• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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• pp.88-89
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• 2013

A variety of influenza A viruses from animal hosts are continuously prevalent throughout the world which cause human epidemics resulting millions of human infections and enormous industrial and economic damages. Thus, early diagnosis of such pathogen is of paramount importance for biomedical examination and public healthcare screening. To approach this issue, here we propose a fully integrated Rotary genetic analysis system, called Rotary Genetic Analyzer, for on-site detection of influenza A viruses with high speed. The Rotary Genetic Analyzer is made up of four parts including a disposable microchip, a servo motor for precise and high rate spinning of the chip, thermal blocks for temperature control, and a miniaturized optical fluorescence detector as shown Fig. 1. A thermal block made from duralumin is integrated with a film heater at the bottom and a resistance temperature detector (RTD) in the middle. For the efficient performance of RT-PCR, three thermal blocks are placed on the Rotary stage and the temperature of each block is corresponded to the thermal cycling, namely $95^{\circ}C$ (denature), $58^{\circ}C$ (annealing), and $72^{\circ}C$ (extension). Rotary RT-PCR was performed to amplify the target gene which was monitored by an optical fluorescent detector above the extension block. A disposable microdevice (10 cm diameter) consists of a solid-phase extraction based sample pretreatment unit, bead chamber, and 4 ${\mu}L$ of the PCR chamber as shown Fig. 2. The microchip is fabricated using a patterned polycarbonate (PC) sheet with 1 mm thickness and a PC film with 130 ${\mu}m$ thickness, which layers are thermally bonded at $138^{\circ}C$ using acetone vapour. Silicatreated microglass beads with 150~212 ${\mu}L$ diameter are introduced into the sample pretreatment chambers and held in place by weir structure for construction of solid-phase extraction system. Fig. 3 shows strobed images of sequential loading of three samples. Three samples were loaded into the reservoir simultaneously (Fig. 3A), then the influenza A H3N2 viral RNA sample was loaded at 5000 RPM for 10 sec (Fig. 3B). Washing buffer was followed at 5000 RPM for 5 min (Fig. 3C), and angular frequency was decreased to 100 RPM for siphon priming of PCR cocktail to the channel as shown in Figure 3D. Finally the PCR cocktail was loaded to the bead chamber at 2000 RPM for 10 sec, and then RPM was increased up to 5000 RPM for 1 min to obtain the as much as PCR cocktail containing the RNA template (Fig. 3E). In this system, the wastes from RNA samples and washing buffer were transported to the waste chamber, which is fully filled to the chamber with precise optimization. Then, the PCR cocktail was able to transport to the PCR chamber. Fig. 3F shows the final image of the sample pretreatment. PCR cocktail containing RNA template is successfully isolated from waste. To detect the influenza A H3N2 virus, the purified RNA with PCR cocktail in the PCR chamber was amplified by using performed the RNA capture on the proposed microdevice. The fluorescence images were described in Figure 4A at the 0, 40 cycles. The fluorescence signal (40 cycle) was drastically increased confirming the influenza A H3N2 virus. The real-time profiles were successfully obtained using the optical fluorescence detector as shown in Figure 4B. The Rotary PCR and off-chip PCR were compared with same amount of influenza A H3N2 virus. The Ct value of Rotary PCR was smaller than the off-chip PCR without contamination. The whole process of the sample pretreatment and RT-PCR could be accomplished in 30 min on the fully integrated Rotary Genetic Analyzer system. We have demonstrated a fully integrated and portable Rotary Genetic Analyzer for detection of the gene expression of influenza A virus, which has 'Sample-in-answer-out' capability including sample pretreatment, rotary amplification, and optical detection. Target gene amplification was real-time monitored using the integrated Rotary Genetic Analyzer system.

• 생태와환경
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• 제48권3호
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• pp.147-152
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• 2015

본 연구는 Kjeldahl 증류법을 이용하여 암모니아성 질소 및 질산성 질소의 안정동위원소 분석법을 연구하였으며, 건조방법 및 시료 농도 범위에 따른 분석값의 변화에 대하여 고찰하였다. 표준시료를 다양한 농도 범위 (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, $10mgL^{-1}$)로 조제하여 질산성 및 암모니아성 질소 안정동위원소비 ($^{15}NH_4-N$, $^{15}NO_3-N$)를 분석한 결과, $^{15}NH_4-N$는 $0.1{\sim}10mgL^{-1}$의 농도 범위에서 측정 가능하였으며 (${\pm}0.2$‰)$^{15}NO_3-N$는 $0.4{\sim}10mgL^{-1}$의 농도 범위에서 측정 가능하였다 (${\pm}0.3$‰). Kjedahl 증류법으로 얻어진 시료를 건조할 경우 오븐건조는 질소 안정동위원소비가 2.2‰의 큰 변화를 보이지만, 동결건조는 0.5‰의 작은 차이를 보이므로 동결건조방법이 적합하였다. 실증연구 일환으로 한강 수계 중권역의 한 지천에서 암모니아성 질소 ($NH_4-N$) 및 질산성 질소 ($NO_3-N$)의 안정동위원소비를 이용하여 질산염의 기원을 추적해 보았다. 지천이 흘러가는 방향을 중심으로 상류, 하수처리 방류장, 하류로 구분하고 각각의 $^{15}NH_4-N$, $^{15}NO_3-N$ 안정동위원소비를 분석하였다. 상류에서 질산염의 $^{15}NO_3-N$, $^{15}NH_4-N$ 값이 가볍게 나타나지만 (2‰, 8‰), 특성이 다른 질소화합물의 방류수 (23‰, 14‰)가 유입되면서 하류 (21‰, 11‰)에 영향을 주는 것으로 여겨진다. 본 연구를 통하여 수행된 $^{15}NH_4-N$, $^{15}NO_3-N$ 안정동위원소비 분석법은 수생태계로 유입되는 다양한 질소 기원을 파악하여 효율적인 수질 관리를 위한 중요 정보를 제공할 수 있을 것으로 사료된다. 다만 이와 같은 기법을 적용하기 위해서는 추후 유역 오염원의 대표값 (end member)의 조사를 통하여 지속적인 자료구축이 이루어져야 할 것이다.