微陣列生物晶片原理及應用. Outline. Principle of microarray chips Types of microarray chips Microarray technologies Applications of microarray chips

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1 微陣列生物晶片原理及應用 Outline Principle of microarray chips Types of microarray chips Microarray technologies Applications of microarray chips 林世章國家同步輻射研究中心 2004/4/16

2 Principle of microarray chips A microarray chip is an array of tens of thousands of biomolecule probes (DNA, antibodies, antigens etc.) in an area of several square centimeters on glasses, polymers or silicon wafers. Biological targets are tagged with a fluorescent dye and incubated with the array. Targets will bind or hybridize to their complementary to probes. Interrogation of the array with a fluorescent scanning device reveals the probe and target that have a complementary match (DNA DNA, Ab Ag etc.).

3 DNA(deoxyribonucleic acid): 由大量的去氧核糖核甘酸所組成的長鏈 去氧核糖核甘酸之構造 : 鹽基 (base): 鹽基順序帶有遺傳訊息 A: 腺嘌呤 G: 鳥糞嘌呤 T: 胸腺嘧啶 C: 胞嘧啶 磷酸鹽基和去氧核糖 : 扮演結構的角色 DNA 為雙螺旋立體構造 鹽基成對 :A=T, G C 雙鏈間以氫鍵結合 雙鏈互補 DNA ( 去氧核糖核酸 )

4 RNA ( 核糖核酸 ) RNA = ribonucleic acid 由大量的核糖核甘酸所組成的長鏈 核糖核甘酸之構造 : 鹽基 (base): 鹽基順序帶有遺傳訊息 A: 腺嘌呤 G: 鳥糞嘌呤 U: 尿嘧啶 C: 胞嘧啶 磷酸鹽基和核糖 : 扮演結構的角色

5 Gene chip: DNA hybridization Fluorescence or color Probe Target

6 DNA Microarray Image

7 Protein chip: antigen-antibody interaction Measurement of Antibody Fluorescenc e or color Chip Chip Chip Coat the surface with antigens Measurement of Antigen Add primary antibodies to be measured Add fluorescent dye-labelled antibodies against primary antibodies Fluorescenc e or color Chip Chip Chip Coat the surface with antibodies Add antigens to be measured Add fluorescent dye-labelled antibodies against a different determinant

8 蛋白質微陣列實例 ( 清大工科所微系統實驗室 )

9 Advantages of microarray chips High-throughput analysis Miniaturization Small volumes of samples & reagents Low cost Disposable or reusable

10 Types of microarray chips Gene chip Protein chip Tissue microarray chip Microfluidic array chip

11 Microarry Technologies 1. Photo-based technologies Photolithography Light-directed synthesis Virtual mask photolithography 2. Noncontact dispensing Piezoelectric ink-jet printing 3. Contact dispensing Pin printing Solid pin Quill and split pin Capillary array printer Micro contact printing Elastic stamp Back-filling stamp Dip-pen nanolithgraphy

12 Photolithography Substrate Substrate Substrate with photoresist Patterned photoresist Substrate Substrate Substate Photoresist Adhesion promoting silane adsorbed to substrate Photoresist removed and adhesion resistant silane adsorbed to substrate Conventional photoresist technology used for metal patterning for microcircuits is readily adapted to biological molecule patterning. Patterning is done using chemical linkers with different pendant groups to create a heterogeneous monolayer. Silane coupling agents have been the choice of linkers. The left figure shows how silanes are used in a positive photoresist scenario. The substrate is spun cast with photoresist, covered with a mask, and exposed to ultraviolet (UV) irradiation. UV light decomposes the photoresist. Allowing it to be dissolved away, exposing defined regions of the substrate. An adhesion promoting silane, usually amino terminated, is bound within these exposed regions, the slide is then sonicated with acetone to remove the remaining photoresist thus exposing the rest of the substrate. The slide is incubated with a hydrophobic or adhesion resistant silane, typically a methyl or alkyl terminated silane, resulting in a mixed monolayer interface. The sequence with which silanes are immobilized also can be reversed. This photoresist technology has been used to pattern protein and control cellular growth. Biomaterials, 1998, 19:

13 Affymetrix Light-Directed synthesis 25 bases need 100 masks.

14 Virtual Mask Photolithography - 1 J. Micromech. Microeng., 2003, 13: Virtual mask photolithography uses a micromirror array (MMA) as a virtual mask to pattern surfaces. The excitation light was switched on or off using the MMA, and the light pattern was transferred using the pattern of switched-on mirrors. The nitroveratryloxycarbonyl (NVOC) group was utilized as a photolabile protecting group for surface patterning, so that biomolecules could be immobilized on a patterned substrate. When illuminated by UV light, the photolabile protecting group was removed by a chemical reaction, and non-illuminated photolabile protecting groups protected the chip surface.

15 Virtual Mask Photolithography - 2 Selective photo deprotection of the NVOC-protected surface and its application to selective immobilization of FITC-conjugated streptavidin 16 X 16 array Mirror size: 50 X 50 um Fluorescent spot: 45 um

16 Perkin-Elmer Ink-Jet Printing - 1 Piezoelectric Crystal Orifice Glass Capillary Piezoelectric printing technology uses a piezoelectric crystal (e.g. ceramic material) closely apposed to the fluid reservoir. One configure places the piezoelectric crystal in contact with a glass capillary, which holds the sample fluid. The sample is drawn up into the reservoir and the crystal is biased with a voltage, which causes the crystal to deform, squeeze the capillary, and eject a small amount of fluid from the tip. The fast response time of the crystal permits fast dispensing rates, on the order of several thousand drops per second. Furthermore, the small deflection of the crystal results in drop volumes on the order of hundreds of picoliters. The main difficulties in implementing piezoelectric dispensing include air bubbles, which reduces the reliability of the system, relatively large sample volume, and problems with sample changing.

17 Perkin-Elmer Ink-Jet Printing - 2

18 Solid Pin The pin tools are dipped into the sample solution, resulting in the transfer of a small volume of fluid onto the tip of the pins. Touching the pins onto the the substrate surface leaves a spot, the diameter of which is determined by the surface energies of the pin, fluid, and substrate. The typical spot volume is in the high picoliter to low nanoliter range. A modification of solid pins involves the use of solid pins with concave bottoms, which have shown to print more efficiently than flat pins in certain cases. Because the loading volume of both types of solid pins is relatively small, only one or a few microarrays can be printed with a single sample loading, making the overall printing process rather low. Solid pins are best used to make low-density arrays because the printing process in inherently slow. Flat tip Concave tip

19 Quill and Split Pin Quill-based arrayers withdraw a small volume of fluid within the pins from a microwell plate by capillary action. A robot moves the quills to the spotting locations, where only a small fraction from that larger fluid volume is deposited. Quills hold larger sample volumes than solid pins and therefore allow more than one array to be printed from a single sample loading. The forces acting on the fluid held in the tweezer must be overcome for the fluid to be released. Accelerating and then decelerating by impacting the quill on the substrate by tapping achieves this. When the tip of the quill hits the substrate, the meniscus is extended beyond the tip and transferred onto the substrate. Because tapping on the surface is required for fluid transfer, the choice of material is critical to ensure a practical life of small tipped quills. The opening at the gap of the quill is be between 10 and 100 µm. There are some shortcomings to the quill design. Clogging of the gap from dust, particulates, evaporated buffer crystals, or other contaminants can be a problem. The tapping on the surface tends to cause the deformation of the tip and therefore replacement cost is an important factor in the use of the quill technology.

20 Genometrix Capillary Array Printer Each capillary originates from a well plate contained within a pressurized manifold. Precise fluid delivery to the glass surface is initiated and maintained through the capillaries by a computer-controlled pressure regulator. Once the capillaries are primed with sample solution under nitrogen pressure, thousands of identical arrays can be rapidly and sequentially produced using this continuous contact process. Depending on the type of print head mounted to the robot, this instrument is capable of printing 16 X 16 element array every second (200 pl per spot). BioTechniques,1999, 27:

21 Elastic Stamp - 1 PDMS PDMS exposed to solution of HS(CH 2 ) 15 CH 3 Au (200 nm) + Ti (5 nm) Alkanethiol SAM (~1 to 2nm) Stamping transfer thiol Si Biomaterials, 1999, 20: Langmuir, 2002, 18:

22 Elastic Stamp - 2 ( 清大工科所微系統實驗室 ) 微印章 蛋白質印台

23 Back-Filling Stamp ( 清大工科所微系統實驗室 ) Pipette 填充式壓印晶片 (1mm x 1mm) Reservoir Biofluid Embedded Micro Channel Array Micro Stamp Array Stamped Array 生物檢測晶片

24 Dip-Pen Nanolithography - 1 分子傳送 原子力顯微鏡探針 掃描方向 水膜 Dip-pen technology: ink on a sharp object is transported to a paper substrate via capillary forces. DPN uses an atomic force microscope (AFM) tip as a nib, a solid-state substrate (in this case, Au) as paper, and molecules with a chemical affinity for the solid-state substrate as ink. Capillary transport of molecules from the AFM tip to the solid substrate is used in DPN to directly write patterns consisting of a relatively small collection of molecules in submicrometer dimensions. 金底材 Science, 1999, 283: 蘸水筆奈米微影法是利用原子力顯微鏡的探針作為奈米筆在金膜上繪出蛋白質奈米陣列 其主要原理在於 : 原子力顯微鏡的探針在掃描樣品時, 探針與樣品之間的極微小距離會形成毛細管, 當空氣中的水氣凝結在探針表面上時, 在探針與樣品表面之間會有液面連接, 若探針表面已預先塗佈特定的物質, 這些物質分子會被溶出, 沿著探針流到被掃描的樣品表面上

25 Dip-Pen Nanolithography - 2 Science, 2002, 295:

26 Gene Chip 基因晶片是指以寡核甘酸或互補核甘酸為探針, 以陣列型態排列在晶片上, 用以和具有互補序列的核酸產生雜交反應 Oligonucleotide chip ( 寡核甘酸 ) 通常長度為二十至二十五個核甘酸 cdna chip (complementary DNA, 互補 DNA, 反向轉錄 DNA) 以 mrna 為模板, 利用反向轉錄酵素於體外合成的單股 DNA mrna 反轉錄 DNA

27 基因表達 ( 蛋白質合成 ) Gene Exon Intron Exon Transcription Pre-mRNA Splicing Mature mrna Translation Protein Transcription ( 轉錄 ) : 合成一條與 DNA 鏈序列互補的 RNA 單鏈 (A U T A G C C G) Translation ( 轉譯 ) : 以 RNA 為模板, 將 RNA 序列翻譯成胺基酸序列, 合成蛋白質

28 Protein Chip Antibody chip Antigen chip Enzyme chip Oligopeptide chip

29 Applications of Gene and Protein Chips 1. Gene Sequencing ( 基因定序 ) 2. Gene Expression ( 基因表現 ) 3. SNP (single nucleotide polymorphism, 單一核酸多型性 ) 4. Immunoassay ( 免疫檢測 ) 5. Drug screening ( 藥物篩選 ) 6. Disease diagnosis 7. Toxicology analysis ( 毒理分析 )

30 SNP (Single Nucleotide Polymorphism) Misspelling of word: BATCH vs. BITCH ANGER vs. ANGEL

31 Tissue Microarray Chip - 1 Human Molecular Genetics, 2001, 10: A tissue microarray (TMA) is an array with hundreds of different normal/malignant tissue spots on one slide. The construction principle is to drill a cylindrical hole in a sample-free parafin block named the recipient block, then to sample a similar core into the sample block, named the donor block, and to transfer this core into the sample-free block, into the hole made earlier.

32 Tissue Microarray Chip - 2 The array construction involves making a hole in the recipient TMA block, acquiring a cylindrical core smaple from the donor tissue block and depositing this core into the TMA block. This process is repeated with a precision instrument to array hundreds of tissue specimens. Biopsies from different tumors are embedded and arrayed in paraffin to create a TMA. The TMA block is sectioned to obtain thin sections with each of the different tumors then being represented as dots on microscope slides. Each TMA block can be sliced into hundreds of consecutive sections of 3-4 um each. All the resulting TMA slides have the same tissues in the same coordinate positions. The individual slides can be used for a variety of molecular analyses either at the DNA, RNA or protein level in the same set of specimens.

33 Tissue Microarray Chip - 3

34 Key issues of Tissue Microarry 1. TMAs offer high-throughput in situ evaluation of candidate genes or proteins in histopathologically well-defined tissue samples. 2. TMAs consist of up to 1000 minute cylindrical tissue samples (usually 0.6 mm in diameter) that are assembled in a regular-sized routine histology paraffin block. Sections are cut from TMA blocks using standard microtomes. 3. TMAs sections allow the simultaneously analysis of up to 1000 tissue samples in a single experiment. They are, therefore, cost-effective and offer an unprecedented degree of standardization as all tissue samples are subjected to exactly the same experimental conditions and batches of reagents. 4. Despite the small size of arrayed samples (diameter 0.6 mm), TMA studies provide highly representive information.

35 What is a microfluidic chip? Microfluidic array chip Miniaturized labs consisting of microchannels and microreactors can manipulate and control small amounts of liquids and allow small-scale biological/medical assays and experiments. Microfluidic chips need only tiny quantities of samples and reagents, promising to reduce time and cost related to traditional macro-scale research methods.

36 Micromosaic Immunoassay Anal. Chem., 2001, 73: 8-12

37 Nanoscale Protein Crystallization Free Interface Diffusion Sam ple Reagent Sam ple / reagent diffusion Crystal PNAS., 2002, 99: 在逐步解讀出各類生命體的基因資訊之後, 生醫研究的下一個挑戰便是要從整體宏觀的角度, 去探討依照基因編碼所建構的蛋白質其結構與功能之間的關係 立體結構的資訊可以提供很多一般定序分析所無法獲得的珍貴信息, 能夠被用來有效地推測蛋白質的功能

38 References 1. Microarray Biochip Technology, edited by Mark Schena, TeleChem International, Inc., Biochip Technology, edited by Jing Cheng and Larry J. Kricka, Taylor & Francis Books, Inc., 2003