200940986 九、發明說明： 【發明所屬之技術領域】 本發明涉及-種生物晶片，特別是一種帶有特徵光譜 識別光敏感測器陣列的生物晶片。本發明還涉及該生物晶 片的製備方法以及使用該生物晶片的裝置。 【先前技術】 、 生物晶片（bioehip)的原型是8()年代 發展至今已成為現代生命科學研" ® W包減目W和蛋技術之—。生物 研、《診斷、蝴域並廣泛應料生物科 現有技術的基因晶片主要 聚合物基片（尼龍膜、硝醆懺二固定在 通過放射顯影技術進行檢測素2==;=’並 ::=，通過與榮光標記== ❹ _，與螢光俨上直接原位合成的寡核苷酸探針 類型的基因行檢^其中，第-種 大、定量檢測不可靠等輕，又低、*_品#°試劑的需求量 的基因晶二常也 :二灿乍用;抗原或抗體可 目前：軚§己的相對應的抗原或抗體進行檢測。 目4泛應_生物W縣均是_螢光檢測的 200940986 方法進行檢測，最常用的檢測方法主要有雷射共 方法（見第一圖）和CCD相機檢測方法。 w “、、卸描 弟圖示出了現有技術的利用生物晶片對基一人 測的一種裝置。第一圖的檢測裝置由雷射源（未示行= 路系統以及光接收裝置構成，其中光接收裝置是弁ϋ 管，而光路系統包括圖中所示的分束器、物鏡、發射= 器、檢測鏡頭、共焦針孔和用於轉折光路的反射鏡等❿美 因晶片置於第一圖所示檢測裝置中的物鏡下，並待制ς ❾ 111樣品與基因晶片接觸。現有技術的基因晶片是製作= $等硬質基底上的基因探針_，當作了$光標記的待測 =因樣品與之接觸時，會發生雜交反應。在激發光（♦射 ，束）的照射下，與基因探針完全互補匹配的位置會:出 最強的螢光。第一圖所示的檢測裝置用雷射共聚焦逐 黑=測昼京光的位置和強度，從而可以對待測基因樣品進行 分析和檢測。這種檢測方式的探測靈敏度很高，但測試速 度很慢，操作也很複雜。一種可行的改進方法是用cCD _ ^機取代光電倍增管，並用寬束雷射照射，這種方法可以 提向檢測速度，但缺點是探測靈敏度很低。 在第一圖所示利用雷射共聚焦掃描方法的檢測裝置 中，由於採用高靈敏度的光電倍增管，所以其探測靈^度 :以很鬲，但設備也很昂責。此外，由於其採用的是逐點 檢，的方法，所以測完整個生物晶片需要耗的時間报長， 不能獲得生物反應的即時資訊。使用c C D相機檢測方法的 優點是檢測速度較快，但需要一套較複雜的成像系統，造 成其有效孔徑較小。並且，由於螢光發射基本是全方位的 球形發射’因此這種方法的探測靈敏度很低。另外，由於 200940986 成像光學像鱗誤差影響，可能會帶來 ，差，尤歧對於高密度基因晶片的檢測更是如此。對於 無論是採用光電倍增管還是CCD相機 勺檢別裝置都$要進行更換光學濾光片的操作，這也 來-定的誤差’且對獲得即時反應f訊造成了限制。 、隹η必要對現有技術的生物晶片及其檢測方法作 進一步的改善。 【發明内容】 Η广 =卜Γ的是提供一種探測靈敏度*、檢測時 、B、U於夕榮光光譜檢測的新型生物晶片。本發明 =:I製備該生物晶片的方法以及利用該生物晶片對生 物樣品進行檢測的裝置。 〃 一根據本發明—種實施例的生物晶片包括至少 早兀以及至少-組生物分子探針，每個檢測料包括至^ :個檢測—和至少—個參考料，每個檢測單元對應於 -組相同的生物分子探針；每個檢測圖元包括_ .導 體光學感測ϋ中的-個半導體光學感·以及陣 的—個光學濾'光片，所述—個光學濾光片完全; 二所述-個半導體光學感測器的表面；每個參考圖元包括 Γϋί導體光學感測器中的一個半導體光學感測器；每 ，先于滤m有特徵波長，使得以所述特徵波長為中心 的預定波長寬度範_的波長能夠經過光學渡光片透 而所述範圍之外的波長不能透射。 上述特徵波長通常就是待測螢光光譜的波長。根 發明，檢_元只接收和測量該榮光光譜的能量；而表 圖元用來探測激發光源的能量，並將兩種圖元的輸出相結 200940986 而使:Γ二 元，因激_、的能量較強 膜。已鑛有先學漁光可以覆蓋有技光學衰減薄 體光學感測器的i面可學=薄膜的陣列式半導 用化學或機械拋光方法將介質保護層’並可以 面。這樣既可以賴加；^辆的完整平 又可的生物分子的液體侵入， ❹ Ο 方法製作紅㈣適#表接^认法或敎合成法等 面’也可以先製作到另—片介質保護層的表 向介質保護層的方式料的基底上，織以面 量單元對應於__二生的表面上。每個測 括生s ^ 4 生物樣品進行檢測的裝置包 例，此時生物’心上二：，貧訊處理部分。以基因檢測為 作了榮光標記二測針即為基因探針。當已 列與基因探針完全互ΐ待測基因樣品的某一序 適當激發光的照射下，、t一、 &一雜父反應最完全，在 光级如果1'、、 廷一位置發出最強螢光，該螢光的 波;寬濾光片的以特徵波長為中心的所述預定 測器中的檢=圖元=穿過光學據光片而被陣列式光學感 分佈資訊，、元會接收到激發光的強度 度和位置資訊資訊處理部分，與榮光強 蛋白質檢測雜也與之類似’只不過反應是基於抗原- 8 200940986 抗體反應。 優選地，根據本發明的生物檢測裝置中還可以加入溫 度控制系統’以幫助控制反應’同時還能即時監測反應過 程，例如，即時聚合酶鏈式反應(PCR)檢測。200940986 IX. Description of the Invention: [Technical Field] The present invention relates to a biochip, and more particularly to a biochip with a characteristic spectral identification photosensor array. The present invention also relates to a method of preparing the biochip and an apparatus using the same. [Prior Art], the prototype of bioehip is the 8 () era. It has become the modern life science research " ® W package reduction eye and egg technology. Bio-research, "diagnosis, butterfly field and widely applied to the bio-technical gene chip main polymer substrate (nylon film, niobium II fixed in the detection by radiography technology 2 ==; = ' and:: =, by the glory label == ❹ _, and the direct synthesis of the oligonucleotide probe type of gene in the direct detection of the gene, which is the first, large, quantitative detection is not reliable, low, *_品#°Reagents of the required amount of the gene crystal is also often used: two can be used; antigen or antibody can be detected: 軚 § the corresponding antigen or antibody for detection. 目 4 pan should be _ biological W county are _ Fluorescence detection 200940986 method for detection, the most commonly used detection methods are mainly laser common method (see the first figure) and CCD camera detection method. w, ", the schematic diagram shows the prior art of the use of bio-chip A device for measuring one person. The detecting device of the first figure is composed of a laser source (not shown = road system and light receiving device, wherein the light receiving device is a manifold, and the optical path system includes the points shown in the figure. Beam, objective, emission = detector, detection lens, total The pinhole and the mirror for the turning optical path are placed under the objective lens in the detecting device shown in the first figure, and the sample of the ❾111 is contacted with the gene wafer. The prior art gene chip is produced = $ The gene probe _ on the hard substrate is treated as a light-labeled test. = Hybridization reaction occurs when the sample is in contact with it. Under the excitation light (♦, beam, beam), the gene probe is completely The position of the complementary matching will be: the strongest fluorescence. The detection device shown in the first figure uses the laser confocal black-by-black = to measure the position and intensity of the light, so that the sample to be tested can be analyzed and detected. The detection method has high detection sensitivity, but the test speed is very slow and the operation is very complicated. A feasible improvement method is to replace the photomultiplier tube with cCD _ ^ machine and use wide beam laser irradiation, this method can improve the detection speed. However, the disadvantage is that the detection sensitivity is very low. In the detection device using the laser confocal scanning method shown in the first figure, since the high-sensitivity photomultiplier tube is used, the detection degree is very high, but it is In addition, because it uses a point-by-point method, it takes a long time to measure the entire biochip, and it is impossible to obtain real-time information of biological reaction. The advantage of using c CD camera detection method is The detection speed is faster, but a more complicated imaging system is needed, resulting in a smaller effective aperture. Moreover, since the fluorescence emission is basically an all-round spherical emission, the detection sensitivity of this method is very low. In addition, due to 200940986 The influence of imaging optical scale error may be brought about, and the difference is especially true for the detection of high-density gene chips. For the use of photomultiplier tube or CCD camera scoop check device, it is necessary to replace the optical filter. The operation, which also comes with a fixed error, and limits the ability to obtain an immediate response. It is necessary to further improve the prior art biochip and its detection method. SUMMARY OF THE INVENTION Η广=卜Γ is a new type of biochip that provides detection sensitivity*, detection time, B, and U glory spectrum detection. The present invention =: I A method of preparing the biochip and a device for detecting a biological sample using the biochip. A biochip according to an embodiment of the present invention includes at least early and at least a set of biomolecular probes, each of which includes to: one detection - and at least one reference material, each detection unit corresponding to - Grouping the same biomolecule probes; each of the detection primitives includes _. a semiconductor optical sensation in the conductor optical sensing · and an optical filter 'optical sheet of the array, the optical filter being complete; The surface of each of the semiconductor optical sensors; each reference picture element comprises a semiconductor optical sensor in the 导体ί conductor optical sensor; each having a characteristic wavelength prior to the filter m such that the characteristic wavelength The wavelength of the predetermined wavelength width range of the center can pass through the optical light-passing sheet and the wavelength outside the range cannot be transmitted. The above characteristic wavelength is usually the wavelength of the fluorescence spectrum to be measured. According to the invention, the _ element only receives and measures the energy of the glory spectrum; and the table element is used to detect the energy of the excitation source, and the output of the two primitives is combined with 200940986 to make: Γ binary, 激 _, The energy is stronger than the membrane. The mine has a pre-learning light that can be covered with a technical optical attenuation thin optical sensor. i-surface can be learned = thin film array semi-conductor. The dielectric protective layer can be made by chemical or mechanical polishing. In this way, it is possible to make a liquid intrusion of a complete and pleasing biomolecule of the vehicle, and to produce a red (four) suitable method, such as a surface or a synthetic method. The surface of the layer is oriented on the substrate of the dielectric protective layer, and the surface unit is woven on the surface of the __. Each device that measures the s ^ 4 biological sample for testing, at this time, the biological 'heart two:, the poor processing part. Gene detection is used as the GEN light marker and the second stylus is the gene probe. When it has been completely collated with the gene probe to illuminate a certain sequence of the gene sample to be tested, the t-, &-----the most common reaction, at the light level, if the position is 1', The strongest fluorescent light, the fluorescent wave; the detecting element in the predetermined detector centered on the characteristic wavelength of the wide filter = the optical information distributed through the optical data sheet, and the element The intensity and position information processing part of the excitation light will be received, which is similar to the glory protein detection. The reaction is based on the antigen- 8 200940986 antibody reaction. Preferably, a temperature control system' can be added to the biodetection device according to the present invention to help control the reaction' while also allowing for immediate monitoring of the reaction process, e.g., real-time polymerase chain reaction (PCR) detection.
根據本發明，可以利用半導體工藝以低成本、高可靠 性、大規模地製造出生物晶片，並可以利用這樣的晶片方 便地製作出探測靈敏度高、檢測速度快、能同時檢測多個 螢光光譜、能即時監測反應過程、且體積極小、成本很低 的生物檢測裝置。 為使能更進一步瞭解本發明之特徵及技術内容，請參 閱以下有關本發明之詳細說明與附圖，然而所附圖式僅提 供茶考與說明用’並非用來對本發明加以限制者。 【實施方式】 下面將'纟σ e附圖對本發明的示例性實施例進行說明， 以2更好地理解本發明的目的、技術方案以及優點。在本 申請的說明中，相同的標號表示相同的元件。 第二圖示出了根據本發明一種實施例的生物晶片ι〇 的局部俯視示意圖。在俯視圖中，生物晶片1〇 檢測單幻00，例如，第二圖的局部俯視= 多H Ϊ㈣檢測單71 1〇G °每個檢測單元刚包括 表老囝70，足些圖元中包括至少一個檢測圖元和至少一個 對多個螢光特㈣長進行檢測的情況 的光學濾；片圖元，並使各組檢測圖元表面所帶 生物晶片^對不同的波長。例如，在本實施例中， 中，，U個測單元⑽包括兩組檢測圖元，其 板測圖凡101用於檢測CY5螢光（其特徵 9 200940986 波長在655nm〜685nm之間、„ , ^ , 用於檢測CY3螢光(豆特忾、、且_欢測圖疋102 士丄— 、将徵波長在565mn〜595mn之間）〇 在本^施例中，每個檢測單元_包括-個參考圖元1Q3。 第一圖7F出了根據本實施例的生物晶片W沿第二圖 I-III線所取的局部剖視示意圖。 =的結構至少包括基底99以及位於基底99上= j 98。優選地，生物晶片1G還可以包括位於 =的保護層刚以及位於保護層顺上方的表面修_ 基底"包括多個半導體光學感測器，這 感測器在第三圖所示座編x_y平面内以陣列 歹i例如，本發明中以陣列方式排列的多個半風 :器可^通過電_合元件（CCD)面陣感測器、 二!ros)面陣感測器、紅外陣列感測器 t朋-極體_感·等方式實現。在這些感測 鲁 極體陣列傳感器具有最高的探測$敏度。但 :遗者數碼相機、可照相手機的迅猛發展，cc :j 面陣感測器的技術已十分成熟，圖元數4 i 擇因此是本發明使用的陣列式的半導體光學感測 第二圖中的每個檢測圖元包括基底99中的— =學感·，每個參考圖元也包括基底99中的^ 2學感測器:其中，每個檢測圖元101、1〇2的表面伫 ^片’該濾、光片能夠完全覆蓋對應的檢咖元叫可 102 優選為使其形狀、大/]、與對應的檢咖元⑼、 表面相同或幾乎相同。 10 200940986 遽波器作用，I01、102上的光學濾光片具有光學帶通 的預定μ 職夠透射叫目躺_波長為中心 能透:「長;而使該範圍之外的其咐^ 低於㈣不能透射」表示相應波長的透射率 n選而要而預定設定的閾值（臨界值），例如，在 :、波長的透射率低於1%、01%或001% = §忍為其不能读结· 月兄下即可 φ ❹ 例，干出測CY3 ®光的濾、光片為 出了濾先片的光譜透射率曲線形狀的一種血 ^在第四圖中’透射的特徵波長中心約為575_，、： =波長寬度（即帶通濾、波器的通帶寬度）約為i4nm = 其相應的激發光（波長為532nm)的透過率小於愤 對於檢測其他波長所用的濾光片，其特徵波長的中0 應地位於所述其他波長處；透射的波長寬度可以， 14mn，也可以是其他數值。各個濾光片透射的波長寬= 選為2nm — 50nm之間的數值。 、又交 參考圖元103用於檢測激發光的強度和分佈。表 元103可以保持為不帶濾、光片的狀態。但是，激發光 度通常較強’有可能使參考圖元⑽的輸—號飽和，= 而參考圖7L 103優選地鍍有中性光學衰減薄膜以減 光強度。這裡「中性光學衰減薄膜」表示可以對半_ 學感測器整個回應範圍内的波長進行大致相同程度 膜層。所述中性光學衰減薄膜例如可以包括適當厚声/么 屬〇、犯、们观及其合金’其透過率例如可以在〇1^〜贈 之間。 ° 由各個檢測圖元101、102上的光學滤光片構成第 令的光譜詹98;在參考圖元1〇3上帶有中性光學衰減^ 200940986 ，下，由各個檢測圖元101、102上的来與清伞H v » 中中性光學衰減^一^成第i圖 第二圖和第三圖所示的生物晶片 圖元。但應當明白%即此堇圖Λ和兩組各12個檢測 ❹ 響 測:元數目和每個檢測單元t包括目實::檢 讀目和參考.數目都可以根 ^讀目、檢測圖 說來，根據生物分子探 命*而要來確定。一般 的檢測單元數目可 2的讀不同，每個生物晶片 可以包括1〜10 Γ ’⑻0個之間。每個檢測單元 讀範圍進行d ’用於對1〜1G個不同的檢測光 特徵波長。IU η母個Λ中的光學遽、光片具有相同的 一個之間：^ 例如可以在w。。個之間'1早兀中包括的參考圖元數目 排列在檢測單元以矩陣方式 元刚位於檢測單⑽ttr的實施例中’參考圖 位置分佈不限於第測圖元和參考圖元的 具體選擇。 ώ所不^況，而可以根據實際情況來 片和:母個檢測圖元101和102上的光學濾光 在半導體光^701G3上的光學衰減薄麟可以直接製作 在+ 表面上，其製作可以完全通過半導體 12 200940986 工藝來進行，或者採用與半導打藝相容的製作卫蔽。例 如，其製作工蟄可以包括化學氣相沉積（CVD)、踐鐘、 真空蒸鑛、或新型的激化輔助濺鍍（Radicai Assi^d S_e_，RAS) I藝等’並可以採用光刻加咖、抬離 (liftoff)、或刻蝕和抬離相結合的方法來進行圖案化， 可以包括必要的清洗、拋光、去膠等步驟。 ❹ φ 所述光學遽光片可以包括由交替排列的高折射率介質 材料層和低折射率介質材料層組成的多層膜結構。、 高折射率介質材料可以包括但不限於下列材料中的^ 種：Ti〇2、Nb2〇5、Ta：»〇;、HfD、vr 料可以包材According to the present invention, a biochip can be manufactured at a low cost, high reliability, and large scale by using a semiconductor process, and the wafer can be easily fabricated with high detection sensitivity, high detection speed, and simultaneous detection of multiple fluorescence spectra. A biological detection device capable of monitoring the reaction process in real time and having a small body and a low cost. For a better understanding of the features and technical aspects of the present invention, reference should be made to the accompanying drawings and drawings. [Embodiment] Hereinafter, an exemplary embodiment of the present invention will be described with reference to the accompanying drawings, and the objects, technical solutions and advantages of the present invention will be better understood. In the description of the present application, the same reference numerals denote the same elements. The second figure shows a partial top view of a biowafer that is in accordance with one embodiment of the present invention. In the top view, the biochip 1〇 detects a single phantom 00, for example, the partial top view of the second figure = multiple H Ϊ (four) detection list 71 1 〇 G ° each detection unit has just included the table 囝 70, including at least some of the elements An optical filter for detecting a picture element and at least one case for detecting a plurality of fluorescent (four) lengths; a picture primitive, and causing each group to detect a biochip on the surface of the picture element to a different wavelength. For example, in the present embodiment, the U measurement units (10) include two sets of detection primitives, and the panel map 101 is used to detect CY5 fluorescence (the characteristic 9 200940986 wavelength is between 655 nm and 685 nm, „, ^ , used to detect CY3 fluorescence (bean 忾, and _ 疋 疋 疋 102 丄 丄 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 565 565 565 565 565 565 565 565 565 565 Reference Figure 1Q3. First Figure 7F is a partial cross-sectional view of the biochip W according to the present embodiment taken along the line I-III of Figure 2. The structure of = includes at least the substrate 99 and is located on the substrate 99 = j 98. Preferably, the bio-chip 1G may further include a protective layer located at = and a surface repairing substrate located above the protective layer, including a plurality of semiconductor optical sensors, the sensor is shown in the third figure. In the x_y plane, an array 歹i, for example, a plurality of semi-winds arranged in an array in the present invention can pass through an electric-coupled component (CCD) area array sensor, a second ros) area array sensor, Infrared array sensors are implemented in the form of poles, poles, etc. In these sensed Lu pole array sensors have High detection of $min. However: the rapid development of digital cameras and camera phones, the technology of cc:j area array sensors is very mature, and the number of elements is therefore the array type used in the present invention. Each of the detection primitives in the second diagram of the semiconductor optical sensing includes -=sense in the substrate 99, and each reference primitive also includes a sensor in the substrate 99: wherein each detection primitive 101, 1 〇 2 surface ' ^ 'The filter, the light sheet can completely cover the corresponding checker, the callable 102 is preferably made to have a shape, large /], and the corresponding checker (9), the surface is the same or almost the same 10 200940986 Chopper function, the optical filter on I01, 102 has a predetermined band of optical bandpass. The transmission is called lie. The wavelength is centered: "long; and the outside of the range is 咐^ Below (4) "non-transmissive" means that the transmittance of the corresponding wavelength is selected to be a predetermined threshold (critical value), for example, at: the transmittance of the wavelength is less than 1%, 01% or 001% = § Can not read the knot · Moon brother can be φ ❹ Example, dry out the CY3 ® light filter, the light film is the filter first light A blood in the shape of a transmittance curve. In the fourth figure, the center of the characteristic wavelength of transmission is about 575_,, : = the width of the wavelength (ie, the bandpass filter, the passband width of the wave filter) is about i4nm = its corresponding excitation light. The transmittance (wavelength of 532 nm) is less than that of the filter used for detecting other wavelengths, and the mid-zero of the characteristic wavelength should be located at the other wavelength; the wavelength of the transmitted wavelength may be 14 nm, or other values. The wavelength of the transmission of each filter is wider = a value between 2 nm and 50 nm. And reference numeral 103 is used to detect the intensity and distribution of the excitation light. The cell 103 can be maintained in a state without a filter or a light sheet. However, the excitation luminosity is generally strong 'there is a possibility of saturating the input signal of the reference picture element (10), while the reference picture 7L 103 is preferably plated with a neutral optical attenuating film to reduce the light intensity. Here, "neutral optical attenuating film" means that the wavelength of the entire response range of the semi-sensing sensor can be approximately the same. The neutral optical attenuating film may, for example, comprise a suitable thick sound/mother, a sin, a smear, an alloy thereof, and a transmittance thereof, for example, between 〇1^~. ° The spectrum of the third order is formed by the optical filters on the respective detection primitives 101, 102; the neutral optical attenuation ^200940986 is provided on the reference primitive 1〇3, and the respective detection primitives 101, 102 are On the come and the umbrella H v » Neutral optical attenuation ^ a ^ into the i-fi diagram of the second and third figures shown in the biochip. However, it should be understood that the % is the map and the two groups of 12 detections: the number of elements and each detection unit t includes the facts: the number of readings and references. The number can be read and read. According to the biomolecular life*, it is necessary to determine. A typical number of detection units can be read differently, and each biochip can include between 1 and 10 ’ ’ (8) 0. Each detection unit read range is performed for d 'for 1 to 1G different detection light characteristic wavelengths. The optical 遽 and the light sheet in the IU η parent 具有 have the same one: ^ For example, it can be at w. . The number of reference primitives included in the '1 early 排列 is arranged in the matrix in which the detection unit is located in the matrix (10) ttr. The reference position distribution is not limited to the specific selection of the first and second reference elements. According to the actual situation, the optical attenuation on the semiconductor light ^701G3 can be directly fabricated on the + surface, and the optical filter on the mother detecting elements 101 and 102 can be directly fabricated on the + surface. It is done entirely by the semiconductor 12 200940986 process, or by a fabrication that is compatible with semi-conductive work. For example, the fabrication process may include chemical vapor deposition (CVD), practice clock, vacuum distillation, or new type of enhanced auxiliary sputtering (Radicai Assi^d S_e_, RAS) I, etc. Patterning by means of liftoff, or a combination of etching and lift-off, may include the necessary steps of cleaning, polishing, stripping, and the like.遽 φ The optical calender sheet may comprise a multilayer film structure consisting of alternating layers of high refractive index dielectric material and low refractive index dielectric material. The high refractive index dielectric material may include, but is not limited to, the following materials: Ti〇2, Nb2〇5, Ta:»〇; HfD, vr material may be packaged
MgF2、Sl〇2。具體的製作卫藝在名稱為「―種用於^作 濾光片陣列的新工藝」的中國專 ' ^ 20〇61_襲.5和名稱為「特徵光譜朗晶片= 法及使用該晶片的檢測裝置」的中國專 請^胤7中已有描述，因此這裡不再進行詳細敍述。. 各檢測圖1()2上的光學渡光片和參考 上的光學哀減薄膜厚度通常是不相同的 膜層之間還可能存在縫隙。因此，生物 ：卜攻些 =會是高低不平的，並可能對此後的生物分子探Γ十= =後，一在===== 層104的表面平坦化，從而使整個生 電路的作用，從而可以避免在生物分 13 200940986 之後將待測樣品滴入其内時，可能造成的液體侵入等不良 影響。 、 ^層1()4優選為透明的敏密介質材料。可以採用例 如屯水加強化學器相沈積（PECvd)工藝萝借MgF2, S1〇2. The specific production of Wei Yi in the name of "a new process for the use of filter arrays" in China's special ' ^ 20 〇 61 _. 5 and the name is "feature spectrum radiant wafer = method and use of the wafer The detection device has been described in the Chinese application, and therefore will not be described in detail here. The thickness of the optical fascia on each of the optical drip sheets and the reference on each of the inspection sheets 1 and 2 is usually different. There may also be gaps between the layers. Therefore, the creature: Bu attack some = will be uneven, and may be after the biomolecules after the test = 10 = one, the surface of the layer 104 is flattened =====, so that the entire circuit, Therefore, it is possible to avoid the adverse effects such as liquid intrusion which may be caused when the sample to be tested is dropped into the sample after the bio-integration 13 200940986. , layer 1 () 4 is preferably a transparent, dense dielectric material. It can be borrowed, for example, by using a water-reinforced chemical phase deposit (PECvd) process.
乍為保護層1〇4,其厚度可以是大㈣5〜二' ，為2〜20Km。但是本發明的製作工藝不限於PECVD， ^疋也可以採用其他適宜的加工工藝來進行，例如錢鑛工 蟄，特別是激化輔助濺鍍工藝，也可以是旋塗等化學涂敷 在形成保護層1〇4中的薄膜後，採用化學或機^抛 f式’例如化學機械減（CMp)卫藝，將整個表面處 理成一個光滑的平面，以利於後續工序的加工。 一在製備生物晶片時，希望基底表面是平整、潔淨、均 I的，而且自發螢光低。用化學試劑對基底表面進行表面 j可以製備表面富含活性基團的基片，這種活性基團適 二Ϊ接生物分子。在本發_上述實闕巾，例如在所述 理3，104是—層二氧化賴的情況下，可以通過酸敍處 ，在這層二氧化矽膜上形成很多矽羥基，從而有利於表 和表面塗敷。其中’表面鍵合是把含反應活性基團 、、=1通過共髓直接連接在W表面上軸具有化學 的單分子層。有機魏化試較—類常用的表面處理 ，予4劑’它們大多是輕聯齊卜例如3_氨基丙基三乙氧基 表面塗敷是通過非共彳請方式在基#材料表面形成 田二。丙烯醯胺、聚賴氨酸、俩纖維素和複脂糖是常 的表面塗敷材料。這些表面塗敷層有時也含反應活性基 如第二圖所不，經過上述表面修飾，已經過拋光的保 14 200940986 表面上可以生成—層由活性有機化合物薄膜構 05，並可以將生物分子探針陣列直 接製作在表面修飾層105上。 且 理㈣基片上製作生物分子探針_ 的過 二 固定」7採用濕式培養、烘㈣ =在夕已處理好的基片上鍵合各種生物分子，如舰、 ❹ ❹ L十L'二、酶、抗原、抗體甚至細胞等。以基因（dna) 固，斤用的方法可以是點樣法或原位合成 晉點樣法_料精密機械手等 指定的位置，並包括接觸式點樣方法: 泛使用的微加工方法，利用 系甲，、 定位置導人㈣光則丨^认膜域鏡技術等方式在預 密度寡聚核魏_。對;步合成高 物晶片分析》（科學出版社， /見1玍 詳，可以參見美國專利5,7二，)這=;= ::ϊ m而本申請中為簡明起見將不對這些方法 到奈而彳*過固定將生物分子探針陣列200結合 進二‘㉟:〇5上後’還可以用一些試劑對所得的晶片 掉，\ ’將晶Μ 4面料固定生物分子雜性基團反應 =低後期晶片使用過程中表面的非特異吸附，提高 敏度。這一過程通常稱之為「封閉」，所用的試劑乍 is the protective layer 1〇4, and its thickness can be large (four) 5~2', which is 2~20Km. However, the manufacturing process of the present invention is not limited to PECVD, and other suitable processing techniques may be used, such as a miner's work, especially an intensified auxiliary sputtering process, or a chemical coating such as spin coating to form a protective layer. After the film in 1〇4, the entire surface is processed into a smooth plane by chemical or mechanical formula, such as chemical mechanical subtraction (CMp), to facilitate the processing of subsequent processes. When preparing a biochip, it is desirable that the surface of the substrate be flat, clean, uniform, and that the spontaneous fluorescence is low. The surface of the substrate can be surfaced with a chemical reagent to prepare a substrate rich in active groups on the surface, and the reactive group is suitable for binding biomolecules. In the case of the above-mentioned real wipes, for example, in the case where the rule 3, 104 is a layer of oxidized lanthanum, a lot of ruthenium hydroxy groups can be formed on the ruthenium dioxide film by the acid narration, thereby facilitating the table. And surface coating. Wherein 'surface bonding is a monolayer having a chemically reactive group, =1 through the common marrow directly connected to the W surface and having a chemical monolayer. Organic Weihua trials are compared to the commonly used surface treatments, and 4 doses of 'most of them are lightly linked. For example, 3_aminopropyltriethoxy surface coating is formed on the surface of the base material by non-co-injection. two. Acrylamide, polylysine, two celluloses, and lipoipose are common surface coating materials. These surface coating layers sometimes also contain reactive groups as shown in the second figure. After the above surface modification, the surface has been polished. The surface of the film can be formed from the active organic compound film 05, and the biomolecule can be The probe array is fabricated directly on the surface modification layer 105. And (4) on the substrate to produce biomolecular probe _ of the two fixed "7" using wet culture, baking (four) = on the substrate has been processed on the substrate to bond a variety of biomolecules, such as ship, ❹ ❹ L L L' two, Enzymes, antigens, antibodies and even cells. The method of fixing the gene (dna) may be a spotting method or an in-situ synthesis of a spotting method, a precise position such as a precision manipulator, and a contact spotting method: a micro-machining method used in general The armor, the position of the person (4) light is 丨 ^ 膜 域 域 域 认 在 在 在 。 。 。 。 。 。 。 。 。 。 。 。 。 For the analysis of step-by-step synthesis of high-quality wafers (Science Press, / see 1 ,, see US Patent 5, 7 2), this ===:ϊϊ m and these methods will not be correct for the sake of simplicity in this application. After the biomolecule probe array 200 is incorporated into the two '35: 〇5, it is also possible to use some reagents to remove the resulting wafer, and the 'crystal wafer 4 fabric is fixed with bio-molecular hetero groups. Reaction = non-specific adsorption of the surface during low-end wafer use to increase sensitivity. This process is often referred to as "closed" and the reagents used.
:為封閉劑。常用的封閉劑有硼氫化鈉、三乙醇胺、BSA 等。 从在上述固疋過私中，在每個檢測單元上固定預定的生 分子探針，並使同—檢測單元上的生物分子探針都是相 15 200940986 f的’而不同檢測單元上 單元上的反應=二：樣= -種==二用：:的生物晶片來進行生物檢剩的 射二極體）11 (通常為固體雷射器或雷 =14後，均勻準直地照 二王息 ❹ 度不^雜^1^，因探針的序列匹配與否’發生程 度的不同’所激發出的螢 士夂认，〜矛 的你Γ /先強度和位置，制參考圖元_的各位置 各卜二度’ 一起輸，到資訊處理部分20 ’再與預知的 D早7G上的基因探針序列相比較，即可得到檢測結 。其中，光學透鏡12和導光板13可以幫助激發光束均 2照射到生物晶片10上。為了進一步提高光照均勻性並 ❿ 兩咼光強度’還可以採用並排設置多個激發光源的方式（如 田射二極體組）。同時，如果採用不同波長的多個激發光源 (例如：激發CY5螢光的035nm波長固體雷射和激發CY3 螢光的532nm波長固體雷射）並排設置的方式，還可以實 現多螢光光譜同時的即時檢測。全息片14可以幫助激發光 束基本準直地照射到基因晶片丨〇上，並抑制斜入射情況的 發生。此外，全息片14的表面還可以鍍有只透過激發光波 長、而對其他光波長有高反射率的光學濾光片，從而可以 將向外發射的螢光也反射回檢測單元，進一步提高檢測效 率。 16 200940986: is a blocking agent. Commonly used blocking agents are sodium borohydride, triethanolamine, BSA, and the like. From the above-mentioned fixation, a predetermined biomolecular probe is immobilized on each detection unit, and the biomolecule probes on the same-detection unit are both on the unit of the different detection unit. Reaction = two: sample = - species = = two with:: biochip to carry out bio-detection of the remaining emitters) 11 (usually a solid laser or mine = 14, after uniformly collimating the two kings The degree of interest is not ^1^^, because the sequence of the probe matches or not 'the difference in the degree of occurrence' is inspired by the phoenix, the spear of your Γ / first strength and position, the reference element _ Each position is divided into two degrees, and the information processing part 20' is compared with the predicted gene probe sequence on the early 7G to obtain a detection knot. The optical lens 12 and the light guide plate 13 can help the excitation. The light beam 2 is irradiated onto the biochip 10. In order to further improve the uniformity of illumination and the intensity of the two lights, it is also possible to adopt a method of arranging a plurality of excitation light sources side by side (for example, a field diode group). Multiple excitation sources (eg, CY5 Firefly) The 035 nm wavelength solid laser and the 532 nm wavelength solid laser that excites CY3 fluorescence are arranged side by side, and simultaneous detection of multiple fluorescence spectra can be realized. The hologram 14 can help the excitation beam to collimate substantially onto the gene chip.丨〇, and suppress the occurrence of oblique incidence. In addition, the surface of the hologram 14 can also be plated with an optical filter that transmits only the wavelength of the excitation light and has a high reflectance to other wavelengths of light, so that it can be emitted outward. The fluorescent light is also reflected back to the detection unit to further improve the detection efficiency. 16 200940986
除了基因檢測外，第五圖的裝置還可以對其他生物 性進行檢測’如蛋白質的抗體-抗原等。對於某些生物特性 檢測，需要監測反應的即時過程，而且反應過程與溫度 係很大，如聚合酶鏈式反應（PCR)等。在此情該 裝置還可以在生物晶片10附近或生物晶片1〇上（例如= 生物晶片10的背面側）加入溫度控制裝置30，對生物晶 片表面的反應溫度進行控制。可以將溫度資料也輸入到資 訊處理部分20並由其進行回饋控制。優選地，溫度控制^ 置30可以是半導體加溫、冷卻裝置，該裝置通過對所加$ 壓的極性進行控制來實現所需的加溫或者冷卻。 現有技術的檢測裝置，例如第一圖所示的裝置，是出 於傳統的醫學檢測方式來構成的，其中激發光源、光路系 統以及接收裝置等集中設置在一個複雜的檢測裝置中，而 生物晶片僅僅作為一種搭載了「試劑」的測試載體置於檢 測裝置中。根據本發明的生物晶片以及檢測裝置提供了與 此完全不同的思路。通過在生物晶片中利用成熟廉價的^ 導體工藝直接製作陣列式半導體光學感測器，第五圖的裝 置可以實現並聯測量方式，而不必通過掃描來測量螢光分 佈，因而可以省去傳統測量裝置中複雜的掃描系統，減小 了體積、成本並大大減少了檢測時間。而且，由於檢測圖 元’、螢光源的距離非常近，因而省去了傳統測量裝置中複 雜的成像系統，這同樣可以減小體積、成本，還可以消除 像差等影響並大大提高探測靈敏度。此外，該裝置還可以 同時實現多螢光光譜的即時檢測’這是其他現有檢測裝置 都不具備的功能。 第五圖的裝置中的激發光源除了固體雷射器外，也可 17 200940986 類型的雷射，或是帶有窄帶濾光片的寬頻光源。 先學透鏡12、導光板13以及全息片14等元件也可以由相 同或類似功能的器件來代替。 需要說明的是，上述實施似是·對本發明進行更 直=朗，本發明的範科局限於這些具體的實施例， 而由申請專利範圍來限定。 、 【圖式簡單說明】 ❹ ❹ 第一圖是現有技術的-種生物晶片掃描器的示意圖。 視示^圖是根據本發明—種實_的生物晶片的局部俯 f三圖是第二圖所示生物晶片的局部剖視示意圖。 第四圖示出了根據本發明的生物晶片 光片的示例性透射率曲線圖。 、先子 第五圖示出了採用本發明的生物晶 的-種示例。 ⑬“料物檢測⑸ 【主要元件符號說明】 1〇 生物晶片 100 檢測單元 1〇1 檢測圖元 102檢測圖元 103 參考圖元 104 保護層 105 表面修飾層 200 生物分子探針陣列 18 200940986 11 激發光源 12 光學透鏡 13 導光板 14 全息片 20 資訊處理部分 30 温度控制裝置 98 光譜層 99 基底In addition to genetic testing, the device of Figure 5 can also detect other organisms such as antibody-antigens of proteins. For some biological property tests, an immediate process of monitoring the reaction is required, and the reaction process is very temperature-dependent, such as polymerase chain reaction (PCR). In this case, the apparatus can also add a temperature control device 30 in the vicinity of the biochip 10 or on the biochip 1 (e.g., the back side of the biochip 10) to control the reaction temperature on the surface of the biochip. The temperature data can also be input to the information processing section 20 and subjected to feedback control. Preferably, temperature control means 30 may be a semiconductor warming, cooling device that achieves the desired warming or cooling by controlling the polarity of the applied pressure. The prior art detecting device, such as the device shown in the first figure, is constructed by a conventional medical detecting method in which an excitation light source, an optical path system, and a receiving device are collectively disposed in a complicated detecting device, and the biochip Only a test carrier equipped with a "reagent" is placed in the detection device. The biochip and the detecting device according to the present invention provide a completely different idea from this. By directly fabricating an arrayed semiconductor optical sensor using a mature and inexpensive ^ conductor process in a biochip, the device of the fifth figure can realize a parallel measurement method without measuring the fluorescence distribution by scanning, thereby eliminating the need for a conventional measuring device. The complex scanning system reduces the size, cost and greatly reduces the detection time. Moreover, since the distance between the detecting element and the fluorescent light source is very close, the complicated imaging system in the conventional measuring device is omitted, which can also reduce the volume and cost, and can also eliminate the influence of aberrations and greatly improve the detection sensitivity. In addition, the device can simultaneously perform simultaneous detection of multiple fluorescence spectra', which is not available in other existing detection devices. In addition to solid-state lasers, the excitation source in the device of Figure 5 can also be a laser of the type 200940986 or a broadband source with a narrow-band filter. Elements such as the lens 12, the light guide plate 13, and the hologram 14 may also be replaced by devices of the same or similar function. It should be noted that the above-described embodiments are intended to be more straightforward, and that the scope of the present invention is limited to these specific embodiments and is defined by the scope of the patent application. [Simple Description of the Drawings] ❹ ❹ The first figure is a schematic diagram of a prior art biochip scanner. The figure is a partial cross-sectional view of the biochip according to the present invention, which is a partial cross-sectional view of the biochip shown in the second figure. The fourth figure shows an exemplary transmittance graph of a biowafer sheet in accordance with the present invention. The fifth figure shows an example of the use of the biocrystal of the present invention. 13 "Material Detection (5) [Description of Main Components] 1〇 Biochip 100 Detection Unit 1〇1 Detection Element 102 Detection Element 103 Reference Element 104 Protective Layer 105 Surface Modification Layer 200 Biomolecular Probe Array 18 200940986 11 Excitation Light source 12 Optical lens 13 Light guide plate 14 Hologram 20 Information processing section 30 Temperature control device 98 Spectral layer 99 Substrate