TWI554612B - Biologic sensing platforms and methods of fabricating the same - Google Patents
Biologic sensing platforms and methods of fabricating the same Download PDFInfo
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本發明係有關一種生物檢測平台，且特別係有關一種可保護脂質雙層膜之生物檢測平台及其製造方法。 The present invention relates to a biological detection platform, and in particular to a biodetection platform capable of protecting a lipid bilayer membrane and a method of manufacturing the same.
於1972年，生物學家辛格(Singer)與尼克森(Nicolson)提出流體鑲嵌模型(Fluid Mosaic Model)，說明生物之細胞膜係由脂質雙層(lipid bilayer)所構成之生物膜。脂質雙層係由兩層具有親水端及親油端之磷脂質分子(phospholipid)，以兩個親油端相向而兩個親水端朝外之排列方式所構成。細胞膜相關蛋白質、脂質及醣類唯有鑲嵌於脂質雙層中才能維持其原生結構與功能。此外，脂質雙層膜表面復具有二維流動性，可使細胞膜相關物質在脂質雙層膜中流動或聚合，因此，脂質雙層被廣泛地運用於生物感測技術，用以再現細胞膜相關生物分子之功能與其它生物分子或藥物間之交互作用。 In 1972, the biologists Singer and Nicholson proposed the Fluid Mosaic Model, which showed that the cell membrane of the organism is a biofilm composed of lipid bilayers. The lipid bilayer consists of two layers of phospholipids having a hydrophilic end and a lipophilic end, which are formed by two oleophilic ends facing each other and two hydrophilic ends facing outward. Cell membrane-associated proteins, lipids, and carbohydrates can only be maintained in their native structure and function by being embedded in a lipid bilayer. In addition, the surface of the lipid bilayer membrane has two-dimensional fluidity, which allows the cell membrane-related substances to flow or polymerize in the lipid bilayer membrane. Therefore, the lipid bilayer is widely used in biosensing technology to reproduce cell membrane-associated organisms. The interaction between the function of a molecule and other biomolecules or drugs.
使用具有脂質雙層膜之生物檢測平台1進行生物檢測，於運送樣品或更換反應溶液時，生物檢測平台中之脂質雙層膜不可避免地會曝露於空氣中，而使脂質雙層膜之 結構及流動性受到破壞。如第1圖所示，當基底10表面上之脂質雙層膜11曝露於空氣時，空氣-水界面100之界面張力會沿著界面遷移方向(如箭頭所示)破壞脂質雙層膜11中親油端間之凡得瓦力，導致脂質雙層膜11自生物檢測平台1之基底10表面剝離。 Bioassay is performed using a bioassay platform 1 having a lipid bilayer membrane. When transporting a sample or changing a reaction solution, the lipid bilayer membrane in the bioassay platform is inevitably exposed to the air, and the lipid bilayer membrane is Structure and fluidity are undermined. As shown in Fig. 1, when the lipid bilayer membrane 11 on the surface of the substrate 10 is exposed to air, the interfacial tension of the air-water interface 100 destroys the lipid bilayer membrane 11 along the interface migration direction (as indicated by the arrow). The van der Waals force between the oleophilic ends causes the lipid bilayer membrane 11 to peel off the surface of the substrate 10 of the bioassay platform 1.
為防止生物檢測平台上之脂質雙層膜不被空氣-水界面破壞，部分已知技術使用化學修飾法修飾脂質雙層膜或基材之表面化學性質，以增加脂質雙層膜中脂質對脂質或脂質雙層膜對基材之作用力，來達到保護脂質雙層膜不被空氣-水破壞之目的；另有已知技術使用他種生物分子覆蓋於脂質雙層膜表面來增加脂質雙層膜之剛性，用以避免脂質雙層膜於通過被空氣-水脂界面時被界面張力自基材上剝離。 In order to prevent the lipid bilayer membrane on the bioassay platform from being destroyed by the air-water interface, some known techniques use chemical modification to modify the surface chemistry of the lipid bilayer membrane or substrate to increase lipid to lipid in the lipid bilayer membrane. Or the action of the lipid bilayer membrane on the substrate to achieve the purpose of protecting the lipid bilayer membrane from air-water destruction; another known technique uses other biomolecules to cover the surface of the lipid bilayer membrane to increase the lipid bilayer. The rigidity of the membrane is used to prevent the lipid bilayer membrane from being peeled off from the substrate by the interfacial tension when passing through the air-water lipid interface.
然而，上述之化學修飾法可能導致脂質雙層膜上之生物分子喪失部分二維流動性；上述使用他種生物分子覆蓋脂質雙層膜表面之方法所產生之空間障礙，使得待測分子無法接近脂質雙層膜和其上物質，進而影響生物檢測的可操作性及準確性。因此，如何解決上述種種問題，即為發展本發明之目的。 However, the above chemical modification method may cause partial loss of partial two-dimensional fluidity of the biomolecule on the lipid bilayer membrane; the above-mentioned spatial barrier caused by the method of covering the surface of the lipid bilayer membrane with other biomolecules makes the molecule to be tested inaccessible The lipid bilayer membrane and the substance thereon affect the operability and accuracy of the bioassay. Therefore, how to solve the above problems is to develop the object of the present invention.
鑑於習知技術之種種問題，本發明提供一種生物檢測平台，包含基底、圖案化結構以及脂質雙層膜。圖案化結構係具有相隔一間距設置於該基底表面之至少二阻障，各該阻障具有一高度且該高度與該間距之高寬比係1/2至 1/250。該脂質雙層膜形成於該等阻障間之該基底表面上。 In view of the various problems of the prior art, the present invention provides a biodetection platform comprising a substrate, a patterned structure, and a lipid bilayer membrane. The patterned structure has at least two barriers disposed at a distance from the surface of the substrate, each of the barriers having a height and an aspect ratio of the height to the pitch is 1/2 to 1/250. The lipid bilayer membrane is formed on the surface of the substrate between the barriers.
於一實施例，該基底之材料係矽或矽化物。 In one embodiment, the material of the substrate is tantalum or telluride.
於一實施例，該圖案化結構之材料係光阻、有機物或金屬。 In one embodiment, the material of the patterned structure is photoresist, organic or metal.
於一實施例，該高度與該間距之高寬比係1/2至1/50。 In one embodiment, the aspect ratio of the height to the pitch is 1/2 to 1/50.
於一實施例，該高度與該間距之高寬比係1/2至1/20。 In one embodiment, the aspect ratio of the height to the pitch is 1/2 to 1/20.
本發明復提供一種生物檢測平台之製造方法，包含下列步驟：提供基底；形成圖案化結構於該基底上，該圖案化結構係具有相隔一間距設置於該基底表面之至少二阻障，各該阻障具有一高度且該高度與該間距之高寬比係1/2至1/250；以及形成脂質雙層膜於該等阻障間之該基底表面上。 The invention provides a method for manufacturing a biological detection platform, comprising the steps of: providing a substrate; forming a patterned structure on the substrate, the patterned structure having at least two barriers disposed at a distance from the surface of the substrate, each of the The barrier has a height and the aspect ratio of the height to the pitch is 1/2 to 1/250; and a lipid bilayer film is formed on the surface of the substrate between the barriers.
於一實施例，形成該圖案化結構之步驟包含：以光阻或金屬材料形成阻層於該基底表面上；以及以微影製程定義該阻層形成該圖案化結構。 In one embodiment, the step of forming the patterned structure comprises: forming a resist layer on the surface of the substrate with a photoresist or a metal material; and defining the resist layer by a lithography process to form the patterned structure.
於一實施例，形成該等圖案化結構之步驟包含：以有機物形成阻層於該基底表面上；以及以酵素水解該阻層形成該圖案化結構。 In one embodiment, the step of forming the patterned structures comprises: forming a resist layer on the surface of the substrate with an organic substance; and hydrolyzing the resist layer with an enzyme to form the patterned structure.
於一實施例，該酵素係磷脂酶。 In one embodiment, the enzyme is a phospholipase.
於一實施例，於形成該等圖案化結構後，復包含：處理該圖案化結構之表面，以增加該等圖案化結構之親水性。 In one embodiment, after forming the patterned structures, the method further comprises: processing the surface of the patterned structure to increase the hydrophilicity of the patterned structures.
於一實施例，該表面處理之圖案化結構表面之水接觸角介於0至90度。 In one embodiment, the surface treated patterned structure surface has a water contact angle between 0 and 90 degrees.
於一實施例，以氬氣電漿處理該圖案化結構之表面。 In one embodiment, the surface of the patterned structure is treated with argon plasma.
因本發明設置圖案化結構於基底上，該圖案化結構可有效防止空氣-水界面破壞該脂質雙層膜，從而確保該生物檢測平台之檢測效能。 Because the present invention provides a patterned structure on the substrate, the patterned structure can effectively prevent the air-water interface from destroying the lipid bilayer film, thereby ensuring the detection performance of the bio-detection platform.
1,2‧‧‧生物檢測平台 1,2‧‧ Biological testing platform
10,20‧‧‧基底 10,20‧‧‧Base
11,22‧‧‧脂質雙層膜 11,22‧‧‧Lipid bilayer membrane
21‧‧‧圖案化結構 21‧‧‧ patterned structure
211‧‧‧阻障 211‧‧‧Resistance
100,200‧‧‧空氣-水界面 100,200‧‧‧Air-water interface
201‧‧‧水溶液(緩衝液) 201‧‧‧Aqueous solution (buffer)
h‧‧‧阻障之高度 h‧‧‧The height of the barrier
w‧‧‧阻障之間距 W‧‧‧distance between obstacles
s‧‧‧接觸界面 s‧‧‧Contact interface
θ 1‧‧‧高寬比(h/w)換算之夾角 θ 1‧‧‧ aspect ratio of h/w conversion
θ 2‧‧‧空氣-水界面與水平面之夾角 θ 2‧‧‧An angle between the air-water interface and the horizontal plane
θ 3‧‧‧接觸界面s與空氣-水界面移動方向之夾角 θ 3‧‧‧An angle between the contact interface s and the moving direction of the air-water interface
第1圖係空氣-水界面通過習知生物檢測平台之剖面示意圖；第2圖係本發明之生物檢測平台之剖面側視圖；以及第3圖係空氣-水界面通過本發明之生物檢測平台之剖面側視圖。 1 is a schematic cross-sectional view of an air-water interface through a conventional biological detection platform; FIG. 2 is a cross-sectional side view of the bio-detection platform of the present invention; and FIG. 3 is an air-water interface through the bio-detection platform of the present invention. Sectional view.
第4圖係空氣-水界面通過本發明之生物檢測平台之俯視圖。 Figure 4 is a top plan view of the air-water interface through the bioassay platform of the present invention.
以下係藉由特定的具體實施例說明本發明之實施方式，熟習此技藝之人士可由本說明書所揭示之內容瞭解本發明之其他優點與功效。本發明也可藉由其他不同的具體實施例加以施行或應用，本說明書中的各項細節亦可基於不同觀點與應用，在不悖離本發明之精神下進行各種修飾與變更。 The embodiments of the present invention are described by way of specific examples, and those skilled in the art can understand the advantages and advantages of the present invention as disclosed in the present disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes may be made without departing from the spirit and scope of the invention.
除非文中另有說明，說明書及所附申請專利範圍中所使用之單數形式「一」及「該」包括複數個體；術語「或」包括「及/或」之含義。 The singular forms "a", "the", and "the"
本發明提供生物檢測平台及其製造方法。本發明之第一實施例係使用酵素水解脂質雙層形成網絡式阻障於基底上，以達到保護脂質雙層膜不被空氣-水界面破壞之目的。 於本發明中，可用於水解脂質雙層形成網絡式阻障之酵素包含各式磷脂酶，例如：鞘磷脂酶、磷脂酶A、磷脂酶B、磷脂酶C、磷脂酶D等，但不以此為限。 The invention provides a biological detection platform and a method of manufacturing the same. The first embodiment of the present invention uses an enzyme hydrolyzed lipid bilayer to form a network barrier on the substrate to achieve the purpose of protecting the lipid bilayer membrane from being destroyed by the air-water interface. In the present invention, the enzyme which can be used to hydrolyze the lipid bilayer to form a network barrier comprises various phospholipases, such as: sphingomyelinase, phospholipase A, phospholipase B, phospholipase C, phospholipase D, etc., but not This is limited.
於本實施例，用於製造脂質雙層之材料係購自Avanti Polar Lipids公司之商品：1,2二油醯基卵磷脂(1,2-dioleoyl-sn-glycero-3-phosphocholine，DOPC)、1,2二棕櫚酸磷脂醯基膽鹼(1,2-dipalmitoyl-sn-glycero-3-phosphocholine，DPPC)及膽固醇(cholesterol，Chol)。磷脂膜染料係購自Life Technologies公司之商品：德克薩斯-紅(Texas-Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt，Texas-Red DHPE)。酵素係購自Sigma-Aldrich公司之商品：磷脂酶A2(phospholipase A2，PLA2)。 In this example, the material used to make the lipid bilayer was purchased from Avanti Polar Lipids: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (cholesterol, Chol). The phospholipid membrane dye was purchased from Life Technologies, Inc., Texas-Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt, Texas-Red DHPE). Enzymes were purchased from Sigma-Aldrich Company Product: phospholipase A 2 (phospholipase A 2, PLA 2).
利用酵素PLA2形成網絡式阻障之方法包含下列步驟：以胞沉積法(vesicle deposition method)將DOPC、DPPC、Chol(40：40：20)材料形成大單層微脂胞(Large unilamellar vesicles，LUVs)；以氬氣電漿清潔玻璃基底10分鐘後，迅速將聚二甲基矽氧烷(polydimethylsiloxane，PDMS)材質之片密封於該玻璃基底上，以形成溶液之微流道(microchannel)；將溶於緩衝液之LUVs加入該微流道，經10分鐘之靜置(incubation)獲得微脂胞沉積(vesicle deposition)；以去離子水洗淨多餘之微脂胞沉積後，以Ca-HEPES緩衝溶液(10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEPES,123mM NaCl,2.5nM CaCl2,PH=7.4)清洗後形成脂質雙層；將3.4微克/毫升濃度之PLA2溶液加入SLB中靜置30分鐘，PLA2水解脂質雙層產生磷膽鹼及脂肪酸(lysophosphocholine and fatty acid)形成網絡式阻障；將含有該網絡式阻障之溶液浸入填充有0.1M十二烷基磺酸鈉(Sodium dodecyl sulfonate，SDS)溶液之容器，用以洗去剩餘之PLA2及脂質雙層而保留該網絡式阻障；以超音波震盪30分鐘去除SDS後，以水深入清洗2分鐘；將樣品反覆幾次通過空氣-水界面，以確保SDS及脂質雙層完全被去除而僅保留該網絡式阻障於該玻璃基底上。 The method for forming a network barrier using the enzyme PLA 2 comprises the steps of forming a DUC, DPPC, Chol (40:40:20) material into a large unilamellar vesicles by a vesicle deposition method. LUVs); after cleaning the glass substrate with argon plasma for 10 minutes, a sheet of polydimethylsiloxane (PDMS) material was quickly sealed on the glass substrate to form a microchannel of the solution; The LUVs dissolved in the buffer were added to the microchannel, and vesicle deposition was obtained after 10 minutes of incubation; after washing the excess microlipid cells with deionized water, Ca-HEPES was used. A buffer solution (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEPES, 123 mM NaCl, 2.5 nM CaCl 2 , pH=7.4) was washed to form a lipid bilayer; a 3.4 μg/ml PLA 2 solution was added to SLB. After standing for 30 minutes, PLA 2 hydrolyzed the lipid bilayer to produce phosphocholine and fatty acid (lysophosphocholine and fatty acid) to form a network barrier; the solution containing the network barrier was immersed in 0.1M dodecylsulfonic acid. Sodium (Sodium dodecyl sulf Onate, SDS) solution container for washing away the remaining PLA 2 and lipid bilayer and retaining the network barrier; after removing SDS by ultrasonic shock for 30 minutes, it is thoroughly washed with water for 2 minutes; Through the air-water interface, it is ensured that the SDS and lipid bilayer are completely removed and only the network barrier is retained on the glass substrate.
製造生物檢測平台包含下列步驟：將含有0.5莫耳%Texas-Red DHPE之DOPC單層微脂胞加入該微流道，靜置10分鐘之後，單層微脂胞可破裂並形成脂質雙層膜於未被該網絡式阻障覆蓋之該玻璃基底表面上；以去離子水洗淨多餘之單層微脂胞後，即完成具有脂質雙層之生物檢測平台。 The manufacturing bioassay platform comprises the steps of: adding DOPC monolayer microlipid containing 0.5 mol% of Texas-Red DHPE to the microchannel, and after standing for 10 minutes, the monolayer of microlipids can be broken and form a lipid bilayer membrane. On the surface of the glass substrate not covered by the network barrier; after washing the excess monolayer of microlipid cells with deionized water, a bioassay platform having a lipid bilayer is completed.
測試空氣-水界面通過本實施例之生物檢測平台包含下列步驟：將該生物檢測平台曝露於空氣中約1小時，使空氣-水界面產生於該生物檢測平台表面；當空氣-水界面完全通過脂質雙層膜表面之時點，定義為乾狀態(dry state)之開始；乾狀態開始後，於特定數個時點將水分加入該生物檢測平台進行回濕(rehydrate)；回濕後，以螢光顯微攝影(fluorescence microscopy)及光漂白螢光回復測試法 (fluorescence recovery after photobleaching，FRAP)分別檢測該支撐式脂質雙層膜之完整度及流動性。 Testing the air-water interface The bioassay platform of the present embodiment comprises the steps of exposing the bio-detection platform to air for about one hour to create an air-water interface on the surface of the bio-detection platform; when the air-water interface is completely passed The time point of the surface of the lipid bilayer membrane is defined as the beginning of the dry state; after the dry state begins, moisture is added to the bioassay platform for rehydration at a certain number of time points; Photomicrography (fluorescence microscopy) and photobleaching fluorescence recovery test (fluorescence recovery after photobleaching, FRAP) was used to detect the integrity and fluidity of the supported lipid bilayer membrane.
通過原子力顯微鏡(Atomic force microscopy)觀察該生物檢測平台，該網絡式阻障具有次微米至微米尺寸之結晶狀結構及約12至20奈米之高度，各該阻障且彼此相隔一間距形成於該玻璃基底表面。脂質雙層膜選擇性形成於外露之該玻璃基底表面，各該阻障之高度數倍於該脂質雙層膜之高度。 The biodetection platform is observed by an atomic force microscopy having a submicron to micron-sized crystalline structure and a height of about 12 to 20 nanometers, each of which is formed at a distance from each other. The surface of the glass substrate. The lipid bilayer membrane is selectively formed on the exposed surface of the glass substrate, and the height of each barrier is several times higher than the height of the lipid bilayer membrane.
分析螢光顯微影像及FRAP之檢視結果，於乾狀態開始後，相鄰之阻障間仍可保留水分；空氣-水界面通過該生物檢測平台後，各該阻障之高度可有效阻斷空氣-水界面直接接觸該脂質雙層膜與該基底之界面，使脂質雙層膜能附著於該玻璃基底表面；該網絡式阻障間所保留之水分可防止空氣-水界面之界面張力將該脂質雙層膜自該玻璃基底表面上剝離，從而使該脂質雙層膜仍完整保持於該玻璃基底上。 Analysis of the fluorescence microscopic image and the FRAP inspection results, after the dry state begins, the water can remain in the adjacent barriers; after the air-water interface passes through the biological detection platform, the height of each barrier can be effectively blocked. The air-water interface directly contacts the interface between the lipid bilayer membrane and the substrate, so that the lipid bilayer membrane can adhere to the surface of the glass substrate; the moisture retained by the network barrier prevents the interfacial tension at the air-water interface from being The lipid bilayer membrane is peeled off from the surface of the glass substrate such that the lipid bilayer membrane remains intact on the glass substrate.
由上述之第一實施例可知，形成於基底上之物理性阻障不會影響脂質雙層膜之原有特性，能有效保護脂質雙層膜，且確保生物檢測平台之效能。據此，本發明進一步以各種圖案化結構形成物理性阻障於基底上，用以製造具有脂質雙層膜之生物檢測平台。 It can be seen from the above first embodiment that the physical barrier formed on the substrate does not affect the original characteristics of the lipid bilayer membrane, can effectively protect the lipid bilayer membrane, and ensures the performance of the bioassay platform. Accordingly, the present invention further forms a physical barrier to the substrate in various patterned structures for fabricating a biodetection platform having a lipid bilayer membrane.
於本發明之第二實施例，用於製造脂質雙層之材料係購自Avanti Polar Lipids公司之商品：1,2二油醯基卵磷脂DOPC。磷脂膜染料購自Life Technologies公司之商品：德 克薩斯-紅Texas-Red、生物素-X(N((6(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-gly cero-3-phosphoethanolamine,triethylammonium salt，Biotin-X DHPE)及Alexa flour 488標記鏈黴親合素(Alexa flour 488 conjugated streptavidin)。用於製造圖案化結構之阻擋材料係購自Microchem公司之商品：SU-8負型光阻。 In a second embodiment of the invention, the material used to make the lipid bilayer is commercially available from Avanti Polar Lipids: 1,2 dioleyl lecithin DOPC. Phospholipid film dyes are purchased from Life Technologies: Texas-Red, N((6(biotinoyl)amino)hexanoyl)-1,2-dihexadecanoyl-sn-gly cero-3-phosphoethanolamine, triethylammonium salt, Biotin-X DHPE) Alexa flour 488 labeled streptavidin (Alexa flour 488 conjugated streptavidin). The barrier material used to make the patterned structure was purchased from Microchem Corporation as a SU-8 negative photoresist.
形成物理性障礙包含下列步驟：以RCA法(H2O：H2O2：NH4OH之比例為5：1：1)清潔基底表面；以3000r.p.m.於各該基底上旋轉塗佈阻擋材料SU-8約30秒後，形成約2微米厚度之SU-8阻層；於90℃軟烤SU-8阻層約1分鐘後，以80mJ/cm2能量之UV光通過圖案化光罩曝光各該基底上之SU-8阻層；於95℃軟烤SU-8膜約2分鐘，以顯影液去除未曝光之SU-8阻層而形成圖案化結構，該圖案化結構係具有相隔一間距設置於該基底表面之至少二阻障；以異丙醇清洗並以氮氣乾燥該基底；於150℃硬烤該基底上之該圖案化結構約1小時，用以增進該圖案化結構與該基底之黏著度。以不同間距之圖案化光罩，重複上述步驟，製成具有不同間距阻障之圖案化結構之基底。 Forming a physical barrier comprises the steps of: cleaning the surface of the substrate by RCA method (H 2 O:H 2 O 2 :NH 4 OH ratio of 5:1:1); spin coating blocking on each of the substrates at 3000 rpm After about 30 seconds of material SU-8, a SU-8 resist layer having a thickness of about 2 μm was formed; after softly baking the SU-8 resist layer at 90 ° C for about 1 minute, UV light having an energy of 80 mJ/cm 2 was passed through the patterned mask. Exposing the SU-8 resist layer on each of the substrates; soft-baking the SU-8 film at 95 ° C for about 2 minutes, removing the unexposed SU-8 resist layer with a developer to form a patterned structure, the patterned structures having a separation Disposing at least two barriers on the surface of the substrate; cleaning with isopropanol and drying the substrate with nitrogen; and hard-baking the patterned structure on the substrate at 150 ° C for about 1 hour to enhance the patterned structure and The adhesion of the substrate. The reticle is patterned at different pitches, and the above steps are repeated to form a substrate having a patterned structure with different pitch barriers.
製造生物檢測平台包含下列步驟：以氯仿溶液混和所需比例之脂質與細胞膜相關物質；先以氮氣再以真空去除氯仿，再加入2mg/ml之HEPES緩衝液(10mM HEPES,123mM NaCl,PH=7.4)並混和均勻後，反覆19次將具有脂質之緩衝液擠壓通過微脂胞擠壓器，以製得大單層脂質微胞(LUVs)溶液；以氬氣電漿清潔該圖案化結構10分鐘， 可改變SU-8材質之親水性及表面粗糙度，表面處理後之圖案化結構之水接觸角介於0至90度；以氧氣電漿處理具有微流道之PDMS晶片(100微米×500微米×1.5公分)30秒後，將該PDMS晶片密合於具有該圖案化結構之該基底上；將LUVs溶液加入微流道，經10分鐘靜置後，微脂胞選擇性沉積並形成脂質雙層膜於外露之該基底表面；以緩衝液洗淨多餘之微脂胞後，即完成具有脂質雙層膜之生物檢測平台。使用具有不同間距阻障之圖案化結構之基底重複上述步驟，製造具有不同圖案化結構之生物檢測平台。 The manufacturing bioassay platform comprises the steps of: mixing the desired proportion of lipid and cell membrane-related substances with a chloroform solution; first removing the chloroform by vacuum with nitrogen, and then adding 2 mg/ml of HEPES buffer (10 mM HEPES, 123 mM NaCl, pH=7.4). After mixing and homogenizing, the lipid buffer was pressed through the microlipid extruder 19 times to prepare a large monolayer lipid microcapsule (LUVs) solution; the patterned structure was cleaned by argon plasma 10 minute, The hydrophilicity and surface roughness of the SU-8 material can be changed. The water contact angle of the surface-treated patterned structure is between 0 and 90 degrees. The PDMS wafer with microchannels is treated with oxygen plasma (100 micron x 500 micron). ×1.5 cm) After 30 seconds, the PDMS wafer was adhered to the substrate having the patterned structure; the LUVs solution was added to the microchannel, and after 10 minutes of standing, the microlipid cells selectively deposited and formed a lipid double The film is exposed on the surface of the substrate; after washing the excess microlipid cells with a buffer, a biological detection platform having a lipid bilayer membrane is completed. The above steps are repeated using substrates having patterned structures with different spacing barriers to fabricate bioassay platforms having different patterned structures.
第2圖係本發明之生物檢測平台之剖面側視圖。如第2圖所示，生物檢測平台2包含基底20、圖案化結構21以及脂質雙層膜22。圖案化結構21具有相隔間距w設置於基底20表面之至少二阻障211，各阻障211具有高度h，且高度h與間距w之高寬比係1/2至1/250(高寬比h/w換算夾角為θ 1)。脂質雙層膜22形成於阻障211間之基底20表面上。 Figure 2 is a cross-sectional side view of the bioassay platform of the present invention. As shown in FIG. 2, the biodetection platform 2 includes a substrate 20, a patterned structure 21, and a lipid bilayer membrane 22. The patterned structure 21 has at least two barriers 211 disposed at a distance w apart from the surface of the substrate 20. Each barrier 211 has a height h, and an aspect ratio of the height h to the pitch w is 1/2 to 1/250 (aspect ratio) The h/w conversion angle is θ 1). A lipid bilayer membrane 22 is formed on the surface of the substrate 20 between the barriers 211.
詳細而言，基底20之材質可選用矽(如：單晶矽或多晶矽等)或矽化物(如：二氧化矽、玻璃、石英玻璃、硼玻璃、鈉鈣玻璃等)，但不以此為限。選用對脂質雙層膜不具親合性之阻擋材料作為圖案化結構之材料，如：光阻(光敏性環氧樹脂、聚甲基丙烯酸酯等)或低反應活性之金屬(金、鉑等)。 In detail, the material of the substrate 20 may be selected from ruthenium (such as: single crystal germanium or polycrystalline germanium) or germanium (such as: germanium dioxide, glass, quartz glass, borosilicate glass, soda lime glass, etc.), but not limit. A barrier material having no affinity for the lipid bilayer membrane is selected as a material for the patterned structure, such as photoresist (photosensitive epoxy resin, polymethacrylate, etc.) or low reactivity metal (gold, platinum, etc.) .
第3圖係空氣-水界面通過本發明之生物檢測平台之剖面側視圖。如第3圖所示，空氣-水界面200通過生物檢 測平台2時，脂質雙層膜22與各阻障211之接觸界面近乎垂直於空氣-水界面之移動方向(圖中箭頭所示)。當阻障211之高寬比(h/w)換算之夾角θ 1大於空氣-水界面與水平面之夾角θ 2時，空氣-水界面200不會直接接觸脂質雙層膜22之表面，所以能有效防止空氣-水界面破壞脂質雙層膜22；當空氣-水界面200通過後，相鄰阻障211間可留存部分之水溶液(或緩衝液)201於脂質雙層膜22上，從而能保持脂質雙層膜22之原有特性，確保脂質雙層膜生物檢測平台2之檢測效能。 Figure 3 is a cross-sectional side view of the air-water interface through the bioassay platform of the present invention. As shown in Figure 3, the air-water interface 200 passes the biopsy When the platform 2 is measured, the contact interface between the lipid bilayer membrane 22 and each of the barriers 211 is nearly perpendicular to the moving direction of the air-water interface (indicated by the arrow in the figure). When the angle θ 1 of the aspect ratio (h/w) converted by the barrier 211 is larger than the angle θ 2 between the air-water interface and the horizontal plane, the air-water interface 200 does not directly contact the surface of the lipid bilayer membrane 22, so Effectively preventing the air-water interface from destroying the lipid bilayer membrane 22; when the air-water interface 200 passes, a portion of the aqueous solution (or buffer) 201 remaining between the adjacent barriers 211 can be retained on the lipid bilayer membrane 22, thereby maintaining The original properties of the lipid bilayer membrane 22 ensure the detection efficiency of the lipid bilayer membrane bioassay platform 2.
第4圖係空氣-水界面通過本發明之生物檢測平台之俯視圖。如第4圖所示，脂質雙層膜22與各阻障211形成接觸界面s，接觸界面s與空氣-水界面移動方向(如箭頭所示方向)之夾角θ 3係介於80至110度，藉此圖案化結構21能有效防止空氣-水界面破壞脂質雙層膜22。 Figure 4 is a top plan view of the air-water interface through the bioassay platform of the present invention. As shown in FIG. 4, the lipid bilayer membrane 22 forms a contact interface s with each of the barriers 211, and the angle θ 3 between the contact interface s and the air-water interface moving direction (as indicated by the arrow) is between 80 and 110 degrees. Thereby, the patterned structure 21 can effectively prevent the air-water interface from destroying the lipid bilayer membrane 22.
以下係藉由空氣-水界面通過本發明與習知技術之生物檢測平台後，各該脂質雙層膜之檢測結果來具體說明本發明之優點與功效。 The advantages and effects of the present invention are specifically described below by the detection results of the lipid bilayer membranes after passing through the air-water interface through the bioassay platform of the present invention and the prior art.
測試空氣-水界面通過本發明之生物檢測平台包含下列步驟：以60毫米/分鐘或60000毫米/分鐘流速將氣泡(air-bubble)打入該微流道，以產生空氣-水界面通過微流道內之脂質雙層膜；當氣泡開始進入微流道之時點，定義為乾狀態之開始；當氣泡完全離開微流道之時點，定義為回濕狀態(rehydrate)之開始。 Testing the Air-Water Interface The bioassay platform of the present invention comprises the steps of: blowing air-bubble into the microchannel at a flow rate of 60 mm/min or 60,000 mm/min to produce an air-water interface through the microfluid The lipid bilayer membrane in the channel; defined as the beginning of the dry state when the bubble begins to enter the microchannel; the point at which the bubble completely leaves the microchannel is defined as the beginning of the rehydrate.
回濕完成後，使用532奈米波長之200mW綠雷射模組 光漂白(photobleaching)脂質雙層膜中之磷脂膜染料Texas-Red約0.1秒；使用具有CCD攝影機之反置式顯微鏡(inverted microscope)紀錄漂白點之回復影像(recovery image)；解析該回復影像及數據以獲得各該生物檢測平台上之該脂質雙層膜之完整度及擴散係數，統計分析結果如下表1。 After the wet back is completed, the 200mW green laser module with a wavelength of 532 nm is used. The phospholipid membrane dye Texas-Red in a photobleaching lipid bilayer membrane was about 0.1 second; a recovery image of the bleaching point was recorded using an inverted microscope with a CCD camera; the recovered image and data were analyzed. The integrity and diffusion coefficient of the lipid bilayer membrane on each of the bioassay platforms were obtained, and the statistical analysis results are shown in Table 1 below.
由表1可知，於本發明之生物檢測平台，圖案化結構之高寬比大於1/250時，圖案化結構即可產生保護與支撐脂質雙層之作用，較佳之高寬比是1/2至1/50，更佳之高寬比是1/2至1/20。 It can be seen from Table 1 that in the bio-detection platform of the present invention, when the aspect ratio of the patterned structure is greater than 1/250, the patterned structure can protect and support the lipid bilayer, and the aspect ratio is preferably 1/2. To 1/50, the better aspect ratio is 1/2 to 1/20.
以光漂白螢光回復測試法FRAP分別檢測具有圖案化結構之生物檢測平台及不具有圖案化結構之生物檢測平台，於氣泡通過前、通過中(開始狀態後5分鐘)及回濕後，各該脂質雙層膜之擴散係數，擴散係數為此領域用來代表脂質雙層膜流動性的指標，一般脂質雙層膜的擴散係數在1-2(微米平方/秒)左右。檢測結果如下表2。 The bio-detection platform with patterned structure and the biological detection platform without patterned structure were respectively detected by photo-bleaching fluorescence recovery test method FRAP, before and after the passage of bubbles (5 minutes after the start state) and after rewetting, The diffusion coefficient of the lipid bilayer membrane and the diffusion coefficient are used to represent the fluidity of the lipid bilayer membrane. Generally, the diffusion coefficient of the lipid bilayer membrane is about 1-2 (micrometer square/second). The test results are shown in Table 2 below.
如表2所示，具有圖案化結構之生物檢測平台於氣泡通過前、通過中及回濕後，該脂質雙層膜均能保持原有之擴散係數；而不具有圖案化結構之脂質雙層膜檢測平台於氣泡通過中擴散係數接近於零，且回濕後該脂質雙層膜之擴散性大幅降低。由此可知，本發明之生物檢測平台，圖案化結構具有維持脂質雙層膜流動性之作用。 As shown in Table 2, the bio-detection platform with the patterned structure can maintain the original diffusion coefficient before, after, and after the bubble passes; the lipid double layer without the patterned structure The membrane detection platform has a diffusion coefficient close to zero in the passage of bubbles, and the diffusivity of the lipid bilayer membrane is greatly reduced after rewetting. It can be seen from the above that the biodetection platform of the present invention has a function of maintaining the fluidity of the lipid bilayer membrane.
以光漂白螢光回復測試法分別檢測本發明、使用化學修飾法及使用他種生物分子覆蓋法所製造之生物檢測平台，於空氣-水界面通過前後，各該脂質雙層膜經隔離、修飾或覆蓋法處理後之擴散係數相對於各該脂質雙層膜之原有擴散係數之比例，此擴散係數之比例代表該檢測平台中脂質雙層膜之流動性在通過空氣-水界面後保留的程度，其中，100%代表脂質雙層膜之流動性在通過空氣-水界面前後並未改變；0%代表脂質雙層膜在通過空氣-水界面後不再具有流動性，測結果如下表3。 The photodetection fluorescence recovery test method is used to detect the present invention, using a chemical modification method and a bioassay platform manufactured by using the biomolecule covering method, and the lipid bilayer membrane is isolated and modified before and after passing through the air-water interface. Or the ratio of the diffusion coefficient after the coverage treatment to the original diffusion coefficient of each of the lipid bilayer membranes, the ratio of the diffusion coefficient representing the retention of the fluidity of the lipid bilayer membrane in the detection platform after passing through the air-water interface Degree, wherein 100% represents that the fluidity of the lipid bilayer membrane does not change before and after passing through the air-water interface; 0% means that the lipid bilayer membrane no longer has fluidity after passing through the air-water interface, and the results are shown in Table 3 below. .
如表3所示，相較於使用化學修飾法及使用他種生物分子覆蓋法所製造之生物檢測平台，本發明之具有圖案化結構之生物檢測平台能有效保持脂質雙層膜之二維流動性。 As shown in Table 3, the biodetection platform with patterned structure of the present invention can effectively maintain the two-dimensional flow of the lipid bilayer membrane compared to the bioassay platform manufactured by using the chemical modification method and using other biomolecule covering methods. Sex.
檢測脂質雙層膜之配體-受體交互作用(ligand-receptor interactions)包含下列步驟：提供DOPC摻雜0.5莫耳%之Biotin-X DHPE之biotinylated DOPC脂質雙層膜檢測平台；以60毫米/分鐘或60000毫米/分鐘流速將氣泡打入微流道；將10微克/毫升之Alexa flour 488 conjugated streptavidin流入該微流道，靜置30分鐘以使其和脂雙層膜上之Biotin產生結合；沖除混和液中過多之streptavidin；藉由標準化螢光強度(扣除背景值)表示streptavidin結合於biotinylated DOPC脂質雙層膜檢測平台之量。 The detection of ligand-receptor interactions of the lipid bilayer membrane comprises the steps of providing a biotinylated DOPC lipid bilayer membrane detection platform with DOPC doping of 0.5 mol% Biotin-X DHPE; A minute or a flow rate of 60,000 mm/min was used to drive air bubbles into the microchannel; 10 μg/ml of Alexa flour 488 conjugated streptavidin was flowed into the microchannel and allowed to stand for 30 minutes to allow binding to Biotin on the lipid bilayer membrane; Excess streptavidin in the mixture was washed away; the amount of streptavidin bound to the biotinylated DOPC lipid bilayer membrane detection platform was quantified by normalized fluorescence intensity (netting background values).
以螢光強度分別檢測具有圖案化結構(100微米間距)之生物檢測平台及不具有圖案化結構之生物檢測平台，於60毫米/分鐘流速之氣泡通過前後，各該脂質雙層膜由螢光強度檢測所得之保留完整度，標準化螢光強度即代表脂雙層膜之保留程度，1代表沒被破壞，0代表完全被破壞沒保留，檢測結果如下表4。 A biological detection platform having a patterned structure (100 micrometer pitch) and a biological detection platform having no patterned structure were respectively detected by fluorescence intensity, and the lipid bilayer membrane was fluorescently irradiated before and after the bubble of a flow rate of 60 mm/min. The retention integrity obtained by the intensity test, the normalized fluorescence intensity represents the retention of the lipid bilayer membrane, 1 represents no destruction, 0 represents complete destruction and no retention, and the test results are shown in Table 4 below.
如表4所示，本發明之具有圖案化結構之生物檢測平台能確保脂質雙層膜之檢測效能，便於使用者運送樣品或更換反應溶液。 As shown in Table 4, the biodetection platform with the patterned structure of the present invention can ensure the detection performance of the lipid bilayer membrane, and is convenient for the user to transport the sample or replace the reaction solution.
綜上所述，當空氣-水界面通過本發明之生物檢測平台時，脂質雙層膜與各阻障之接觸界面近乎垂直於空氣-水界面之移動方向，圖案化結構使空氣-水界面不會直接接觸脂質雙層膜之表面，所以能有效防止於空氣-水界面破壞脂質雙層膜；當空氣-水界面通過後，阻障間可留存部分之水溶液或緩衝液於脂質雙層膜上，從而確保該生物檢測平台之檢測效能。 In summary, when the air-water interface passes through the bio-detection platform of the present invention, the contact interface between the lipid bilayer membrane and each barrier is nearly perpendicular to the moving direction of the air-water interface, and the patterned structure makes the air-water interface not It will directly contact the surface of the lipid bilayer membrane, so it can effectively prevent the lipid bilayer membrane from being destroyed at the air-water interface; when the air-water interface passes, a part of the aqueous solution or buffer can be retained between the barriers on the lipid bilayer membrane. To ensure the detection performance of the bioassay platform.
上述實施例僅例示性說明，而非用於限制本發明。任何熟習此項技藝之人士均可在不違背本發明之精神及範疇下，對上述實施例進行修飾與改變。因此，本發明之權利保護範圍，應本案所附之申請專利範圍所載。 The above embodiments are illustrative only and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, the scope of the invention should be construed as being included in the scope of the appended claims.
2‧‧‧生物檢測平台 2‧‧‧Biological testing platform
20‧‧‧基底 20‧‧‧Base
21‧‧‧圖案化結構 21‧‧‧ patterned structure
211‧‧‧阻障 211‧‧‧Resistance
22‧‧‧脂質雙層膜 22‧‧‧Lipid bilayer membrane
200‧‧‧空氣-水界面 200‧‧‧Air-water interface
201‧‧‧水溶液(緩衝液) 201‧‧‧Aqueous solution (buffer)
h‧‧‧阻障之高度 h‧‧‧The height of the barrier
w‧‧‧阻障之間距 W‧‧‧distance between obstacles
θ 1‧‧‧高寬比(h/w)換算之夾角 θ 1‧‧‧ aspect ratio of h/w conversion
θ 2‧‧‧空氣-水界面與水平面之夾角 θ 2‧‧‧An angle between the air-water interface and the horizontal plane
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