초록 ▼

Ⅲ. 연구내용 및 결과요약
1. 연구 내용
ㅇ 일회용 wire-guided droplet PCR 장치 개발
- 세미 벤치탑 시스템 개발
- 핸드헬드 PCR 장치 설계 및 제작
- 프로토타입 장치에 모델 병원체를 적용하여 real-time RT-PCR 실험
ㅇ 병원체 검출용 일회용 PCR 장치 개선
- 핸드헬드 장치 개선
- 개선 PCR 장치와 모델 병원체를 이용하여 real-time RT-PCR 성능 평가
ㅇ 일회용 wire-guided droplet PCR 장치 효능 평가
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Ⅲ. 연구내용 및 결과요약
1. 연구 내용
ㅇ 일회용 wire-guided droplet PCR 장치 개발
- 세미 벤치탑 시스템 개발
- 핸드헬드 PCR 장치 설계 및 제작
- 프로토타입 장치에 모델 병원체를 적용하여 real-time RT-PCR 실험
ㅇ 병원체 검출용 일회용 PCR 장치 개선
- 핸드헬드 장치 개선
- 개선 PCR 장치와 모델 병원체를 이용하여 real-time RT-PCR 성능 평가
ㅇ 일회용 wire-guided droplet PCR 장치 효능 평가
- 일회용 PCR 장치 효능 평가

2. 결과 요약
ㅇ 교차오염을 방지하기 위하여 heater chamber를 일회용 원형카트리지로 설계하였고 wire-guided droplet manipulation(WDM)에 기반한 챔버형 PCR 장치의 프로토타입 버전 1(1차년도), 버전 2(2차년도)를 설계·제작·개선하였음
ㅇ 챔버형 PCR장치 프로토타입 버전 2를 이용한 PCR 증폭실험을 진행한 결과 target을 정확히 증폭할 수 있음을 확인하였음
ㅇ 일회용 PCR장치의 유전자 증폭 성능 개선에 대한 원리를 파악하였음
- 챔버와 droplet내의 실측 온도가 다름을 확인
- 표면장력 분석을 통해 전혈이 유전자 증폭 성능을 개선시킨다는 점을 파악함
- Wire-guided Droplet Manipiilation(WDM)의 대류 열 전달 계수가 커서 가열과 냉각에 필요한 시간은 크게 축소되어, 빠른 PCR 증폭을 이룰 수 있음을 파악함
- Droplet 내부에 가검물의 불필요한 성분과 증폭 시약성분이 효과적으로 분리되어, 별다른 분리·정제 과정 없이 혈액·조직 샘플을 그대로 두고 증폭을 수행할 수 있음을 파악함
ㅇ 빠른 증폭 속도를 위해서는 copy number가 높거나 게놈 사이즈가 크지 않아야 함을 확인하였음
ㅇ 장치 소형화와 더불어 droplet의 100% 안정성을 확보하기 위하여 syringe needle 사용대신에 thermocouple hook를 이용하여 챔버형 PCR 장치 버전 3을 설계하고 제작하여 시운용한 결과 우수한 시험성적을 확보하였음
ㅇ 스마트폰용 형광현미경을 자체 개발하여 시험한 결과 증폭한 산물의 형광이미지 분석을 통해 PCR 증폭 유무를 판단할 수 있었으며, 챔버형 PCR 장치 버전 3에 장착하였음
ㅇ 가검물로부터 핵산을 자동 주줄하여 현장 사용성을 용이하기 위하여 paper microfluidics 방법을 고안하여 살모넬라와 인플루엔자 유전자 증폭에 적용실험을 실시한 결과 유효성을 확인하였음
ㅇ 직선형 PCR 장치 제작 및 개발하여 최대 소형화와 실용성을 겸비하도록 장치를 구현하였음
ㅇ 챔버형 및 직선형 PCR 장치에 대한 평가를 진행하여 표준시료에서 target 증폭 유무 및 유효성을 확인하였음


(출처 : 요약문 5P)

Abstract ▼

The first prototype ( “prototype 1” ) of portable PCR device was designed and fabricated using wire-guided droplet manipulation (WDM). The prototype 1 was designed by CAD (computer aided design) and the parts were fabricated using a CNC-based (computer numerical control) milling machine and a 3D pri

The first prototype ( “prototype 1” ) of portable PCR device was designed and fabricated using wire-guided droplet manipulation (WDM). The prototype 1 was designed by CAD (computer aided design) and the parts were fabricated using a CNC-based (computer numerical control) milling machine and a 3D printer. The droplet (containing the sample and PCR mixture) was made to move over three different heating chambers required for PCR thermocycling (denaturation, annealing and extension). In this manner, the time required for heating and cooling could be significantly reduced. The droplet was in direct contact with oil immersion and made to move within the chamber, maximizing the convective heat transfer (compared to the conductive heat transfer through a hewing block and a plastic tube in conventional thermocycling). The heating chambers were aligned in a circular format, and made as a cartridge that can be replaced after each assay. The temperature of each heating chamber (oil immersion) was maintained by a PCB (printed circuit board) trace heater and a temperature sensor installed within the chamber, and an appropriate PID control logic was incorporated into an Arduino microcontroller (written in C code). Preliminary experiments were performed in amplifying the synthetic oligonucleotide of GAPDH gene, and extremely fast PCR thermocycling were performed (~5 min for 30 cycles). Similar experiments were performed in amplifying the 16S rRNA gene from the 5.3 Mb genome of Klebsiella pneumoniae (~20 min for 30cycles). Amplifications were more successful when 10% whole blood is added to the PCR mixture, which i s generally considered as PCR inhibitor. The components from whole blood were believed to be self-assembled at the oil-water interface, effectively separating the sample matrix (PCR inhibitors) from the target gene and PCR mixture. This indicates that the proposed method can be used with minimal sample pre-processing (purification, extraction, etc.), which is appropriate for field applications. Reproducibility (low success rate) and detection limit (10⁵ copies per uL) remained as major problems.
A couple of issues were identified for prototype 1:1) heat loss from the droplet to air through a metal syringe needle; 2) air was deliberately trapped within the needle as a thermal insulator to resolve this issue; however, this air expanded or shrunk upon heating/cooling, making the droplet to be sucked in or fallen off from the needle; 3) the droplet was sucked in to the needle during the movement from one heating chamber to the other, to prevent the droplet from falling off from the needle. This procedure was repeated for 3 times per each cycle, i.e. 90 times total for 30 cycles. A small offset in each procedure made a huge impact after 30 cycles - either the droplet was completely sucked into the needle or fallen off from the needle. To resolve these issues, the syringe needle was replaced by a thermocouple hook. All previous issues were resolved, while it provided an additional advantage - we could measure the actual temperature within the droplet. The C code was rewritten to make the droplet to leave the heating chamber only after the desired temperature was achieved. The device was significantly simplified (shorter height and elimination of one motor). In addition, we developed a smartphone-based fluorescent microscope for real—time quantification of PCR products and attached it to the system. We referred this new device as “prototype 2.” Both reproducibility and detection limit were significantly improved with this prototype, in amplifying 16S rRNA gene from Escherichia coli. The temporary circuits on a bread board were integrated into a printed circuit board, 5 packs of rechargeable batteries were introduced, and the enclosure for the entire device was designed and fabricated. Additionally, an alternative concept ( “prototype B” ) was conceived and tested : 1) the heating chambers were laid out in a linear fashion; 2) the droplet was made to be “dragged” over the heater surface for faster heating. Several methods were tested, while the addition of Teflon tape on the heater surface provided the most satisfactory results. The contact angle of a droplet on Teflon tape was close to 180 degrees, since the contact angles of Teflon and oil were about the same, eliminating the potential possibility of surface contamination. “Thermocouple loop” was alsoused to ensures table droplet manipulation as well as measuring the actual temperature from the droplet. Again, reproducibility was significantly improved (100%) and the detection limit was as low as a few copies per uL.


(출처 : SUMMARY 15P)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• SUMMARY ... 15
• 목차 ... 17
• Ⅰ. 서언 ... 18
• Ⅱ.재료 및 방법 ... 21
• 1. Polymerase Chain Reaction ... 21
• 2. Droplet Thermocycling and Microfluidics ... 21
• 3. Thermocycling temperature profile measurement ... 23
• 4. Gel Electrophoresis ... 23
• 5. Interfacial Tension Measurements ... 23
• 6. Microfluidic Gene Extraction ... 24
• 7. Real-Time Quantification ... 24
• Ⅲ. 결과 및 고찰 ... 24
• 〈Year 1〉 ... 24
• 1. Convective heat transfer allows rapid heating and cooling of PCR reaction mixture ... 24
• 2. Blood increases droplet interfacial surface tension (IFT) ... 25
• 3. Whole blood increases the reaction efficiency in droplet thermocycling ... 27
• 4. Droplet thermocycling enables amplification directly from whole blood ... 27
• 5. Extremely fast amplification demonstrated for clean and dirty samples ... 28
• 〈Year 2〉 ... 29
• 1. Improvements on WDM (Wire-guided Droplet Manipulation) PCR Device ... 29
• 2. Update on Prototype B ... 37
• Ⅳ. 종합결과 ... 39
• Ⅴ. 적요 ... 41
• Ⅵ. 인용문헌 ... 43
• 끝페이지 ... 45