微流道晶片在數十年來有著廣泛的應用及發展，其中分子生物學中極重要的聚合酶連鎖反應(polymerase chain reaction ,PCR)技術也被大量應用在微流道晶片中。在此，我們製作一可快速製造106個獨立液體之微流道晶片。現今市面上偵測及分析環境或者食品上的細菌是藉由培養計數法，但是此方法需要有合適的培養環境、精確的培養環境控制且也需要2至3天才可以得到結果。PCR及定量PCR (quantitative polymerase chain reaction, qPCR)亦被常用來使用在偵測細菌上，但需要CT值或者標準曲線才可作相對定量。設計Digital PCR藉由將PCR反應試劑分為許多小等份給予了絕對濃度定量並提高偵測靈敏度及改善訊號雜訊比(S/N ratio)。
此論文中，我們製作一可使用digital PCR的高動態偵測範圍的微流體晶片，此晶片可以快速的將樣品分為1,147,040個等份，每一等份為785 fL。此小等份的液體含有目標DNA及足夠的PCR反應物可提高S/N ratio。本論文中，我們利用低濃度的真實細菌樣品去展現此晶片的可行性。但，我們發現在使用10：1 (w/w)的PDMS比例時，經由PCR後孔洞會塌陷或變形；而當我們使用不同的比例混合時，例如：10：2 (w/w)、10：3 (w/w)時，孔洞塌陷或變形的情況會改善。基於此，我們設計的digital PCR晶片不僅可以快速的將液體等分並提高S/N ratio及靈敏度，亦藉由簡單的操作可增強低濃度樣品的偵測準確度。

Here, we describe a digital PCR (polymerase chain reaction) microfluidic chip capable of generating more than 106 droplets within a minute for the quantification of bacteria. Conventionally, bacteria detection and analysis for monitoring the environmental pollution or food contamination are conducted by using the bacteria plate count method, which requires a suitable culture medium, a precise control of culture environment, and takes 2-3 days for collecting the result. PCR and qPCR (quantitative polymerase chain reaction) have also been used for bacteria detection. However, their fluorescence-based readouts need CT values or a standard curve to provide a relative quantification. Therefore, digital PCR (dPCR) is often the method of choice for analyzing low-concentration or complex samples. dPCR provides the absolute DNA concentration with a high sensitivity and an improved signal-to-noise (S/N) ratio by partitioning the PCR reaction into many individual reactions, and its dynamic range is dependent on the number of partitions.
In this study, we have designed and fabricated microfluidic chip to be used in digital PCR that could rapidly separate the sample solution into more than a million (~1,147,040) droplets, each of which is 785- fL in volume. The small volume of each reaction droplet increases the S/N ratio in droplets with positive target DNA templates and allows for efficient PCR using crude bacteria lysate without nucleic acid purification, while the high number of droplets provides a dynamic range of more than six orders of magnitude. We demonstrated the applicability of this chip to quantify the concentration of Escherichia coli (E. Coli). However, we found that many microwells were collapsed or deformed after PCR cycles when the chip was made of polydimethylsiloxane (PDMS) using a typical 10:1 (w/w) monomer to cross-linker ratio. When we used PDMS of different mixing ratios, such as 10:2 (w/w) or 10:3 (w/w), this problem is fixed. Based on these results, we describe a digital PCR microfluidic chip that can rapidly separate the small volume of sample and increase the S/N ratio. Furthermore, digital PCR enhances the accuracy for bacterial detection with simple operation and provides a dynamic range of more than six orders of magnitude.

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