1336399 99. 01.28第96111791號專利說明書及申請專利範圍修正本 九、發明說明： 【發明所屬之技術頜威】 本發明係關於一種微流體晶片系統之多波長螢光偵 測方法，特別是關於利用—光學儀器之一連續波長光源激 發一微流體晶片系統發出螢光，並對該螢光進行偵測，以 提升偵測效率、準確度、簡化設備及降低成本之微流體晶 片系統之多波長螢光偵測方法。 【先前技術】 習用微流體晶片糸統之螢光偵測方法，如中華民國公 告第490558號「具有晶片式電泳裝置之樣品分析系統」發 明專利，其包含自純樣裝置、―晶片、—電源供應器 偵測單兀'一訊號擷取單元及一訊號處理單元。 動進樣震置係用以將-樣品填充或導人該晶片巾。 係設置有數個槽道，且其係供該自動進樣裝=片 ，該樣品進行分離。該電源供應器係用以提供 該::片中，進㈣順電·使該樣品於該日日日片中電屋於 :她i單元係選擇為一榮光偵測單元，其俜用=分離 =難生之魏。職號_單元制 ^讀 =測=品的訊號。該訊號處理單元係將軍 片、錢光_單元係包含—光源、出。 放㈣發據 田利用该習用微流體晶片夺统" 樣品進行偵測時，首先，^二t :先偵測方法對讀 入該晶片令。接著百:用；;該自動進樣裝置將該樣品導 接者·该電源供應器施加—電璧^ —6 s 1336399 99. 01.28第96111791號專利說明書及申請專利範圍修正本 片上並產生電滲透流〔electr00Sm0Sis fimv〕，以影響該樣 品中各成分於各槽道中之流動速率，進而產生分離。接著 於該槽迢中預設一偵測位置。待樣品流至該偵測位置後， 利用δ玄光源發出一激發光通過該激發濾片’以過濾並得到 一特定波長之激發光’再以該分光鏡反射該激發光，並經 由該透鏡將該激發光聚焦至該偵測位置，以利用該聚焦後 之激發光照射位於該偵測位置之樣品。經該激發光照射後1336399 99. 01.28 Patent No. 96111791 and the scope of the patent application. The present invention relates to a multi-wavelength fluorescence detection method for a microfluidic wafer system, in particular, - A continuous-wavelength source of optical instruments excites a microfluidic wafer system to emit fluorescence and detect the fluorescent light to improve detection efficiency, accuracy, simplify equipment, and reduce the cost of microfluidic wafer systems. Light detection method. [Prior Art] A fluorescent detection method for a conventional microfluidic wafer system, such as the invention patent of "The Sample Analysis System with Wafer Electrophoresis Device" of the Republic of China Announcement No. 490558, which includes a self-purity device, a wafer, a power supply The supplier detects a single signal acquisition unit and a signal processing unit. The motion sensor is used to fill or guide the wafer to the sample. The system is provided with a plurality of channels, and the system is provided for the automatic injection device, and the sample is separated. The power supply is used to provide:: in the film, into the (four) power supply, so that the sample is in the day and day of the film house: her i unit is selected as a glory detection unit, the use of = separation = Wei Wei. Job number _ unit system ^ read = test = product signal. The signal processing unit is a military film, a Qianguang_unit system, including a light source and an output unit. (4) The data is used in the field to detect the sample using the microfluidic wafer. First, the second detection method is used to read the wafer order. Then, the automatic sample introduction device applies the sample to the power supply device to the power supply device. The patent specification and the patent application scope are corrected on the film and the electricity is generated. The permeate flow [electr00Sm0Sis fimv] is used to influence the flow rate of each component in the sample in each channel, thereby generating separation. A detection position is then preset in the slot. After the sample flows to the detection position, an excitation light is emitted through the excitation filter to filter and obtain an excitation light of a specific wavelength, and the excitation light is reflected by the spectroscope, and the lens is reflected by the lens. The excitation light is focused to the detection position to illuminate the sample located at the detection position by using the focused excitation light. After irradiation with the excitation light
之樣品將產生螢光放射。接著將該樣品所產生之螢光由該 透鏡收集後依序經該分光鏡、放射濾片及一針孔至該光電 倍增管，以利用該光電倍增管放大該樣品所產生之螢光訊 唬，之後經該訊號擷取單元將類比訊號轉為數位訊號後， 由虎處理單元輸出樣品分析後之數據。如此便完成該 樣品之偵測。 一般而言，上述習用微流體晶片系統之螢光偵測方法 具有下列魏，例如：錢發光通常係直接射人該訊號操 ，早兀：」又該激發光之強度大於該樣品所發出之螢光甚 夕使得該玄光之汛號相對較不明顯，如此將降低量測準 1度°因此’該習用偵測方法f另設置該激發濾片，以過 ，慮出之激發光，造成其具有成本昂貴及設備複雜 ^ ^，該制偵測方法受限於單—波長的激發與 二之多波長之螢光放射’使得其需對應該 t之京以發波長調整該激發光之波長並重複 〇則’除&加樣^損耗量外亦增加檢啊間，盆旦 偵測效率财之缺點。同時，因該制偵測方法之單二波 —7 — 1336399 卯·〇1‘28第96111791號專利說明書及申請專利範圍修正本 長激發與偵測的限制，使得所偵測之數據範圍較小，後續 所求得之量化數據準確性不足。基於上述原因，有必要進 步改良上述習用微流體晶片系統之多波長螢光谓測方法 〇 有鑑於此，本發明改良上述之缺點，其係利用一光學 儀森之連續波長光源激發一微流體晶片系統發出螢光，並 利用一光學分析單元對該螢光進行偵測。藉此，本發明確 實能提升偵測效率、準確度、降低成本及簡化設備之微流 體晶片系統之多波長螢光偵測方法。 【發明内容】 本發明主要目的係提供一種微流體晶片系統之多波 長#光偵測方法，其係利用一光學儀器之連續波長光源激 發一微流體晶片系統發出螢光，並利用一光學分析單元進 行侦測’使得本發明具有提升偵測效率之功效。 本發明次要目的係提供一種微流體晶片系統之多波 長營光伯測方法，其中該光學儀器係為一暗視野光學組， 使得本發明具有降低成本及 簡化設備之功效。 本發明另—目的係提供一種微流體晶片系統之多波 長®光债測方法，其中該微流體晶片系統之微流體晶片上 係设置一參考槽道，以求得一背景值，進而使得本發明具 有提升偵測準確性之功效。 根據本發明之微流體晶片系統之多波長螢光偵測方 法，其包含步驟：提供一微流體晶片具有一基板、一進樣 匕道、一分離槽道及一參考槽道，並將一樣品導入與該分 · 8 一 1336399 99. 01. 28第96111791號專利說明書及申請專利範圍修正本 離槽道相連通之進樣槽道中；提供一電壓於該微流體晶片 上，以驅使該樣品移動至該分離槽道上之一偵測位置；經 由一光學儀器提供一連續波長光源及一接收單元，以利用 該連續波長光源發出之一激發光激發該樣品發射一螢光， 並經由該接收單元接收該螢光，其中，該激發光並未直接 照射該接收單元；及將該光學儀器所接收之螢光訊號傳至 該光學分析單元並進行分析；其中，該參考槽道係用以測 定一背景值，以進一步校正該樣品所測得之數據。 【實施方式】 為讓本發明之上述及其他目的、特徵、優點能更明顯 易懂，下文特舉本發明之較佳實施例，並配合所附圖式， 作詳細說明如下： 請參照第1及2圖所示，本發明較佳實施例之微流體 晶片系統之多波長螢光偵測方法的第一步驟S1係：提供一 微流體晶片1具有一基板11、一進樣槽道12、一分離槽道 13及一參考槽道14，並將一樣品〔未繪示〕導入與該分離 槽道13相連通之進樣槽道12中。更詳言之，該樣品係選 擇由至少一待測物與一背景溶液混合配製而成。該待測物 〔未繪示〕係預先以一螢光染劑〔未繪示〕對該待測物進 行標定，以利後續螢光激發之進行。該基板11係選擇為一 玻璃基板。該進樣槽道12係供該樣品導入，且長度係選擇 為0.8cm。該分離槽道13係用以分離該樣品，且其係與該 進樣槽道12相連通並相交形成一十字形。該分離槽道13 上係預設一偵測位置a，且該分離槽道13之長度係選擇為 —9 — 1336399 99. 01.28第96111791號專利說明書及申請專利範圍修正本 •ί則 5cm。該參考槽道14係平行設置於該分離槽道u ，以供該背景溶液導入並偵測其螢光訊號作為後碎之 析之一背景值，進而減少偵測誤差並提升偵測準=數據分 參考槽道14距該分離槽道13之距離係撰埋’岐。讀 流體晶片1較佳係另設置一刻度線15微 且邊到度線ί ς / 之移動 選擇 平行設置於該分離槽道13之一側，以利判斷 係 —k品之 位置。其中，各槽道之寬度係選擇為1〇〇以爪，深度係 為36从m。 ，、The sample will produce fluorescence emissions. Then, the fluorescent light generated by the sample is collected by the lens, and then sequentially passed through the spectroscope, the radiation filter and a pinhole to the photomultiplier tube to amplify the fluorescence signal generated by the sample by using the photomultiplier tube. After the signal acquisition unit converts the analog signal into a digital signal, the tiger processing unit outputs the sample analyzed data. This completes the detection of the sample. In general, the fluorescent detection method of the conventional microfluidic wafer system has the following Wei, for example, the money illumination is usually directed to the signal operation, and the intensity of the excitation light is greater than the firefly emitted by the sample. The light eclipse makes the nickname of the black light relatively inconspicuous, so that the measurement is reduced by 1 degree. Therefore, the conventional detection method f sets the excitation filter to over-examine the excitation light, thereby causing it to have Costly and complicated equipment ^ ^, the detection method is limited by single-wavelength excitation and two-wavelength fluorescence emission', so that it needs to adjust the wavelength of the excitation light and repeat it. 〇 ' 'In addition to & plus sample ^ loss amount also increased the inspection, the pottery detection efficiency of financial shortcomings. At the same time, due to the detection method of the single-wave method - 7 - 1336399 卯 · 〇 1 '28 No. 96111791 patent specification and the scope of the patent application, the limitation of the excitation and detection is corrected, so that the detected data range is small. The accuracy of the subsequent quantitative data is insufficient. For the above reasons, it is necessary to advance the multi-wavelength fluorescence prediction method for improving the conventional microfluidic wafer system. In view of the above, the present invention improves the above-mentioned disadvantages by exciting a microfluidic wafer using an optical wavelength source of an optical instrument. The system emits fluorescence and detects the fluorescence using an optical analysis unit. Thereby, the present invention can improve the detection efficiency, accuracy, cost reduction and simplification of the multi-wavelength fluorescence detection method of the microfluidic wafer system of the device. SUMMARY OF THE INVENTION The main object of the present invention is to provide a multi-wavelength #light detection method for a microfluidic wafer system, which uses a continuous wavelength light source of an optical instrument to excite a microfluidic wafer system to emit fluorescence, and utilizes an optical analysis unit. Performing detection' makes the invention have the effect of improving detection efficiency. A secondary object of the present invention is to provide a multi-wavelength camping optical measurement method for a microfluidic wafer system, wherein the optical instrument is a dark field optical group, which enables the present invention to reduce cost and simplify the efficiency of the device. Another object of the present invention is to provide a multi-wavelength® optical debt measurement method for a microfluidic wafer system, wherein a reference channel is provided on the microfluidic wafer of the microfluidic wafer system to obtain a background value, thereby enabling the present invention It has the effect of improving detection accuracy. A multi-wavelength fluorescence detecting method for a microfluidic wafer system according to the present invention, comprising the steps of: providing a microfluidic wafer having a substrate, a sampling tunnel, a separation channel, and a reference channel, and a sample The invention is incorporated in the sampling channel in communication with the channel; a voltage is applied to the microfluidic wafer to drive the sample to move. Providing a detection position to the separation channel; providing a continuous wavelength light source and a receiving unit via an optical instrument to excite the sample to emit a fluorescent light by using the continuous wavelength light source, and receiving the fluorescent light through the receiving unit The fluorescent light, wherein the excitation light is not directly irradiated to the receiving unit; and the fluorescent signal received by the optical instrument is transmitted to the optical analysis unit for analysis; wherein the reference channel is used to determine a background Value to further correct the data measured for the sample. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, and advantages of the present invention will become more <RTIgt; And the first step S1 of the multi-wavelength fluorescence detecting method of the microfluidic wafer system of the preferred embodiment of the present invention is: providing a microfluidic wafer 1 having a substrate 11, a sampling channel 12, A separation channel 13 and a reference channel 14 are introduced, and a sample (not shown) is introduced into the sampling channel 12 communicating with the separation channel 13. More specifically, the sample is selected from a mixture of at least one analyte and a background solution. The object to be tested (not shown) is previously calibrated with a fluorescent dye (not shown) to facilitate subsequent fluorescence excitation. The substrate 11 is selected as a glass substrate. The sample channel 12 was introduced for the sample and the length was selected to be 0.8 cm. The separation channel 13 is for separating the sample and is in communication with the injection channel 12 and intersecting to form a cross. The separation channel 13 is preset with a detection position a, and the length of the separation channel 13 is selected to be -9 - 1336399 99. 01.28 Patent No. 96111791 and the patent application scope revision. The reference channel 14 is disposed in parallel to the separation channel u for the background solution to be introduced and detecting the fluorescent signal as a background value of the post-cracking analysis, thereby reducing detection error and improving detection accuracy = data The distance from the sub-channel 14 to the separation channel 13 is buried. The read fluid wafer 1 is preferably provided with a scale line 15 micro and the edge to line ί ς / movement selection is arranged in parallel on one side of the separation channel 13 to determine the position of the product. Among them, the width of each channel is selected to be 1 〇〇 claw and depth is 36 from m. , ,
h再參照弟1及2騎示，本發職佳實施例中… 進樣槽道12之相對兩端係分別形成一第—通孔Η〗’马 與該進樣槽道12相通，該第一通孔121俾 ' 121 另-第-通孔_'__健一=^= 之相對兩端係分別形成一第二通孔131、·! ^ 只·该分離本h, referring to the brothers 1 and 2, in the preferred embodiment of the present invention, the opposite ends of the injection channel 12 respectively form a first through hole Η 'the horse communicates with the injection channel 12, the first A through hole 121 俾 ' 121 another - first through hole _ '__ Jianyi = ^ = opposite ends are respectively formed a second through hole 131, ·! ^ only · the separation
道13相通。該參考槽道14之相對兩端係分別形成一第二 通孔141、14Γ與該參考槽道14相通，該第三通孔l4i ^ 供該背景溶液導入以測定該背景值，另一第三通孔141，’ 用以排除多餘之背景溶液。 ’ 請再參照第1及2圖所示，本發明較佳實施例之微流 體晶片系統之多波長螢光偵測方法的第二步驟s 2係：提: %壓於该微流體晶片1上，以驅使該樣品移動至該分離 夂道13上之一偵測位置a。更詳言之，該電壓係施加於該 乃離槽道13之兩端以形成一電場〔未緣示〕並產生一電滅 逯流〔electroosmosis flow〕。由於該樣品中之各成分受讀 電滲透流之影響各不相同，因此可經由該電滲透流驅動言亥 —10 — 99意28第96111791號專利說明書及申請專利範圍修正本 樣品中之各成分，使得該楛σ — 離並移動細貞測位置卜 钱分賴道13進行分 請再參照第1至3圖所干，士 體晶片系統之多波長營明較佳實施例之微流 -光學儀器2提供第三步㈣係:經由 以利用該連續波長光源叫^;1 f-接收單元22, 發射一螢光，並經由該接收X單 ^先211激發該樣品 激發光则未直接照===其中，該 施，光學儀器2係選擇但不受限於―』野H且= 先學勤2係包含-連續波長光源2卜 、且" 擋板23、一聚光單元24及 =收早❿、一 該連續波長光源21及接。抓品較佳係介於 2!係可多料^ 22之^該連續波長光源 又乾圍之激發光2η ,以激發位 測位置a之樣品發出營光〔未繪示〕。藉由 =偵 21可同時激發多種不同整光、九 曰人、員/長光源 該連續波長光界2m 進而增進偵測效率。 源21係選擇但不受限為 。_ 22係用以接收該樣品所發出之螢光。該激發光犯之= 所發出之螢光甚多，若該接收單元Μ同時接： 因卜11與螢光之訊號’則該螢光之訊號則相對較不 月喊。因贼連續波長絲21崎出 單元22，以避免該接收單元22接收該= 匕強之糾光211 ’而影響該樣品 號強度。該接收單元22係選擇為一物^。之赏先的相封讯 月再〜、第1至3圖所示，本發明較佳實施例中，該 —11 — 1336399 01.28帛96111791號專利說明書及申請專利範圍修正本 連~波長光源21係選擇設置於該接從單元^之 避免域續波長光源21直接照㈣純單n目心 忒達續波長光源21與該接收單 ' 兀22之間的水平方向設置 光源21所發出之激發光扣。 於4擋板23阻擋後，該連續波長 成-中空管狀之激發光211。辭板23=之先^形 擋板23與接收單元22之間係 光，為®形。該 該聚光私24將該中空管狀之^ 早兀24，以利用 該樣品發出螢光。該聚光單元24y選擇激發 。該樣品係位於該接收單元22〒單、9視野聚光鏡 孔25是設置於該接收單元22之上^早几24之間。該針 接收該樣㈣麵後，經由她 光學分析單元3以進行訊號之分析。 ^ = =所發出之激發㈣經由該擔板23阻』= 空管狀之激發光211。接著再經成中 光2Π形成一中空錐形之激發^ 24㈣激發 曰y „ , X先211，並聚焦於該微流體 =激發光211使得位於該偵測 置a之樣4出螢先，且該榮光係由該接 :由於該激發光則形成該中空錐形之二 邊錐形頂點係位於該微流體晶片丨 且 光2U直接進入該接收單元22 ^匕可避免該激發 ^ , T卫'僅有少數受到折-¾ 反射之激發光211可進入該接收單元22中。該中j或 激發光211較佳係聚焦於該偵測位置二’ ^之 請再參照第1至3圖所示’本發明較佳實施例之微流 1336399 99. 01.28第96111791號專利說明書及申請專利範圍修正本 體晶片系統之多波長螢光偵測方法的第四步驟S4係：將該 光學儀器2所接收之螢光訊號傳至該光學分析單元3並進 行分析。更詳言之，該光學儀器2所接收之螢光訊號係選 擇經由一光纖〔optical fiber〕將該螢光訊號傳至該光學分 析單元3以進行分析。該螢光訊號亦可選擇經由一光電倍 增管〔未繪示〕放大後，再傳至該光學分析單元3。該光 學分析單元3較佳係選擇為一紫外-可見-近紅外光之光譜 分析儀，以便同時偵測各種不同波長範圍之榮光。該營光 訊號經由該光學分析單元3分析後，可得到時間〔秒〕、波 長〔nm〕及穿透率〔％〕之相對變化關係，亦可進一步求 得更加精準之量化數據。另外，請參照第1圖所示，為使 所測得之數據更為精準，較佳係於上述步驟完成後，另進 行下列步驟以取得該背景值：將該背景溶液導入該參考槽 道14之第三通孔141中，並施加電壓驅使該背景溶液移動 至該參考槽道14上之一參考位置b ;利用該激發光211製 造位於該參考位置b之背景光，並經由該接收單元22接收 背景光；將該背景溶液之光訊號傳至該光學分析單元3， 以分析並取得該背景值。其中該參考位置b係位於該參考 槽道14上，且係對應該偵測位置a。最後再將該樣品分析 所得之螢光數據扣除該背景值之數據，如此便可校正該樣 品所測得之螢光數據，亦可進一步得到更精準之量化數據 〇 請再參照1至3圖所示，當實行本發明較佳實施例時 ，首先進行該第一步驟S1 :將經由螢光染劑標定後之樣品 —13 — 1336399 99. 01. 28第96111791號專利說明書及申請專利範圍修正本 · L未繪示〕注入該微流體晶片1之進樣槽道〗2中。待該樣 . 品逐漸流動至該進樣槽道12與分離槽道13之交點後；接 . 著進行該第二步驟S2 :提供一電壓於該微流體晶片丨，以 驅使該樣品沿著該分離槽道13移動至該偵測位置a;再進 行該第二步驟S3 :利用該連續波長光源21發射出多波長 之激發光211，再依序經由該擋板23及聚光單元％使該 激發光211形成-中空錐形之激發光211，並聚焦於該偵. 測位置a，以激發位於該偵測位置a之樣品發出螢光。該 樣所如出之里光將被该接收單元22接收，且該激發光 鲁 211並未直接照射該接收單元22，以避免影響該螢光訊號 之相對強度；最後進行該第四步驟S4:將該光學儀器2所 接收之螢光訊號經由該光纖傳至該光學分析單元3並進行 分析’以得到時間、波長及穿透率之相對變化關係。如此 ，便完成本發明較佳實施例之微流體晶片系統之多波長偵 測方法。藉此，本發明確實具有提升偵測效率、準確度、 降低成本及簡化設備之功效。 又 請參照第4圖所示，本發明實施例中，係選擇以打丁匚 φ 、RhB及Att〇610作為該樣品之螢光染劑。第4圖係為各 該螢光染劑經由本發明之多波長螢光偵測方法所測得之吸 收及發射光譜圖形。圖中虛線係指各該螢光染劑之吸收光_ 波長，而實線係指各該螢光染劑之發射光波長。可得知 · FITC、RhB及Atto610之最大吸收光波長係分別為4卯 〔奈米〕、553nm及614nm，而最大放射光波長係分別為 514nm、578nm及633nm。各該螢光染劑之最大放射光波 —14 ~ U0b399 . 99. 8第95111791號專利說明書及申請專利範園修正本 長係為後續螢光分析之重要指標。 測方、丨Μ 3、5及6圖所示，第5㈣為f用$亦 r=:;r 先放 偵測。第=二;不_光放射波長之登光染心 後所得之犯:之螢紐據扣除該背景值之數據 :確表達各該螢光染劑所放射之螢心== 對變化關係。該穿透率係用以判斷該螢光：強二 ®之本發明較佳實施例，由於該激發光〕 : 接進入該接收單元22,因此無須裝設任 ^ 、’未直 即可取得相對·^、、 可叩貝的光學濾片 # h 纟之螢光放射，且由㈣連以 奸種1之設置使得本發明僅需進行―:欠偵測，即可^時 仃各種不同波長之螢光偵測。 螢光6,所示，糊f光_方法對該 取得量化二Γ第5圖中之波峰作體積分，以 最大營光二;：;所測得之數據僅為該榮光染劑之 據準確性得取得之量化數 之各波峰作體積分，如此所取;該料面上 。如此可驗證本發明確實具有提升偵測效Ϊ的 低成本及簡化設備之功致。 卞、準確度、降 如上所述，相較於習用微流體晶片系統之多波長登光 15 — 1336399 99. 01. 28第9611 1791號專利說明書及申請專利範圍修正本 偵測方法需另設置該激發濾片，以過濾出特定波長之激發 光，造成其具有成本昂貴及設備複雜之缺點。再者，該習 用偵測方法受限於單一波長的激發與偵測，無法同時偵測 多波長之螢光放射，造成其具有偵測效率低落之缺點。同 時，因該習用偵測方法之單一波長激發與偵測的限制，使 得其具有所求得之量化數據準確性不足等缺點。反觀第1 圖之本發明係利用該光學儀器2之連續波長光源21激發該 微流體晶片1發出螢光，並利用該光學分析單元3進行偵 測，同時經由該參考槽道14求得該背景值以校正該樣品之 螢光分析數據。藉此，本發明確實能提升偵測效率、準確 度、降低成本及簡化設備之功效。 雖然本發明已利用上述較佳實施例揭示，然其並非用 以限定本發明，任何熟習此技藝者在不脫離本發明之精神 和範圍之内，相對上述實施例進行各種更動與修改仍屬本 發明所保護之技術範疇，因此本發明之保護範圍當視後附 之申請專利範圍所界定者為準。 16 — 1336399 99. 01.28第961Π791號專利說明書及申請專利範圍修正本 【圖式簡單說明】 第1圖：本發明較佳實施例之微流體晶#系統之多波長 螢光偵測方法的流程方塊圖。 第2圖：本發明較佳實施例之微流體晶片之立體圖。 第3圖：本發明較佳實施例之微流體晶片系統之多波長 螢光偵測方法的示意圖。 第4圖：本發明較佳實施例之各該螢光染劑之吸收及放 射光譜圖。 第5圖：本發明較佳實施例之微流體晶片系統之多波長 營光彳貞測方法的3D電泳圖。 第6圖：習用微流體晶片系統之螢光偵測方法所測得之 時間相對穿透率〔％〕之分析結果。 【主要元件符號說明】 1 微流體晶片 12 進樣槽道 12Γ第一通孔 131第二通孔 14 參考槽道 141，第三通孔 2 光學儀器 211激發光 23 擋板 25 針孔 11 基板 121第一通孔 13 分離槽道 13Γ第二通孔 141第三通孔 15 刻度線 21 連績波長光源 22 接收單元 24 聚光單元 3 光學分析單元 17 — 1336399 99. 01.28第961Π791號專利說明書及申請專利範圍修正本 a 偵測位置 b 參考位置 S1 第 一步驟 S2 第二步驟 S3 第 三步驟 S4 第四步驟 —18 —Road 13 is connected. The opposite ends of the reference channel 14 respectively form a second through hole 141, 14 相 communicating with the reference channel 14, the third through hole l4i ^ for the background solution to be measured to determine the background value, and the third Through holes 141,' are used to remove excess background solution. Referring again to Figures 1 and 2, a second step s 2 of the multi-wavelength fluorescence detection method of the microfluidic wafer system of the preferred embodiment of the present invention is as follows: % is pressed onto the microfluidic wafer 1 To drive the sample to a detection position a on the separation channel 13. More specifically, the voltage is applied to the ends of the channel 13 to form an electric field (not shown) and to generate an electroosmosis flow. Since the components in the sample are affected by the read electroosmotic flow, the components in the sample can be modified by the electroosmotic flow to drive the patent specification and the scope of application of the patent. , so that the 楛 σ - separate and move the fine measurement position, the money is divided into 13 points, please refer to the first to third figures, the multi-wavelength scheme of the body wafer system The instrument 2 provides a third step (four) system: by using the continuous wavelength light source called ^; 1 f-receiving unit 22, emitting a fluorescent light, and exciting the sample excitation light via the receiving X single first 211 is not directly photographed = == Among them, the optical instrument 2 is selected but not limited to ““H” and “Xueqian 2” includes – continuous wavelength light source 2, and " baffle 23, a concentrating unit 24 and = Early, one continuous wavelength source 21 and connected. Preferably, the sample is between 2 and 2, and the continuous wavelength source is further surrounded by the excitation light 2η to excite the sample at the position a to emit a camping light (not shown). By using Detective 21, a variety of different illuminating, nine-person, and long-light sources can be simultaneously excited to increase the detection efficiency by 2m. Source 21 is selected but not limited to . _ 22 is used to receive the fluorescent light emitted by the sample. The excitation light is made by the number of fluorescent lights emitted. If the receiving unit is connected at the same time: the signal of the Infrared 11 and the fluorescent light, the fluorescent signal is relatively less screaming. Since the thief continuous wavelength wire 21 is out of the unit 22, the receiving unit 22 is prevented from receiving the = reluctant light correction 211' to affect the sample number intensity. The receiving unit 22 is selected as a substance. In the preferred embodiment of the present invention, the patent specification and the scope of the patent application are modified by the first embodiment of the present invention. Selecting the setting source in the receiving unit to avoid the continuous wavelength source 21 direct illumination (four) pure single n-eye centering wavelength source 21 and the receiving unit ' 兀 22 between the horizontal direction of the light source 21 to emit the excitation light buckle . After the baffle 23 is blocked, the continuous wavelength becomes a hollow tubular excitation light 211. The stencil 23=the first shape is the light between the baffle 23 and the receiving unit 22, which is a о shape. The concentrating light 24 passes the hollow tubular tube 24 to emit fluorescence using the sample. The concentrating unit 24y selects excitation. The sample is located in the receiving unit 22, and the nine-field concentrating mirror aperture 25 is disposed between the receiving unit 22 and a few 24 seconds. After receiving the sample (four), the needle passes through her optical analysis unit 3 for signal analysis. ^ = = the emitted excitation (4) is blocked by the support plate 23 = the empty tubular excitation light 211. Then, through the formation of a hollow cone, a hollow cone-shaped excitation ^ 24 (four) excitation 曰 y „, X first 211, and focusing on the microfluid=excitation light 211, so that the detection is set to a The glory is connected by: the excitation light forms the two-sided cone apex of the hollow cone located on the microfluidic wafer, and the light 2U directly enters the receiving unit 22 匕 to avoid the excitation ^, T Wei' only A small amount of excitation light 211 that is deflected by -3 can enter the receiving unit 22. The medium j or the excitation light 211 is preferably focused on the detection position two '^ Please refer to the figures 1 to 3 again. The fourth step S4 of the multi-wavelength fluorescence detecting method for correcting the body wafer system of the preferred embodiment of the present invention is a microflow 1336399 99. 01.28 No. 96111791 and the patent scope of the invention. The optical signal is transmitted to the optical analysis unit 3 and analyzed. In more detail, the fluorescent signal received by the optical device 2 is selected to transmit the fluorescent signal to the optical analysis unit 3 via an optical fiber. Analyze. The fluorescent signal can also After being amplified by a photomultiplier tube (not shown), it is transmitted to the optical analysis unit 3. The optical analysis unit 3 is preferably selected as an ultraviolet-visible-near-infrared spectrum analyzer for simultaneous detection. The glory of various wavelength ranges. After the optical signal is analyzed by the optical analysis unit 3, the relative change relationship of time [second], wavelength [nm] and transmittance [%] can be obtained, and further precision can be obtained. Quantitative data. In addition, please refer to Figure 1, in order to make the measured data more accurate, preferably after the above steps are completed, the following steps are further performed to obtain the background value: the background solution is introduced into the Referring to the third through hole 141 of the channel 14 and applying a voltage to drive the background solution to a reference position b on the reference channel 14; the excitation light 211 is used to fabricate the background light at the reference position b, and The receiving unit 22 receives the background light, and transmits the optical signal of the background solution to the optical analysis unit 3 to analyze and obtain the background value, wherein the reference position b is located on the reference channel 14 And the corresponding position should be detected. Finally, the fluorescence data obtained by analyzing the sample is deducted from the background value data, so that the fluorescence data measured by the sample can be corrected, and more accurate quantitative data can be further obtained. Referring again to Figures 1 to 3, when the preferred embodiment of the present invention is implemented, the first step S1 is first performed: the sample to be calibrated via the fluorescent dye - 13 - 1336399 99. 01. 28, 96111791 The patent specification and the patent application scope revision L·not shown] are injected into the injection channel of the microfluidic wafer 1. In this case, the product gradually flows to the injection channel 12 and the separation channel 13 After the intersection, the second step S2 is performed: a voltage is applied to the microfluidic wafer cassette to drive the sample to move along the separation channel 13 to the detection position a; and the second step S3 is performed: The excitation light 211 of the multi-wavelength is emitted by the continuous wavelength light source 21, and the excitation light 211 is formed into the hollow cone-shaped excitation light 211 through the baffle 23 and the concentrating unit %, and is focused on the detection and measurement. Position a to excite the sample located at the detection position a Fluorescent. The illuminating light is received by the receiving unit 22, and the excitation light 211 is not directly irradiated to the receiving unit 22 to avoid affecting the relative intensity of the fluorescent signal; finally, the fourth step S4 is performed: The fluorescent signal received by the optical device 2 is transmitted to the optical analysis unit 3 via the optical fiber and analyzed 'to obtain a relative change in time, wavelength, and transmittance. Thus, the multi-wavelength detection method of the microfluidic wafer system of the preferred embodiment of the present invention is completed. Thereby, the present invention has the advantages of improving detection efficiency, accuracy, reducing cost, and simplifying equipment. Referring to Fig. 4, in the embodiment of the present invention, butyl φ, RhB and Att 610 are selected as the fluorescent dye of the sample. Figure 4 is a graph of the absorption and emission spectra of each of the phosphor dyes measured by the multi-wavelength fluorescence detection method of the present invention. The dotted line in the figure refers to the absorption light_wavelength of each of the fluorescent dyes, and the solid line refers to the wavelength of the emitted light of each of the fluorescent dyes. It can be seen that the maximum absorption wavelengths of FITC, RhB and Atto610 are 4 〔 [nano], 553 nm and 614 nm, respectively, and the maximum emission wavelengths are 514 nm, 578 nm and 633 nm, respectively. The maximum emission of each of the fluorescent dyes is 14-14 U0b399. 99. 8 Patent No. 95111791 and the patent application revision are the important indicators for subsequent fluorescence analysis. The test, 丨Μ 3, 5 and 6 are shown, the fifth (four) is f with $ also r =:; r first put detection. No. = two; no _ light radiation wavelength of the light after the heart of the crime: the light of the subtraction of the background value of the data: the expression of each of the fluorescent dyes emitted by the heart of the heart == change relationship. The transmittance is used to determine the preferred embodiment of the present invention: the excitation light: since the excitation light is connected to the receiving unit 22, so that it is not necessary to install any of the two, · ^, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , Fluorescence detection. Fluorescent 6, as shown, the paste f light _ method to obtain the quantified peak of the peak in Fig. 5, to the maximum camp light two;:; the measured data is only the accuracy of the glory dye The peaks of the quantified numbers obtained are taken as volume fractions, and thus taken; As such, it can be verified that the present invention does have the low cost of improving the detection effect and the simplification of the device.卞, accuracy, and drop as described above, compared to the conventional microfluidic wafer system, multi-wavelength Dengguang 15 - 1336399 99. 01. 28 No. 9611 1791 Patent Specification and Patent Remedy Correction This detection method requires another setting The filter is excited to filter out excitation light of a specific wavelength, which causes it to be expensive and complicated. Moreover, the conventional detection method is limited by the excitation and detection of a single wavelength, and the multi-wavelength fluorescence emission cannot be detected at the same time, which causes the detection efficiency to be low. At the same time, due to the limitation of single wavelength excitation and detection of the conventional detection method, it has the disadvantages of insufficient accuracy of the obtained quantized data. In contrast, the invention of FIG. 1 uses the continuous wavelength light source 21 of the optical instrument 2 to excite the microfluidic wafer 1 to emit fluorescence, and the optical analysis unit 3 performs detection, and the background is obtained through the reference channel 14. Value to correct the fluorescence analysis data for the sample. Thereby, the present invention can improve the detection efficiency, accuracy, cost reduction and simplification of the device. While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims. 16 — 1336399 99. 01.28 Patent Specification No. 961 791 and the scope of the patent application. [Simplified description of the drawings] FIG. 1 is a flow block of a multi-wavelength fluorescence detection method of the microfluidic crystal system of the preferred embodiment of the present invention. Figure. Figure 2 is a perspective view of a microfluidic wafer in accordance with a preferred embodiment of the present invention. Figure 3 is a schematic illustration of a multi-wavelength fluorescence detection method for a microfluidic wafer system in accordance with a preferred embodiment of the present invention. Figure 4 is a graph showing the absorption and emission spectra of each of the fluorescent dyes of the preferred embodiment of the present invention. Figure 5 is a 3D electropherogram of a multi-wavelength camping spectrometry method for a microfluidic wafer system in accordance with a preferred embodiment of the present invention. Figure 6: Analysis of the time-to-penetration [%] measured by the fluorescence detection method of the conventional microfluidic wafer system. [Main component symbol description] 1 microfluidic wafer 12 injection channel 12 Γ first through hole 131 second through hole 14 reference channel 141, third through hole 2 optical instrument 211 excitation light 23 baffle 25 pinhole 11 substrate 121 First through hole 13 separation channel 13 Γ second through hole 141 third through hole 15 tick mark 21 continuous wavelength light source 22 receiving unit 24 concentrating unit 3 optical analysis unit 17 - 1336399 99. 01.28 No. 961 791 patent specification and application Patent range revision a A detection position b reference position S1 first step S2 second step S3 third step S4 fourth step - 18 -