DNA Molecules Stretched and Embedded in Polymer Nanofibers by Near-Field Electrospinning Microfluidic Chip for Applications of Fiber- Fish Sequencing

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Project title

Code/計畫編號

NSC97-2218-E019-001-MY2

Translated Name/計畫中文名

近場電紡微流體晶片製作奈米DNA纖維於原位雜交定序之應用

Project Coordinator/計畫主持人

Shih-Hao Huang

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Mechanical and Mechatronic Engineering

Website

https://www.grb.gov.tw/search/planDetail?id=1622932

Year

2008

Start date/計畫起

01-01-2008

Expected Completion/計畫迄

01-07-2008

Bugetid/研究經費

643千元

ResearchField/研究領域

醫學技術

"DNA 纖維螢光原位雜交（Fiber-FISH, fiber based fluorescent in situ hybridization）技術，是近年來發展起來的一項直接在DNA 纖維上進行螢光原位雜交的技術，其原理是將具有螢光標記的DNA 探針直接原位雜交到DNA 纖維上，用來檢測DNA 序列在DNA 纖維上的空間定位。因此，本研究設計一近場電紡微流體晶片製作奈米DNA 纖維於原位雜交定序之應用，藉由近場電紡技術(near-filed electrospinning process)，將單一DNA 分子置入聚合物奈米纖維(nanofibers)內，產生直徑100 至350 nm 的奈米纖維。利用近場電紡噴流中的展伸力可使DNA 分子幾乎完全伸展開來且彼此分離，再者，完全展伸的DNA 分子嵌入在聚合物奈米纖維中，可永久維持展伸的構形且侷限在奈米尺度的聚合物纖維空間內，可不受外界環境影響，再藉由電滲透流或是使用表面張力驅動流體方式帶動具有DNA 分子嵌入的奈米纖維，以便觀察特定DNA 分子探針的序列。不同於傳統電紡技術，採用固定或旋轉基板方式，本研究創新之處在於採用近場方式(探針和收集板距離<1 cm)、以及利用微流體方式收集與傳輸聚合物奈米纖維，使聚合物奈米纖維以10μm/s 的速度朝向光學偵測的區域流動，光學偵測將採用單一分子螢光偵測法的方式，利用共軛焦顯微鏡與單光子感光度的多組崩潰式光偵測器來進行螢光訊號的檢測，以線性方式讀取DNA 纖維上螢光標記的DNA 探針訊號，可快速地、準確地辨認 DNA 分子的序列、長度、與DNA 分子中特異序列的存在…等。另外，藉由表面修飾技術可利用流體自我組裝方式將具有DNA 分子嵌入的奈米纖維排列具有功能性的結構陣列，以利後續光學的偵測。利用近場電紡微流體晶片進行Fiber-FISH DNA 序列的定位，可在只需消耗極少量的檢體的情形下，達到即時、快速、自動化檢測的目的，對於人類及動物基因組的研究具有顯著的助益。" "Fiber-FISH is a technique recently developed to detect directly DNA sequences on extended DNA fibers by fluorescence in situ hybridization (FISH), the resolution and sensitivity of which are improved to 1-2 kb and 200 bp respectively. A rapid molecular mapping technology—Direct Linear Analysis (DLA)—on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags is a practicable method. In this study, we propose a near-field electrospinning microfluidic chip for applications of Fiber- Fish sequencing. Based on the near-field electrospinning technique, the proposed microfluidic chip can encapsulate single molecules of DNA inside polymer nanofibres by an electrospinning process. The DNA molecules were stretched to nearly their full length. The resulting nanofibers were 100 - 350 nm in diameter. The strong elongational forces present in electrospinning jets could serve to stretch DNA into their full length. The stretched DNA molecules encapsulated in a protective nanofiber can be subsequently collected, manipulated, and driven by electroosmotic flow or surface-tension flow to optically analyze the probe sequences. Instead of the conventional electrospinning technique using a conductively rotational plate for collection, we use the near-field electrospinning technique, i.e. the distance between collected plate and probe below 1 cm, and microfluidics to collect and transport nanofibers to the optical detection area at 10μm/s. A detection system capable of single-fluorophore sensitivity is used to detect at sequence-specific motif sites on the stretched DNA molecules. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. Besides, by using the techniques of surface-patterned modification and microfluidic self-alignment, we can align nanofibers into a functional structure for the subsequently optical detection. The proposed near-field electrospinning microfluidic chip for applications of Fiber- Fish sequencing is a quick, inexpensive, and straightforward method to obtain many stretched molecules allowing for single molecule analysis, which can be a useful tool in human genome mapping."

Keyword(s)

DNA 纖維螢光原位雜交
近場電紡
奈米纖維
Fiber-FISH
Near-field electrospinning
nanofiber

DSpace CRIS

DNA Molecules Stretched and Embedded in Polymer Nanofibers by Near-Field Electrospinning Microfluidic Chip for Applications of Fiber- Fish Sequencing

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