization. The self aggregated sol-gel matrix gave good solid support for the antibody-antigen interaction even under the fast flow rate. Since releasing the captured target cell without altering its biological property is important for the down -stream detection, such as analysis at cellular level, we employed non-chemical method of target cell release. To have non-chemical, biocompatible release of the target capture, photocleavable linker was conjugated with antibody. This photocleavable linker was cleaved under the UV irradiation, and amount of the UV irradiated upon the sample does not affect antibody or cell activity. Also, micropump enabled automation of the sample delivery and flow rate control. The sample pretreatment device was optimized with three different pumping scheme and pumping time; 3step, 5 step, and 7 step. After the sample operation, captured cells were lysed and their genomic DNA was successfully extracted. The ability to preconcentrate the cell was quantitatively measured with real-time PCR curves. The fastest flow rate was driven under the 3 step with 100 ms actuation time, but capture yield was higher under 7 step pump scheme with 300 ms pumping time. Target pathogen, Staphylococcus aureus sample was best captured at slower flow rate, due to the longer antigen-antibody reaction time. The device showed limits of the detection up to 1 cell present in 100 μL with total 20 minutes of on-chip sample preparation. This novel microdevice can be further integrated with various detection methods on a single wafer to realize true sample-in-answer-out format for future point-of-care system in medical applications.
샘플 전처리란 병원균 검출을 하기 위한 필수 과정으로, PCR, ELISA 등 다양한 분석 방법에 맞게 타겟 샘플을 농축시켜 디텍션 감도를 높여주는 준비 과정이다. 병원균 검출은 빠르고 정확한 기술이 요구되는데 이는 개인의 질병 진단에서부터 국가적 보안문제까지 병원균 검출에 대한 필요성이 매우 커지고 있기 때문이다. 이러한 병원균 검출을 위해 지금까지 연구는 칩에서 이루어지는 검출 방법이 최근 연구되어 빠른 시간 내에 더욱 정확한 검출을 가능하게 하였으나, 칩에서 이루어지는 검출 실험은 대부분 아직 오프칩을 이용한 샘플 전처리에 의존하고 있다. 이러한 요구에 따라 개발된 랩온어칩 샘플 전처리 또한 아직 평면을 이용한 채널이기 때문에 실제 상황에서 발생하는 대량 샘플을 처리하는 데에 많은 문제점이 제시된다. 그러므로 본 연구에서는 많은 샘플을 빠르게 처리하며, 디텍션 감도는 높여줄수 있는 샘플 전처리 소자 개발을 연구하였다. 석영과 PDMS를 이용한 멀티레이어 랩온어 칩은 자동으로 작동하는 마이크로 펌프를 이용하여 샘플 흐름 속도 조절을 가능하게 하였으며, 이를 이용, 소자의 최적화 조건을 찾게 되었다. 이러한 최적화 조건을 가지고 실험 한 결과 100 uL 에 포함된 1개의 박테리아도 성공적으로 샘플 전처리가 되어 RT-PCR로 확인 가능하게 하였다. 이 샘플 전처리 소자의 개발은 RT-PCR 뿐만 아니라 앞으로 다른 다양한 랩온어칩 기술에 융합되어 의료, 식품, 범죄자 판명 등 활용분야가 확대될 것으로 기대된다.