1281947 玖、發明說明： 【發明所屬之技術領域】 5 適用 本發明係關於-種生物晶片及其檢測裝置，尤指__種 於DNA分子檢測用之生物晶片及檢測裝置。 【先前技術】 生物晶片係指在玻璃、矽片、塑膠等材質上，整合微 機電技術，完成生物化學樣品之分析或製備。近年來生物 晶片迅速發展，其優點為分析速度快、所用樣品及試劑少， 10並可於短時間内得到整體性的分析數據；而其關鍵技術在 於一個晶片上所能整合之功能，以及分析結果之可信度與 精確性。因此，樣品需要量少、功能多的生物曰曰曰Μ，二及 準確靈敏的偵測裝置便十分重要。目前生物晶片按其用途 大致了刀為一種類型·微流體晶片（Micr〇nuidic chip)、微 15陣列晶片（Micr〇_array chip)，以及微晶片實驗室 (Lab-on-a-chip)，可應用於樣品製備、生化反應與結果檢 測等領域。其中，微流體晶片與微晶片實驗室係結合反應 單元與分離單元於一晶片上，可藉由程式控制反應流程， 再用各種驅動方式使反應完成之樣品進入分離單元進行樣 20品之分析與製備。此種設計大幅縮短反應所需時間，且在 短時間内可獲得大量分析資料。 生物ΒΘ片之分析關鍵技術之一在於樣品偵測的精確 度與可信度，因此，如何降低背景值之干擾便是此領域所 欲突破之技術。在生物樣品偵測方面常用光學或電學方式 1281947 偵測，其中一種生物分子常用的光學偵測方法係為螢光偵 測’其優點為靈敏度高、所需樣品量少，且不會破壞生物 樣品。因此，實驗室中廣泛應用螢光偵測方法，亦發展至 生物晶片之偵測分析上。 5 習知之生物晶片及其螢光偵測裝置如第1圖。係以雷 射光源110作為激發（excitation)光源，入射光經分光鏡 120 ( beam splitter)作全反射，透過目鏡13〇聚焦照至樣品 140上’引發樣品發射（emissi〇n)出螢光14ι ;再由目鏡 130收集樣品140所發出之螢光141，此螢光141可穿透分光 10鏡120 ’經針孔（pinh〇ie ) 150聚焦放大，再經濾光鏡ι6〇 濾去原激發光之干擾，傳送至偵測器17〇偵測。然此裝置 中，激發光與螢光行進同一路徑，激發光對於偵測結果干 擾非常大；因此，分光鏡與濾光鏡的選擇相當重要：分光 鏡需有效區隔出激發光與螢光，同時必須使螢光穿透率 15高；濾光鏡則必須只允許小範圍波長通過。此種設計相當 繁複且有諸多限制。此外，一般所使用之分光器效果大多 不盡理想，致使激發光之干擾仍相當大。之後，便有設計 將激發光源入射路徑與螢光偵測路徑分開，發現在兩光線 路徑成45度角時，可降低激發光源所造成的干擾。但此方 2〇法須將整個偵測裝置固定於某一角度，在操作上相當不方 便。 另外，由於螢光相當微弱，因此若有一光線收集裝置 能降低螢光之散失，便可大幅增加偵測訊號。先前技藝如 第2圖，係以黃光製程於基板21〇上钱刻出凹槽，並注入適 1281947 ^緩衝/容液形成電泳分離單元240 ,並將光纖230以黃光 ^ 口，於基板210上之樣品出口處附近，以收集樣品所發 古之螢光’ Α法雖可增加偵測訊?虎，然黃光製程之成本過 且須將光纖與樣品出口處對準，過程相當複雜，不符 而要因此’目丽仍需要一個可提高偵測訊 干擾，义同時操作方便之摘測裝置。 ^月厅、 、"人爰因於此，本於積極發明之精神，亟思一種可 ίο 15 上述問題之「生物晶片及其檢測裝置」，幾經研究 貫驗、、至完成此項嘉惠世人之發明。 【發明内容】 具有一分 本發明之主要目的係在提供—種生物晶片 離早凡，可快速分離生物樣品。 同時兼具 本發明之另_目的係在提供_種生物晶片.粟 口口 乂之刀離單元與一 Dna分子反庫輩开-ρ Α 之製備與分析。 刀子反應早几，可-次完成樣 置，二之又一目的係在提供-種生物晶片檢測裝 置俾此提鬲偵测訊號、降低昔旦 ^ ^ m 丨令低月不干擾、降低檢測成本、 工間使用彈性大，同時提供—操作方便之裝置。 係包:達本發明「生物晶片及其檢測裝置」 孫…、 具有溝槽；-樣品載入單元， 上，_八输口口一 樣口口刀離早兀，係直立於該基板 〜刀離早兀之一端係位於該溝槽上。 本發明亦關於一種適用於分離嶋分子之生物晶 20 1281947 片，包含：一基板，該基板上具有溝槽；一樣品載入單元， 係位於該溝槽上；一樣品分離單元，係直立於該基板上， 該分離單元之一端係位於該溝槽上；以及—dna分子反應 單元，亦位於該溝槽上。 5 本發明亦關於一種生物晶片檢測裝置，係配合一生物 晶片，包含：一基台，以固定該生物晶片；一光纖，係位 於该生物晶片之上方，且該光纖之一末端與該生物晶片欲 偵測之表面形成一介於25至90度之夾角，以偵測該生物晶 片表面之光變化；其中該光纖之末端不與該生物晶片直接 10 接觸。 由於本發明構造新穎，能提供產業上利用，且確有增 進功效，故依法申請發明專利。 【實施方式】 15 為月b讓貝審查委員能更瞭解本發明之技術内容，特 舉一較佳具體實施例說明如下。 如第3圖所示，本裝置包含一生物晶片基板3 1〇，基板 3 1〇上刻有溝槽3 11與3 12，該溝槽3 11與該溝槽3 12交會形成 T型。該基板310上具有一 PCR反應單元34〇、一樣品載入單 20元32〇，以及一電泳單元330。其中該PCR反應單元係位於 "亥溝槽311上之樣品載入單元負極321處；該PCR反應單元 包含一可更換之毛細管341、一上蓋342以及一溫度控制單 元’其中該毛細管341外緣塗覆有避光隔熱物質，且長度在 1 〇 a刀以下（視所使用之核酸分子長度而定）。而該樣品載 1281947 入單元320亦形成於該溝槽3 12上，係於溝槽3 12之兩端分別 置有負極321與正極322，用以提供樣品載入所須之驅動電 壓；溝槽内並填充有緩衝溶液如PBS，用以帶動樣品到達 分離單元。該電泳單元係位於於溝槽312上，其包含一組驅 5 動電極，分別為負極332與正極333，以及一提供電泳分離 環境之毛細管331，該毛細管33 1係直立於該溝槽3 11與該溝 槽312交界處，其上方有一上蓋334;且該毛細管331中填充 有適合DN A分子分離之聚丙烯醯胺（Polyacrylamide)。本發 明更包含一光學偵測單元，係於樣品出口處（毛細管電泳正 10 極333附近）附近置有一雷射光源351與一光纖352 ;其中該 雷射光源351與該光纖352形成90度角。 以DNA分子定序工作為例。首先係先將DNA定序所須 之PCR反應所須之各種試劑、酵素與樣品混合準備好，再 以虹吸原理將前述混合樣品吸入毛細管341中，並蓋上上蓋 15 342，避免加溫過程中使水分蒸散流失，同時可維持溫度。 之後再藉由溫度控制單元控制溫度升降程式，進行PCR反 應。反應完成後，樣品載入單元之電壓開啟，此時PCR反 應單元中之DNA樣品恰位於負極321，受到電壓驅動，樣 品往正極322方向移動；待樣品移動至溝槽311與溝槽312 20 交界處時，正好是毛細管電泳331之入口，此時便有另一組 分離單元之電壓啟動，此電壓遠高於載入電壓，約為 5000V。此時樣品開始進行分離，由於毛細管331中填充有 適當濃度之聚丙烯醯胺（Polyacrylamide)，可進行解析度相 當高（1-2個base)之DNA分子分離工作。待電泳分離完成 1281947 後’ 7 口處，即正極333附近，進行樣品_步驟。本例 偵測，、由雷射光源351提供連續式雷射光束作為激 :，數發樣品發出螢光，再由光纖352收集此螢光訊 邊。该雷射光源35卜毛細管331與光纖352之 以第4圖作更進一步之解說。 、…第圖係本發明生物晶片之痛測裝置之上視圖。其中 10 15 〇/、忒毛細官430形成之直線，與該光纖420與該毛 心430$成之直線呈9〇度角或是25度（布魯斯特角），意即 雷射光源所射出之光線，與光纖所收集的螢光方向呈9〇度 角或2續角，如此便可降低雷射光源對於螢光分析之干擾^ 、’不上所述，本發明提供了一種可快速製備、快速分析 且偵測準確之生物晶片及檢測裝置。就反應單元而言，在 PCR反應中最重要需突破的技術便在於溫度之控制，·而本 發明以短小之毛細管作為反應環境，相較於傳統PCR反應 =而言，本單元溫度控制相當準確，並可於數秒内完成升 溫與降溫動作’使原本需要數小時之反應可縮短為數十分 鐘，效果十分顯著。本發明亦加上一上蓋預防水分之蒸散， 維持反應之品質；如前所述，此單元所需之樣品量相當少， 只須零點幾微升（μ1)之樣品量即足夠。此種反應裝置兼具 20操作方便、使用樣品量少以及反應時間短、反應品質控制 佳之優點。 就分離單元而言，本裝置使用lOOOV/cm之高電壓梯 度’使樣品分離時間大幅縮短，且經實驗證明解析度相當 好。由於所使用之毛細管長度不超過十公分，管徑亦只有 1281947 幾微米，所須之樣品量亦相當少。相較於傳統製程，相當 省時及節省原料。且本裝置之分離單元主要係配合該偵； 裝置，其相對位置對於降低背景值相當重要，因此該分離 單元並非與習知為流體晶片一樣，為水平置放於晶片上， 5而疋直立於曰曰片上，此種設計考量未曾見於習知技藝中， 且經實驗證實此種設計確實可降低激發光源之干擾，择 偵測訊號之準確度。 就偵測單元而言，本裝置利用光纖固定某一角度收集 螢光，佔龐大體積與重量之偵測器位置不受限制，僅需移 H)動輕小之光纖對準即可，空間使用彈性大。且以光纖傳輸 螢光訊號，訊號衰減小。且光纖不須黏附至晶片上，省去 育光製程之成本與繁靖步驟，並可重複使用光纖，兼呈成 本低廉與使用方便操作簡單之優點。上減如上述，此侦測裝 置不僅可使用於微流體晶片或微晶片實驗室，亦可應用於 15微陣列晶片，只要是使用螢光偵測方式皆可使用，應用範 圍更廣。此外，本發明全程可以微電路控制。結合上述優 點，本發明除了能達到傳統之毛細管電泳或一般生物分子 分離系統之效果外，更具備快速、自動化、系統整合以及 更加靈敏之優點。 20 、综上所陳，本發明無論就目的、手段及功效，在在均 顯不其迥異於習知技術之特徵，為「生物晶片及其檢測裝 置」之-大突破，懇請早日賜准專利，俾嘉惠社會，實感 乙便I·隹應庄思的疋，上述諸多實施例僅係為了便於說明 1281947 而舉例而已，本發明所主張之權利範圍自應以申請專利範 圍所述為準，而非僅限於上述實施例。 & 【圖式簡單說明】 5第1圖係習知生物晶片之示意圖。 第2圖係習知生物晶片之示意圖。 第3圖係本發明生物晶片及其偵測裝置之示意圖。 第4圖係本發明生物晶片偵測裝置之上視圖。 10 【圖 號說明】 110 雷射光源 111 渡鏡 120 分光鏡 130 目鏡 140 生物樣品 141 螢光 150 針孔 160 渡鏡 170 光學偵測裝置 210 基板 220 雷射光源 230 光纖 310 基板 311 溝槽 312 溝槽 320 樣品載入單元 321 樣品載入驅 動322 樣品載入驅動 電極負極 電極正極 330 電泳單元 331 毛細管 332 電泳單元驅動 電極負極 333 電泳單元驅動 334 上蓋 340 PCR反應單元 電極正極 341 毛細管 342 上蓋 350 偵測單元 351 雷射光源 352 光纖 121281947 发明Invention Description: [Technical Field of the Invention] 5 Application The present invention relates to a biochip and a detection device thereof, and more particularly to a biochip and a detection device for DNA molecule detection. [Prior Art] Biochip refers to the integration of MEMS technology on glass, enamel, plastic and other materials to complete the analysis or preparation of biochemical samples. In recent years, biochips have developed rapidly. The advantages are fast analysis, low sample and reagents, and comprehensive analysis data in a short time. The key technology is the integration of functions on a wafer and analysis. The credibility and accuracy of the results. Therefore, it is important to have a small amount of sample, a large number of functional biopsies, and an accurate and sensitive detection device. At present, biochips are roughly classified into a type of microfluidic chip, a Micr〇_array chip, and a Lab-on-a-chip. It can be applied to the fields of sample preparation, biochemical reaction and result detection. Wherein, the microfluidic wafer and the microchip laboratory are combined with the reaction unit and the separation unit on a wafer, and the reaction flow can be controlled by a program, and then the sample of the reaction is entered into the separation unit by various driving methods to perform analysis of the sample 20 and preparation. This design drastically reduces the time required for the reaction and provides a large amount of analytical data in a short period of time. One of the key techniques for the analysis of bio-slices is the accuracy and credibility of sample detection. Therefore, how to reduce the interference of background values is the technology to be broken in this field. In the detection of biological samples, optical or electrical methods are commonly used for 1281947 detection. One of the commonly used optical detection methods for biomolecules is fluorescence detection. The advantages are high sensitivity, low sample volume, and no damage to biological samples. . Therefore, the fluorescence detection method is widely used in the laboratory and has also been developed to detect and analyze biochips. 5 The conventional biochip and its fluorescent detecting device are as shown in Fig. 1. The laser light source 110 is used as an excitation light source, and the incident light is totally reflected by a beam splitter 120, and is focused on the sample 140 through the eyepiece 13 ' 'initiating sample emission (emissi〇n) out of the fluorescent light 14 The eyepiece 130 collects the fluorescent light 141 emitted by the sample 140, and the fluorescent light 141 can penetrate the splitting light 10 mirror 120' through the pinhole (pinh〇ie) 150 for focusing and amplification, and then filtering through the filter ι6 去 to the original excitation The interference of light is transmitted to the detector 17 for detection. However, in this device, the excitation light and the fluorescent light travel in the same path, and the excitation light interferes greatly with the detection result; therefore, the selection of the beam splitter and the filter is very important: the beam splitter needs to effectively separate the excitation light and the fluorescent light. At the same time, the fluorescence transmittance must be 15 high; the filter must allow only a small range of wavelengths to pass. This design is quite complicated and has many limitations. In addition, the effects of the spectroscopes generally used are mostly unsatisfactory, so that the interference of the excitation light is still quite large. Later, there is a design to separate the incident path of the excitation light from the fluorescent detection path, and it is found that the interference caused by the excitation light source can be reduced when the two light paths are at an angle of 45 degrees. However, this method must fix the entire detecting device at an angle, which is quite inconvenient in operation. In addition, since the fluorescence is rather weak, if a light collecting device can reduce the loss of fluorescence, the detection signal can be greatly increased. In the prior art, as shown in FIG. 2, a groove is formed on the substrate 21 by a yellow light process, and an electrophoresis separation unit 240 is formed by injecting 1281947 ^ buffer/capacity, and the optical fiber 230 is yellowed on the substrate. Near the exit of the sample on the 210, the ancient fluorescent method of collecting samples can increase the detection signal. The cost of the yellow light process must be aligned with the fiber exit. The process is quite complicated. Therefore, it is necessary to have a measuring device that can improve the detection interference and facilitate the operation at the same time. ^月厅,,"人爰 Because of this, in the spirit of active invention, I have thought of a "biochip and its detection device" that can solve the above problems. After several studies, the completion of this benefit The invention of the world. SUMMARY OF THE INVENTION One of the main objects of the present invention is to provide a biological wafer that is relatively early and can rapidly separate biological samples. At the same time, the other object of the present invention is to provide a preparation and analysis of a biochip, a knife-to-cell, and a Dna molecule. The knives react a few times earlier, and the sample can be completed. The second purpose is to provide a biochip detection device, which can detect the signal and reduce the smear. The work space is flexible and provides a convenient device for operation. The bag: the invention of the "bio-wafer and its detection device" Sun..., with a groove; - sample loading unit, upper, _ eight-port mouth like mouth knife away from early, is standing on the substrate ~ knife away One of the early ends is located on the groove. The invention also relates to a biocrystal 20 1281947 sheet suitable for separating ruthenium molecules, comprising: a substrate having a groove thereon; a sample loading unit located on the groove; and a sample separation unit erected on the substrate On the substrate, one end of the separation unit is located on the groove; and a -dna molecular reaction unit is also located on the groove. 5 The present invention also relates to a biochip detecting device, which is coupled to a biochip, comprising: a base for fixing the biochip; an optical fiber located above the biochip, and one end of the optical fiber and the biochip The surface to be detected forms an angle of between 25 and 90 degrees to detect a change in light on the surface of the biochip; wherein the end of the fiber is not in direct contact with the biochip 10. Since the invention has novel construction, can provide industrial utilization, and has an improvement effect, it applies for an invention patent according to law. [Embodiment] 15 For the month b, the board review committee can better understand the technical contents of the present invention, and a preferred embodiment will be described below. As shown in Fig. 3, the apparatus comprises a biochip substrate 3 1 , and the substrate 3 1 is engraved with grooves 3 11 and 3 12 which intersect with the groove 3 12 to form a T-shape. The substrate 310 has a PCR reaction unit 34, a sample loading unit of 20 Å, and an electrophoresis unit 330. Wherein the PCR reaction unit is located at the negative electrode 321 of the sample loading unit on the trench 311; the PCR reaction unit comprises a replaceable capillary 341, an upper cover 342 and a temperature control unit, wherein the outer edge of the capillary 341 It is coated with a light-proof and heat-insulating substance and has a length of 1 〇a or less (depending on the length of the nucleic acid molecule used). The sample carrying 1281947 inlet unit 320 is also formed on the trench 3 12 , and has a negative electrode 321 and a positive electrode 322 respectively disposed at two ends of the trench 3 12 for providing a driving voltage required for loading the sample; The inside is filled with a buffer solution such as PBS to drive the sample to the separation unit. The electrophoresis unit is located on the trench 312, and comprises a set of driving electrodes, which are a negative electrode 332 and a positive electrode 333, respectively, and a capillary 331 for providing an electrophoretic separation environment. The capillary 33 1 is erected on the groove 3 11 . At the junction with the trench 312, there is an upper cover 334 thereon; and the capillary 331 is filled with polyacrylamide suitable for molecular separation of DN A. The invention further comprises an optical detecting unit, which is provided with a laser light source 351 and an optical fiber 352 near the sample exit (near the capillary electrophoresis positive 10 pole 333); wherein the laser light source 351 forms a 90 degree angle with the optical fiber 352. . Take the DNA molecular sequencing work as an example. First, the various reagents, enzymes and samples necessary for the PCR reaction required for DNA sequencing are mixed and prepared, and then the mixed sample is sucked into the capillary 341 by the siphon principle, and the upper cover 15 342 is covered to avoid the heating process. Allows water to evade and drain while maintaining temperature. The temperature reaction program is then controlled by the temperature control unit to perform a PCR reaction. After the reaction is completed, the voltage of the sample loading unit is turned on. At this time, the DNA sample in the PCR reaction unit is located at the negative electrode 321, and is driven by the voltage, and the sample moves toward the positive electrode 322; the sample is moved to the boundary between the groove 311 and the groove 31220. At the moment, it is exactly the entrance of capillary electrophoresis 331, at which point there is another group of separation unit voltages starting, this voltage is much higher than the loading voltage, about 5000V. At this time, the sample starts to be separated, and since the capillary 331 is filled with an appropriate concentration of polyacrylamide, separation of DNA molecules having a relatively high resolution (1-2 bases) can be performed. After the electrophoresis separation is completed, after 1281947, at the 7th port, that is, near the positive electrode 333, the sample_step is performed. In this example, a continuous laser beam is provided by the laser source 351 as a laser: a plurality of samples are emitted, and the fluorescent signal is collected by the optical fiber 352. The laser source 35, the capillary 331 and the optical fiber 352, are further illustrated in Fig. 4. The figure is a top view of the pain test device of the biochip of the present invention. The straight line formed by 10 15 〇/, 忒 细 细 430, and the straight line of the optical fiber 420 and the 440 430 is 9 degrees or 25 degrees (Brewster angle), which means that the laser light source emits The light is at a 9-degree angle or a 2-degree angle with the direction of the fluorescence collected by the optical fiber, so that the interference of the laser source with the fluorescence analysis can be reduced, and the present invention provides a rapid preparation, Rapid analysis and detection of accurate biochips and inspection devices. As far as the reaction unit is concerned, the most important technology to be broken in the PCR reaction is the control of temperature. However, the present invention uses a short capillary as the reaction environment, and the temperature control of the unit is quite accurate compared to the conventional PCR reaction. And can complete the heating and cooling action in a few seconds' so that the reaction that would otherwise take hours can be shortened to tens of minutes, the effect is very significant. The present invention also incorporates an upper cover to prevent moisture evapotranspiration and maintain the quality of the reaction; as previously mentioned, the amount of sample required for the unit is relatively small, and a sample amount of only a few microliters (μ1) is sufficient. The reaction apparatus has the advantages of convenient operation, small sample amount, short reaction time, and good reaction quality control. In the case of the separation unit, the apparatus uses a high voltage gradient of 100 Ω/cm to greatly shorten the sample separation time, and the resolution is proved to be quite good by experiments. Since the capillary length used is no more than ten centimeters, the diameter of the capillary is only 1281947 micrometers, and the amount of sample required is also relatively small. Compared with the traditional process, it is quite time-saving and saves raw materials. And the separation unit of the device mainly cooperates with the detection device; the relative position of the device is very important for reducing the background value, so the separation unit is not placed on the wafer horizontally like the conventional fluid wafer, and the 疋 is erected on the On the cymbal, this design consideration has not been seen in the prior art, and it has been experimentally confirmed that this design can reduce the interference of the excitation light source and select the accuracy of the detection signal. In terms of the detecting unit, the device uses the optical fiber to fix the fluorescent light at a certain angle, and the position of the detector occupying a large volume and weight is not limited, and only needs to be moved by moving the light and small fiber, and the space is used. Very flexible. And the optical signal is transmitted by the optical fiber, and the signal attenuation is small. Moreover, the optical fiber does not need to be adhered to the wafer, the cost and the stimulating step of the cultivating process can be omitted, and the optical fiber can be reused, and the utility model has the advantages of low cost, convenient use and simple operation. As described above, the detection device can be used not only in microfluidic wafer or microchip laboratories, but also in 15 microarray wafers, as long as it can be used by using fluorescence detection, and the application range is wider. Moreover, the present invention can be controlled throughout the microcircuit. In combination with the above advantages, the present invention has the advantages of being fast, automated, system integrated, and more sensitive, in addition to the effects of conventional capillary electrophoresis or general biomolecular separation systems. 20 In summary, the present invention, regardless of its purpose, means and efficacy, is not surprisingly different from the characteristics of the prior art, and is a big breakthrough for "biochips and their detection devices". , 俾 惠 社会 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , It is not limited to the above embodiment. & [Simple diagram of the diagram] 5 Figure 1 is a schematic diagram of a conventional biochip. Figure 2 is a schematic representation of a conventional biochip. Figure 3 is a schematic view of the biochip of the present invention and its detecting device. Figure 4 is a top view of the biochip detecting device of the present invention. 10 [Description of the number] 110 Laser light source 111 Ferro mirror 120 Beam splitter 130 Eyepiece 140 Biological sample 141 Fluorescent 150 Pinhole 160 Ferro mirror 170 Optical detection device 210 Substrate 220 Laser light source 230 Optical fiber 310 Substrate 311 Groove 312 Groove Slot 320 Sample Loading Unit 321 Sample Loading Drive 322 Sample Loading Drive Electrode Negative Electrode 330 Electrophoresis Unit 331 Capillary 332 Electrophoresis Unit Drive Electrode Negative 333 Electrophoresis Unit Drive 334 Top Cover 340 PCR Reaction Unit Electrode Positive 341 Capillary 342 Upper Cover 350 Detection Unit 351 laser source 352 fiber 12