201102648 六、發明說明： 【發明所屬之技術領域】 本發明是有關於一種微流體晶片，特別是指一種具拉 曼光譜增顯功能之微流體晶片。 【先前技術】201102648 VI. Description of the Invention: [Technical Field] The present invention relates to a microfluidic wafer, and more particularly to a microfluidic wafer having a Raman spectral enhancement function. [Prior Art]
傳統微生物鑑定方法主要有培養法與DNA鑑定法，典 養法一般需要耗時2〜7天使細菌生長到足夠的量，再進行 革蘭氏染色或選擇性培養基生長，往往緩不濟急。DNA鑑 定法雖然較快，但需要經過細胞裂解、聚合酵素鏈鎖反應 放大與DNA雜交反應等繁雜的步驟，一般需耗時6〜1〇小 時，且需要有經過訓練的專業人員與繁雜的操作手續才有 辦法順利檢測。ELISA g然更為快速，但靈敏度不佳，且 需要有特異性蛋白才可檢測。 相季又於現今拉曼光譜之檢測方Traditional microbial identification methods mainly include culture method and DNA identification method. The cultivation method generally takes 2~7 angels to grow bacteria to a sufficient amount, and then Gram staining or selective medium growth is often slow. Although the DNA identification method is faster, it requires complicated steps such as cell lysis, polymerization enzyme chain reaction amplification and DNA hybridization reaction, which usually takes 6 to 1 hour, and requires trained professionals and complicated operations. There are ways to successfully check the procedures. ELISA g is faster, but it is less sensitive and requires specific proteins to detect. The season is also the detection of current Raman spectroscopy
川、又/U δ百个欢次，J 可快速鑑定細菌，且不需繁雜的前處理，但檢測訊號通常 >田弱、”田菌“紋不谷易辨識，且細菌濃度必須要相當 > (0 CFU/ml以上）’雷射光才有辦法打到細菌。近來有 =學者發展拉曼增顯的方式，如以奈米金屬粒子與細菌 進^亍結合而達到增顯科JB. y , θ<.，'員效果，但此方式常因奈米金屬粒子無 全麗：的分布在細菌表面’而造成增顯效果不-，且奈米 電於細菌表面時’將造成細菌整體粒徑與表面 以致於無法以介電泳力或其他電控方式進行 刀離或#控。近來也有部八m 射光激發時，於㈣基材表面造成雷 -、金屬表面的共振增強而達到增顯的 201102648 方法與技術都仍然需要相當高的細菌濃度(約 m且須以非常昂貴的儀器（例如雷射鉗，laser —Γ)將細菌固定才有辦法偵測，使用上仍相當不便。 【發明内容】 因此，本發明之目的 功能之微流體晶片。 即在提供一種具拉曼光譜增顯 本發明具拉曼光譜增顯功能之微流體晶片，可 捕捉集中及鑑別連續流體巾之不同生物分子， 於是 用以分離 該微流體晶片包含—可供用以激發產生拉曼光譜之光子束 穿透的晶片本體’及分別設置於日日目片本體之—分選電極單 元、-捕捉電極單元與多數遮蔽電極。該晶片本體包括由 下往上依序疊接之一基板層、一流道層與一覆蓋層，且該 基板層、流道層與覆蓋層相配合界定出—微流道，該微流 道具有-分選段’及至少：分別連通於該分選段末端之捕 捉段。該分選電極單元是外露於該分選段中，並可被預定 電訊號驅動’而將分選段中被連續流體帶動通過之不同生 ，分子分離並分別導引入預定捕捉段中。該捕捉電極單元 是外露於該等捕捉段中，並可被狀電訊號驅動，而將該 等捕捉段中被連續流體帶動之特定生物分子捕捉集中於該 捕捉^又之預疋區域。该等遮蔽電極是可防止光子束往上穿 透地被覆於覆蓋層底面，且分別外露於該等捕捉段中，並 分別位於該等捕捉電極分別將生物分子捕捉集中之預定區 域上方，且每一遮蔽電極具有一面向被捕捉集中之生物分 子並可使拉曼光譜產生磁場共振增顯之粗糙狀粗糙面。 201102648 本發明之功效：透過於該晶片本體之微流道中設置該 分選電極單元、捕捉電極單元與該等遮蔽電極的結構設計 ，使得該微流體晶片可於生物分子之分離與捕捉集中過程 中’即時進行拉曼光譜偵測，並藉由該粗糙化遮蔽電極， 使拉曼光譜訊號產生磁場共振而增強數十倍，可大幅提高 訊號的辨識度並縮短偵測時間。 【實施方式】 有關本發明之前述及其他技術内容、特點與功效，在 以下配合參考圖式之一個較佳實施例的詳細說明中將可 清楚的呈現。 如圖1、2所示，本發明具拉曼光譜增顯功能之微流體 晶片的較佳實施例，適用於分離並捕捉集中檢體中之不同 生物分子，例如血球細胞與細菌，或分離不同之細菌，且 可以拉曼光譜偵測即時對微流體晶片中被集中後之生物分 子進行鑑定。 該微流體晶片包括一晶片本體3，及分別設置於晶片本 體3中之一分選電極單元4、多數捕捉電極單元5與多數遮 蔽電極6。在本實施例中，該微流體晶片是以介電泳微流體 晶片為例進行說明，但實施時，微流體晶片類型不以此為 限0 該晶片本體3包括由下往上依序疊接之一基板層31、 一 道層32與一覆蓋層33，且該基板層31、流道層32與 覆蓋層33相配合界定出一穿設於流道層32之微流道3〇， 該微流道30具有一左右延伸之分選段3〇1、多數前後排列 201102648 地分別連通於該分選段301右端之捕捉段302，及多數間隔 在下貫穿該覆蓋層33而分別連通於分選段左端與該等 捕捉段302右端之儲液段303。 6亥基板層31與覆蓋層33皆採用可供用以激發拉曼光 ☆訊號之光子束穿透的玻璃材質，在本實施例中，該光子 束為雷射光’該基板層31為〇17麵蓋玻片，其目的是為 了減少光子束穿過時所產生之螢光雜訊造成的干擾，但實 施時’亦可以石英玻璃代^覆蓋層33為載玻片該流道 J疋由JSR光阻經過微影方式成型製成，並經由熱壓 接合而固接於該基板層31與覆蓋層33間。但實施時，該 晶片本體3之材質與製作方式不以此為限。 /分選電極單元4包括四個上下兩兩對稱地分別被覆 固疋於及基板層31頂面與覆蓋層33底面之分選電極“， 且該等成對分選電極41是分別沿該分選段3()1長度方向間 ㈣列’且分職料露於分選段3Ql巾，而分別位於分 選& 3〇1上、下側，其中，左側成對分選電極41是往右後 方延伸人分選段3G1中，右側成對分選電極41則是往右前 方延伸入分選段3〇1中，並朝其中一捕捉段3〇2開口處延 伸。 勺該捕捉電極單元5是設置外露於該等捕捉段302中， :括二上下對稱地分別被覆於基板層31頂面與覆蓋層Μ =捕捉電極51，且該等捕捉電極51分別具有二分別位 於5亥4捕捉段302中且呈開口朝力夕v〜 1朝左之V字型的捕捉部511。 該等遮蔽電極6是呈三角形，且分別被覆於該覆蓋層 201102648 33底面，並分別外露於該等捕捉段3〇2中，而分別鄰近相 對應捕捉部511左側，且該等遮蔽電極6可防止光子束往上 穿透進入覆蓋層33,每一遮蔽電極6具有一面向捕捉段 302並可使拉曼光譜產生共振增顯之粗糙化粗糙面η，且 對應不同大小之待鑑定生物分子，該等遮蔽電極61會分別 採用不同粗縫度之粗經面61。Chuan, and / U δ hundred happy times, J can quickly identify bacteria, and does not require complicated pre-treatment, but the detection signal is usually > field weak, "field bacteria" is not easy to identify, and the bacterial concentration must be equivalent > (0 CFU/ml or more) 'The laser light has a way to hit the bacteria. Recently, there have been scholars who have developed Raman enhancement methods, such as the combination of nano metal particles and bacteria into the JB. y, θ<., 'member effect, but this method is often due to nano metal particles. No Quanli: The distribution on the surface of the bacteria 'does not increase the effect, and the nano-electricity on the surface of the bacteria' will cause the overall particle size and surface of the bacteria to be able to be separated by dielectrophoresis or other electronic control. Or #控. Recently, there are also eight-m light excitations, which cause a resonance of the thunder- and metal surface on the surface of the substrate. The method and technology still require a relatively high bacterial concentration (about m and must be very expensive instruments). (For example, laser clamp, laser-Γ) can fix the bacteria in a way to detect it, and it is still quite inconvenient to use. [Invention] Therefore, the microfluidic wafer of the purpose of the present invention is provided with a Raman spectrum increase. The invention discloses a microfluidic wafer with Raman spectral enhancement function for capturing and identifying different biomolecules of a continuous fluid towel, and thus separating the microfluidic wafer comprises: a photon beam traversing for exciting Raman spectra The transparent wafer body 'and the separation electrode unit, the capture electrode unit and the plurality of shielding electrodes respectively disposed on the sunday film body. The wafer body includes a substrate layer and a first-order layer layer sequentially stacked from bottom to top. And a cover layer, and the substrate layer, the flow channel layer and the cover layer cooperate to define a micro flow channel, the micro flow channel has a -segment segment and at least: respectively a capture section at the end of the sorting section. The sorting electrode unit is exposed in the sorting section and can be driven by a predetermined electrical signal to separate the molecules in the sorting section by continuous fluid, and the molecules are separated and guided separately. Into the predetermined capture segment, the capture electrode unit is exposed in the capture segments and can be driven by the electrical signal, and the specific biomolecules driven by the continuous fluid in the capture segments are concentrated in the capture. The masking electrode is configured to prevent the photon beam from being penetratingly overlying the bottom surface of the cover layer, and is respectively exposed in the capturing segments, and respectively located at predetermined regions where the capturing electrodes respectively capture the concentration of biomolecules Above, and each of the shielding electrodes has a rough rough surface facing the biomolecules that are concentrated and can cause the Raman spectrum to generate a magnetic field resonance. 201102648 The effect of the present invention: the micro-channel is disposed in the micro-channel of the wafer body The structural design of the sorting electrode unit, the trapping electrode unit and the shielding electrodes enables the microfluidic wafer to be separated and captured by biomolecules In the process of concentration, the Raman spectroscopy is performed instantaneously, and the Raman spectroscopy electrode is used to make the magnetic field resonance of the Raman spectroscopy signal to be enhanced by several tens of times, which can greatly improve the identification of the signal and shorten the detection time. The above and other technical contents, features and effects of the present invention will be apparent from the following detailed description of a preferred embodiment of the drawings. FIG. A preferred embodiment of a microfluidic wafer with a Mann spectral enhancement function is suitable for separating and capturing different biomolecules in a concentrated sample, such as blood cells and bacteria, or separating different bacteria, and can detect Raman spectra instantly. The microfluidic wafer is identified by the concentrated biomolecule. The microfluidic wafer comprises a wafer body 3, and one sorting electrode unit 4, a plurality of trapping electrode units 5 and a plurality of shielding electrodes 6 respectively disposed in the wafer body 3. In this embodiment, the microfluidic wafer is described by taking a dielectrophoretic microfluidic wafer as an example. However, when implemented, the microfluidic wafer type is not limited thereto. The wafer body 3 includes sequentially stacked from bottom to top. a substrate layer 31, a layer 32 and a cover layer 33, and the substrate layer 31 and the channel layer 32 cooperate with the cover layer 33 to define a micro flow channel 3〇 passing through the flow channel layer 32, the microflow The track 30 has a left and right extension section 3〇1, a plurality of front and rear arrays 201102648 are respectively connected to the capture section 302 of the right end of the sorting section 301, and a plurality of intervals are respectively penetrated through the cover layer 33 and are respectively connected to the left end of the sorting section and the same The liquid storage section 303 at the right end of the segment 302 is captured. Both the substrate layer 31 and the cover layer 33 are made of a glass material for exciting the photon beam penetration of the Raman light ☆ signal. In this embodiment, the photon beam is laser light, and the substrate layer 31 is a 〇17 surface. The cover glass is designed to reduce the interference caused by the fluorescent noise generated when the photon beam passes through, but in practice, the quartz glass can be used as the cover glass. The flow path J is blocked by the JSR. It is formed by lithography, and is fixed between the substrate layer 31 and the cover layer 33 by thermocompression bonding. However, the material and manufacturing method of the wafer body 3 are not limited thereto. The sorting electrode unit 4 includes four sorting electrodes symmetrically and respectively affixed to the top surface of the substrate layer 31 and the bottom surface of the cover layer 33, and the pair of sorting electrodes 41 are respectively along the points Select paragraph 3 () 1 between the length direction (four) column 'and the sub-worker is exposed in the sorting section 3Ql towel, and respectively located on the sorting & 3〇1 upper and lower sides, wherein the left pair of sorting electrodes 41 are to the right rear In the extended person sorting section 3G1, the right paired sorting electrode 41 extends into the sorting section 3〇1 to the right front and extends toward the opening of one of the capturing sections 3〇2. The scooping of the capturing electrode unit 5 is set to be exposed. In the capture segments 302, the top surface of the substrate layer 31 and the cover layer 捕捉=capture electrode 51 are respectively symmetrically disposed, and the capture electrodes 51 are respectively located in the capture section 302 of the 5H 4 and The V-shaped capturing portion 511 is opened to the left of the v-to-left. The shielding electrodes 6 are triangular and are respectively covered on the bottom surface of the covering layer 201102648 33 and are respectively exposed to the capturing segments 3〇2. Medium, respectively adjacent to the left side of the corresponding capturing portion 511, and the shielding electrodes 6 are preventable The photon beam penetrates into the cover layer 33, and each of the shielding electrodes 6 has a roughened rough surface η facing the capturing section 302 and causing resonance enhancement of the Raman spectrum, and corresponding to different sizes of biomolecules to be identified, The masking electrodes 61 will respectively adopt the rough surface 61 of different rough degrees.
在本實施例中，該等遮蔽電極6被覆於覆蓋層33時， 是先對該覆蓋層33底面進行B〇E (buffered 〇xide灿）化 學钱刻處理，利用B0E對玻璃触刻時殘留於玻璃上的反應 物所U成之不均句性餘刻，並透過控制姓刻時間，先於該 覆蓋層33底面預^部位得到數個分別具有不同粗縫度的粗 ，表面#著，再以真空電子束蒸鑛的方式將構成該遮 蔽電極6之金屬分別沉積於該等粗糖表面上，而形成一具 有粗糙面61的薄膜狀三角形遮蔽電極6。 仁實粑時，该遮蔽電極6之製作方式不以此為限，亦 :利用其他的微奈米製程技術或奈米壓印技術，先於該覆 “層精確的1作出不同大小程度的粗糖表面後，再於該 粗糙表面被覆上該遮蔽電極6 ’以便因應不同大小程度的生 物分子’例如數微米至數百奈米大小之細胞1菌、細菌 ，以及數奈米至數十奈米的病毒、DNA、RNA等。 在本實施例中 極51於微流道3〇 、捕捉與集中連續 δ亥等成對分選電極 ’主要是利用該等分選電極41與捕捉電 内之連續流體產生之介電泳力，來分離 流體中具有不同介電特性之生物分子。 41與捕捉電極51可分別被施加預定頻 201102648 率之交流電，而分別於分選段301與捕捉段3〇2之連續流 體中產生吸引或排斥不同介電特性之生物分子的介電泳力 ’並配合連續流體流動產生之推送力4，將連續流體中之 生物分子分離並分別導引入預定的捕捉段3〇2 +，再由該 等捕捉電極51產生之介電泳力作用’將特定介電特性的生 物分子擋止於該等捕捉部511左方之V字型開口中，並隨 著連續流體的持續通過，而逐漸聚集集中。由於該等分選 電極41與捕捉電極51分離、導引與捕捉集中不同介電特 生之生物分子的技術為習知技術，因此不再詳述。 當要以拉曼光譜訊號來個別鑑定檢體中之特定生物分 子時，可先將檢體混合於特定之介電泳液後，將該介電泳 液持續導入該微流道30巾，並分別於該等分選電極41與 捕捉電極51施加一預定頻率之交流電，先將待鑑定生物分 子分離並操控至預定捕捉段3〇2中，並經由該等捕捉電極 51擋止聚集於捕捉段302後，便可以光子束往上射向該捕 捉段302中之遮蔽電極6’透過該遮蔽電極6具有預定粗縫 度之粗糙面61設計，使拉曼光譜訊號產生磁場共振而增強 數十倍，提高生物分子的鑑定準確度。 以下先直接將金黃色葡萄球菌【&〇c〇ccwi ⑼， BCRC 14957】置於具有不同粗糙化之遮蔽電極6上進行鑑 疋為例，說明不同粗糙度之遮蔽電極6對拉曼光譜訊號增 顯的作用’其中，遮蔽電極6之材質為金（Au)。 如圖2〜4所示，以三種不同粗糙度之遮蔽電極6進行 測°式’其中一遮蔽電極6是在未經BOE餘刻之覆蓋層33上 201102648 製成的平坦狀鍍金電極，表面粗糙度約為2〜3 (波峰與 波谷之高度差），由於該覆蓋層33為一般市售之載玻片， 其表面之波鋒對波鋒的間距非常不規則，在同一片載玻片 中由數百奈米至數百微米都有，但因高低差只冑2〜3 nm， 故其波鋒與波鋒之間距已經變得相當不重要，已接近於平 坦，所以一般市售載玻片的粗糙度是以高低差來定義。 另外兩個遮蔽電極6則是在經過B〇E蝕刻之覆蓋層33 上製成的粗糙狀鍍金電極，分別標示為粗糙度（較緻密與 較小粗糙度，如圖3所示）與粗糙度_2 (較大粗糙度，如圖 4所示）。其中，粗糙度-1之遮蔽電極ό的粗糙面61凹凸起 伏的深度約為60〜80 nm，波谷寬度約為卜丨5 μιη ,波峰寬 度約為0.1〜0.3 μηι ;粗糙度_2之遮蔽電極6的粗糙面61凹 凸起伏深度約為80〜1〇〇 nm，波鋒與波谷寬度皆約為 1.5〜2.5 μιη。In the present embodiment, when the shielding electrodes 6 are applied to the cover layer 33, the bottom surface of the cover layer 33 is first subjected to a B〇E (buffered 〇xide) chemical etching process, and the B0E is left to be etched when the glass is inscribed. The reactants on the glass are formed into a non-sequential sentence, and by controlling the time of the surname, a plurality of coarse portions having different rough seams are obtained before the bottom portion of the cover layer 33, and the surface is The metal constituting the shielding electrode 6 is deposited on the surface of the coarse sugars by vacuum electron beam evaporation to form a film-shaped triangular shielding electrode 6 having a rough surface 61. In the case of Renshi, the shielding electrode 6 is not limited to this. It is also possible to use other micro-nano process technology or nano-imprint technology to make different sizes of raw sugar before the layer is accurately After the surface, the masking electrode 6' is further coated on the rough surface so as to cope with different levels of biomolecules, such as cells of several micrometers to hundreds of nanometers, bacteria, and several nanometers to several tens of nanometers. Virus, DNA, RNA, etc. In the present embodiment, the pole 51 is in the microchannel 3〇, capturing and concentrating the continuous δ hai, etc. The pair of sorting electrodes 'is mainly using the sorting electrode 41 and the continuous fluid in the trapping electric current. The dielectrophoretic force is generated to separate biomolecules having different dielectric properties in the fluid. 41 and the trapping electrode 51 can be respectively applied with alternating current of a predetermined frequency of 201102648, and continuous fluids of the sorting section 301 and the capturing section 3〇2, respectively. The dielectrophoretic force of a biomolecule that attracts or repels different dielectric properties' and the push force 4 generated by the continuous fluid flow separates and separates the biomolecules in the continuous fluid into a predetermined trap Segment 3〇2 +, and then the dielectrophoretic force generated by the capture electrodes 51 acts to block biomolecules of specific dielectric properties in the V-shaped opening to the left of the capture portion 511, and with the continuous fluid The technique of continuous separation and gradual concentration. Since the separation electrode 41 is separated from the capture electrode 51, and the technique of guiding and capturing biomolecules with different dielectric properties is a conventional technique, it will not be described in detail. When the specific biomolecule in the sample is individually identified by the Raman spectroscopy signal, the sample may be first mixed into the specific dielectrophoresis solution, and the dielectrophoresis solution is continuously introduced into the microchannel 30, and respectively The sorting electrode 41 and the trapping electrode 51 apply an alternating current of a predetermined frequency, first separating and manipulating the biomolecule to be identified into the predetermined capturing section 3〇2, and stopping the gathering of the capturing section 302 via the capturing electrodes 51, The masking electrode 6' that can be directed upward into the capturing section 302 through the photon beam has a rough surface 61 having a predetermined roughness through the shielding electrode 6, so that the Raman spectrum signal generates a magnetic field resonance and is enhanced by several tens of times, and the biomolecule is enhanced. The accuracy of the identification. First, the Staphylococcus aureus [& 〇c〇ccwi (9), BCRC 14957] is directly placed on the shielding electrode 6 with different roughening as an example to illustrate the shielding electrode 6 with different roughness. The effect of Raman spectral signal enhancement is shown in which the material of the shielding electrode 6 is gold (Au). As shown in Figures 2 to 4, the shielding electrode 6 of three different roughness is used to measure the type of one of the shielding electrodes 6 It is a flat gold-plated electrode made of 201102648 on the cover layer 33 without BOE, and the surface roughness is about 2~3 (the height difference between the peak and the trough), since the cover layer 33 is a generally commercially available glass carrier. The surface of the wave front is very irregular with respect to the wave front. It is from hundreds of nanometers to hundreds of micrometers in the same slide, but the height difference is only 〜2~3 nm, so its wave front The distance from the wave front has become quite unimportant and is close to flat, so the roughness of a commercially available slide is defined by the height difference. The other two shielding electrodes 6 are rough gold-plated electrodes formed on the B layer E-etched cover layer 33, respectively marked as roughness (more dense and less roughness, as shown in Figure 3) and roughness _2 (larger roughness, as shown in Figure 4). Wherein, the rough surface 61 of the roughness electrode of the roughness-1 has a depth of relief of about 60 to 80 nm, a valley width of about 5 μm, a peak width of about 0.1 to 0.3 μm, and a masking electrode of roughness _2. The rough surface 61 of 6 has an undulating depth of about 80 to 1 〇〇 nm, and the wave front and the trough width are both about 1.5 to 2.5 μm.
光子束為514 nm Argon雷射光’雷射光強度約為1 mW ，雷射光斑約為8 μιη ’金黃色葡萄球菌濃度為1 〇8 cFu/ml 〇 配合圖5，於平坦遮蔽電極6表面上測得之拉曼光譜訊 號中’只出現二個微小的峰值訊號’分別在1158、1522 與2940 cm·1，以1158 cm·1的峰值訊號為基準，其訊號強度 約為250 counts。於粗糙度-2的遮蔽電極6所量得之訊號中 ’出現了八根較明顯的峰值訊號，分別在1 、1158、 1288、1523、1588、2313、2670 與 2938 cm·1，其中，1158 cm 1的峰值訊號強度約為1500 counts。於粗链度_1的遮蔽 201102648 電極6表面所量得之訊號中，則出現了十二根可供辨識的 峰值訊號’分別在 960、1007、1199、1158、1288、1442、 1522、1589、2313、2535、2670 與 3035 cm·1，且 1158 cnT1 的峰值訊號強度高達7000 counts ’相較於平坦狀遮蔽電極6 表面’拉曼光譜訊號增強了約三十倍左右，證實本發明粗 糙化之遮蔽電極6的設計，確實可有效的增顯拉曼光譜訊 號。 接著’再以混合有血球細胞與細菌之檢體於本發明微 流體晶片中直接進行分離、捕捉集中與拉曼光譜鏗定。 _ 如圖2、6、7所示，採用之細菌為金黃色葡萄球菌【 &叩BCRC 14957】，濃度為 1〇6 CFU/ml， 施加於該等分選電極41之電壓為12 Vpp、頻率為5〇〇 kHz ，捕捉電極電壓為20 Vpp、頻率為500 kHz，流體流速為ι pL/min，當帶有血球細胞與細菌之連續流體依序流經該等 分選電極41時，血球細胞與細菌會分別被操控分離至不同 的捕捉段302中，血球細胞與細菌之操控移動與分離方向 分別如圖6、7鍵頭符號所示方向，並經該等捕捉電極5ι φ 的捕捉聚集二分鐘後’於微流體晶片上即時進行拉曼光级 偵測。 曰 配合圖8，當微流體晶片未設置該遮蔽電極6時由於 螢光雜訊過大，即使將拉曼圖譜經過基準線校正後，也气 忐看到2940 cm處有一根微小的peak，大部分的拉曼光级 訊號都已被螢光雜訊掩蓋。 3 當微流體晶片中設有平坦狀遞蔽電極6時，其拉曼圖 10 201102648 譜在1008、1158、1520、2670與2940 cm.1具有勉強可辨 識的訊號’其訊號略大於直接將檢體滴於一般平坦鑛金表 面上所偵測到之拉曼光譜訊號（圖未示），預估應該是因 為細菌於微流體晶片中被局部集中於該遮蔽電極6區域後 ’使得細菌濃度相對提高，所以經過微流體晶片集中後所 偵測的訊號會略大於一般平坦鍍金表面之訊號。 當微流體晶片中設有粗棱化的遮蔽電極6時，其拉曼 圖譜在 958、1007、1199、1158、1286、1448、1522、1588 、2313、2536、2670與2933 cm·1皆產生足以辨識的訊號， 且相較於平坦遮蔽電極6之微流體晶片，其訊號強度增顯 了十倍左右。 且經實驗證實，在連續流體的帶動與該微流體晶片產 生之介電泳力捕捉濃縮作用下，可相對減少檢體所需之樣 本濃度’當細菌濃度約1〇6 CFU/ml時，從分離、捕捉集中 到拉曼光譜偵測的整個過程僅需三分鐘，當細菌濃度約1〇5 CFU/ml時，則約30分鐘即可完整分離、捕捉集中與拉曼 光譜偵測流程。 综上所述，透過於該晶片本體3之微流道3〇中設置該 分選電極單70 4、捕捉電極單元5與該等遮蔽電極6的結構 设计，可先利用分選電極單元4與捕捉電極單元5產生之 ^電泳力，對檢體中具有不同介電特性之生物分子進行分 離，並操控制預定捕捉段302中後，再將分離後之生物分 子捕捉集中於遮蔽電極6下方，如此一來，即使是低濃度 的生物檢體，也可透過該等捕捉電極51之捕捉集中，而使 201102648 得該遮蔽電# 6所在區域有極高的待測生物分子濃度，解 決了現今拉曼光學檢測無法適用於低濃度檢體的瓶頸。 此外，透過上述結構設計，該微流體晶片還可於生物 分子之分離與捕捉集中過程中，即時進行拉曼光譜偵測， 並藉由該《化遮蔽電極6’使拉曼光譜訊號產生磁場共振 而增強數十倍’除了可大大的增加訊號的辨識度與縮短偵 測時間外’對生物分子進行鑑定時，亦可改用較低強度的 雷射光（約1 mW)進行量測，以避免破壞生物樣本的活性， 再藉由該粗糙化遮蔽電極6的增顯應效應，得到足以辨識 的訊號。 因此，本發明具有快速分離與捕捉集中功能，並結合 表面增顯拉曼光譜㈣之微流體晶片’將可廣泛應用於臨 床致病菌的分離與鑑定，以及畜牧業、食品業的致病菌與 品質的快速檢測等。確實可達到本發明之目的。 惟以上所述者，僅為本發明之較佳實施例而已，當不 能以此限定本發明實施之範圍，即大凡依本發明申請專利 範圍及發明說明内容所作之簡單的等效變化與修飾，皆仍 屬本發明專利涵蓋之範圍内。 【圖式簡單說明】 圖1是本發明具拉曼光譜增顯功能之微流體晶片之一 較佳實施例的立體分解圖； 圖2是該較佳實施例的組合仰視示意圖，其中一基板 層已移除，並說明一分選電極單元、一捕捉電極單元、多 數遮蔽電極與微流道間之相對位置關係； 12 201102648 遮蔽電極的粗糙面之原子力 圖3是該較佳實施例之 顯微鏡影像； 圖4是類似圖3之視圖； 圖5是直接將金黃色㈣球菌置於遮㈣極上進行拉 支光5普偵蜊所得的拉曼光譜圖； 胞二^^之?部顯微影像圖，說〜 沪著分1之操控導引與連續流體帶動下， /〇者刀選電極延伸方向移動的情況； 下 圖7是類似圖6之視圖， 球細胞與細菌分離時的情況；及 成對分選電極將血 圖8是該較#, 行拉曼光譜檢測所得的^^離自没合桧體中之細菌進 13 201102648 【主要元件符號說明】 3....... •…晶片本體 4....... ....分選電極單元 30 … ....微流道 41 ..... ....分選電極 301.... ....分選段 5....... ....捕捉電極單元 302.… ....捕捉段 51 ..... ....捕捉電極 303.... ....儲液段 511.··· ....捕捉部 31 ..… ....基板層 6....... ....遮蔽電極 32 …·· ....流道層 61 "… ....粗糙面 33 ..... ....覆蓋層The photon beam is 514 nm Argon laser light 'the intensity of the laser light is about 1 mW, the laser spot is about 8 μιηη', the concentration of Staphylococcus aureus is 1 〇8 cFu/ml 〇 with Figure 5, measured on the surface of the flat shielding electrode 6 In the Raman spectral signal, 'only two tiny peak signals appear' are 1158, 1522 and 2940 cm·1, respectively, based on the peak signal of 1158 cm·1, and the signal strength is about 250 counts. In the signal measured by the masking electrode 6 of the roughness-2, eight distinct peak signals appear, which are 1, 1158, 1288, 1523, 1588, 2313, 2670 and 2938 cm·1, of which 1158 The peak signal strength of cm 1 is approximately 1500 counts. In the signal of the surface of the electrode 6 of the cover 201102648 of the thick chain _1, twelve identification peak signals appearing at 960, 1007, 1199, 1158, 1288, 1442, 1522, 1589, respectively. 2313, 2535, 2670 and 3035 cm·1, and the peak signal intensity of 1158 cnT1 is as high as 7000 counts'. Compared with the surface of the flat shielding electrode 6, the Raman spectral signal is enhanced by about 30 times, which proves that the invention is roughened. The design of the shielding electrode 6 can effectively increase the Raman spectral signal. Then, the sample mixed with the blood cells and the bacteria is directly separated into the microfluidic wafer of the present invention to capture concentration and Raman spectroscopy. _ As shown in Figures 2, 6, and 7, the bacteria used are Staphylococcus aureus [& BCRC 14957] at a concentration of 1 〇 6 CFU/ml, and the voltage applied to the sorting electrodes 41 is 12 Vpp. The frequency is 5 〇〇 kHz, the capture electrode voltage is 20 Vpp, the frequency is 500 kHz, and the fluid flow rate is ι pL/min. When a continuous fluid with blood cells and bacteria flows through the sorting electrodes 41 in sequence, the blood cells The cells and bacteria are separately manipulated and separated into different capture segments 302, and the manipulation and separation directions of the blood cells and bacteria are respectively shown in the directions indicated by the key symbols in Figures 6 and 7, and captured by the capture electrodes 5 ι φ. Immediately after two minutes, Raman light level detection was performed on the microfluidic wafer.曰With FIG. 8, when the microfluidic chip is not provided with the shielding electrode 6, since the fluorescent noise is too large, even if the Raman spectrum is corrected by the reference line, the gas is seen to have a tiny peak at 2940 cm, most of which is The Raman light level signal has been covered by fluorescent noise. 3 When the flat-shaped divergent electrode 6 is provided in the microfluidic wafer, its Raman diagram 10 201102648 spectrum has a barely identifiable signal at 1008, 1158, 1520, 2670 and 2940 cm.1, and its signal is slightly larger than the direct inspection. The Raman spectroscopy signal (not shown) detected on the surface of a generally flat gold deposit is estimated to be because the bacteria are locally concentrated in the region of the occluding electrode 6 in the microfluidic wafer. Increased, so the signal detected after the concentration of the microfluidic wafer is slightly larger than the signal of the generally flat gold-plated surface. When the coarse-grained shielding electrode 6 is provided in the microfluidic wafer, the Raman spectrum is sufficient at 958, 1007, 1199, 1158, 1286, 1448, 1522, 1588, 2313, 2536, 2670, and 2933 cm·1. The signal is recognized, and the signal intensity is increased by about ten times compared to the microfluidic wafer of the flat shielding electrode 6. And it has been experimentally confirmed that under the action of continuous fluid and the dielectrophoretic force capture and concentration generated by the microfluidic wafer, the sample concentration required for the sample can be relatively reduced'. When the bacterial concentration is about 1〇6 CFU/ml, the separation is performed. It takes only three minutes to capture the entire process of Raman spectroscopy. When the concentration of bacteria is about 1〇5 CFU/ml, the process of concentration and Raman spectroscopy can be completely separated and captured in about 30 minutes. In summary, the structural design of the sorting electrode unit 70 4 , the capturing electrode unit 5 and the shielding electrodes 6 is provided in the micro flow channel 3 of the wafer body 3, and the sorting electrode unit 4 can be used first. Capturing the electrophoretic force generated by the electrode unit 5, separating biomolecules having different dielectric properties in the sample, and controlling the predetermined capturing segment 302, and then collecting the separated biomolecules under the shielding electrode 6, In this way, even a low-concentration biological sample can be concentrated by the capture electrodes 51, so that the region where the shielding electricity #6 is located has a very high concentration of the biological molecules to be tested, thereby solving the current pull. Mann optical inspection cannot be applied to the bottleneck of low-concentration samples. In addition, through the above structural design, the microfluidic wafer can also perform Raman spectroscopy detection in the process of separation and capture concentration of biomolecules, and the Raman spectroscopy signal generates magnetic field resonance by the shisha shielding electrode 6'. The enhancement of dozens of times 'in addition to greatly increasing the signal identification and shortening the detection time', when identifying biomolecules, it can also be measured with lower intensity laser light (about 1 mW) to avoid The activity of the biological sample is destroyed, and the signal of sufficient identification is obtained by the effect of the roughening shielding electrode 6. Therefore, the present invention has the functions of rapid separation and capture concentration, and combined with surface-enhanced Raman spectroscopy (4) microfluidic wafers, which can be widely used in the isolation and identification of clinical pathogenic bacteria, as well as pathogenic bacteria in animal husbandry and food industries. With rapid detection of quality, etc. The object of the invention can indeed be achieved. The above is only the preferred embodiment of the present invention, and the scope of the invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All remain within the scope of the invention patent. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an exploded perspective view of a preferred embodiment of a microfluidic wafer having Raman spectral enhancement function of the present invention; FIG. 2 is a schematic bottom view of the preferred embodiment, wherein a substrate layer Removed, and illustrates the relative positional relationship between a sorting electrode unit, a trapping electrode unit, a plurality of shielding electrodes, and a microchannel; 12 201102648 Atomic force of the rough surface of the shielding electrode FIG. 3 is a microscope image of the preferred embodiment Fig. 4 is a view similar to Fig. 3; Fig. 5 is a Raman spectrum obtained by directly placing golden yellow (tetra) cocci on the cover (four) pole for pulling light, and detecting the microscopic image of the cell; , saying that ~ the control of the Shanghai branch is controlled by the continuous fluid, and the movement of the electrode is selected to move in the direction of extension; Figure 7 is a view similar to Figure 6, when the ball cells are separated from the bacteria; For the sorting electrode, the blood is shown in Figure 8. The Raman spectroscopy is used to detect the bacteria from the corpus callosum. 13 201102648 [Main component symbol description] 3....... •... Wafer body 4..... Sorting electrode unit 30 ..... micro flow channel 41 ..... .... sorting electrode 301 ..... sorting segment 5....... .... capture electrode unit 302.... ....capture segment 51 ..... .... capture electrode 303..... reservoir segment 511.···.. capture portion 31 ..... .... substrate Layer 6.............shading electrode 32 ...··.. runner layer 61 "....rough surface 33 ..... .... covering layer
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