五、新型說明： 【新型所屬之技術領域】 · 本創作使用傳統細胞培養於孔盤的方式，利用磷光相 位差耗氧檢測系統，完成生醫感測裝置，此裝置能準確分 析細胞的狀況變化，可應用在新藥物的研發以及生物醫學 檢測上。 【先前技術】 1.目前已有許多細胞定位方式以及耗氧感測之方式被提出，僅針 對相關於本創作之裝置分析比較，歸納於下： 2· 2007年’ David A· Chang-Yen等人將耗氧縣粒子Ru與高分 子材料混合，旋塗至玻璃表面，形成一層奈米薄膜，完成此耗 氧感測裝置。此裝置通人待職體後，將波長為45()nm的藍光 LED作為燈職玻片旁照射，光在玻璃裡會產生全反射，藉此 激發玻璃表面上的螢光，最後接收某範圍表面的螢綠號，利 用Stern- VolmerS程式將所得到的螢光訊號與勒免氧氣濃度 相互換算。通入_ 100%的氧氣濃度於裝置裡，分別感測其 螢光強度在不同氧濃度下之間的關係、，當氧氣濃度為1%以下 時，螢光強度與氧濃度仍可_線性的_，所以文獻利用此 耗氧感測裝置可量測到氧濃度的小變化量，未來將可應用在生 物代謝研究、環境的檢測及細胞的觀察上。 3. 2007年，Ylna Kuang等人將市售直徑1⑽⑽的聚苯乙烯奈 米粒子，使其產生帶正電極性的根，然後讓螢光粒子 mtheniumdi)包覆在奈料奸表面上，最後帶正電極性的奈 米粒子在轉啤瞒物縣面上，雜福絲子貼覆細胞 表面，做連續性的耗氧榮光強度量測。這個帶有榮光的聚苯乙 稀奈米粒子具有非侵入性’不會因被細胞吞嗔而造成細胞的死 亡.且具有可逆性’必要時可以將貼覆於細胞的耗氧營光粒子 排除；因為貼覆的夠近，可以精確即時感測細胞的耗氧情況。 4. 2009年，Timothy W. Molter等人在玻璃基板上製作出微井陣 列後’將螢光粒子配成溶液散佈在微井陣列裝置上，當裝置上 盖下愿’排除多餘的溶液，只留下微井陣列裡的溶液，螢光粒 子便會在微井陣列裡沉殿，裝置上蓋打開後將溶液烤乾，榮光 粒子自動在微井底部形成環狀結構，最後再燒結固定。耗氧螢 光偵測方面，本文獻選擇量測其碟光壽命（lifetime)，當激發 光源關閉後’螢光粒子會有短暫_光散發時間，在碟光降逝 、言夺門裡拍了兩張曝光時間不同的螢光畫面，再將兩張螢 光旦面相除當作某氧氣濃度下的無因次比值，當氧氣濃度不同 ^ ’所得到兩張照片強度無因次比值就會相對應的改變。鱗光 可〒不會因為螢光粒子的多寡而改變，只會因為環境的氧氣濃 度而有所長短’故利用此方式克服傳統量測螢光強度時，營光 粒子濃度會影響營光強弱之問題，並且降低螢光淬媳效應 (Ph〇t〇bleach) ’延長此耗氧感測裝置的使用時間。 【新型内容】 M400480 本創作設計之感測裝置，將細胞培養於孔盤的方式，加入氧偵 測磷光粒子並密封孔盤，在結合磷光相位差耗氧檢測系統，量測 孔盤内孔洞的溶氧量。此系統主要由液晶顯示器，透過本專利之 光學架設，可控制不同的激發光源圖形’達到單一孔洞磷光激發 或是不同孔洞間的磷光激發，進而比較不同孔洞間細胞活性的差 異性，增加觀察上的自由度。耗氧量測原理以磷光相位量測方式， 其原理是建立於磷光壽命的產生。一般當激發光照射填光物質， _碟光物質被激發出碟光，而當激發光停止照射，填光會維持一段 時間，這段時間被稱為破光壽命’若將激發光與碌光記錄下來形 成波形’則磷光之波峰會延遲一段時間而形成相位差，兩訊號相 比較後得到的相位差占便會符合tan( 5 ) = 2;τ I» r，從此式亦 可以推得球光壽命之變化。占為相位差，u為激發光頻率，r為 磷光壽命。此一相位差便可配合激發光頻率，利用公式計算出磷 光寿命。在由 Stern - Volmer 方程式 1。/1= td/t =1 + 鲁kq：r〇[P〇2] ’式巾1〇、r。小r分別表示沒有氧氣和有氧氣存在 下的螢光(磷光）強度和壽命，kq為雙分子淬熄常數，[p〇2]為氧 氣濃度。測得溶氧濃度〇mg/L與待測物的填光壽命，代入r。/ ^ 則可以得到待測物溶氧濃度。因此相位差越大則螢光壽命越長， 氧氣濃度也越低，藉此判斷細胞耗氧量變化。量測系統可以拆解 為兩個部分’首先是觸發與接收部分由電腦程式LabView控制資 料擷取卡送出訊號’觸發LED Driver，LED Driver依據所給予之 訊號頻率同時觸發LED，LED之光源經過液晶顯示器定義出激發光 m4〇〇48〇 源圖形，在透過分光鏡一部分光源由光強度偵測器接收訊號作為 參考訊號，另一部分則照射至待量測的磷光粒子激發出磷光，由 光強度偵測器接收訊號作為磷光訊號。而訊號紀錄、計算、輪出 的部分，將兩光強度偵測器之訊號送到資料擷取卡後，資料擷取 卡以每秒500k筆數據的紀錄速度，記錄兩光強度偵測器所測得的 光訊號’訊號傳送到本專利所撰寫的程式中，以一階Butter w〇rth 帶通濾波，此一濾波動作可有效去除外界光源以及電流雜訊所帶 來的衫響。濾、除雜訊後，程式分別以快速傅立葉、鎖相兩種方式 分別計算出相位差，相位計算每秒鐘進行1〇次，1〇次計算結果平 均後輸出，則可崎到該秒之相健果。本專利提出光學碟光相 位差1測系統裝置，期望可藉由分析細胞内的耗氧量，得知細胞 的存活、細胞内粒線體障礙、藥物或化學物對細胞的毒化衝擊、 以及致病病毒感染或疾病造成的細胞代謝變化…等。 本創作之特點於下： 1. 將細胞培養於孔盤的方式，可鮮朗_化之目的。此方法 無需任何的製程且材料取得容易，便於操作。 2. 使用被祕的⑽酬絲子，感耻湖氧濃度來判斷細胞 的呼吸’不與細胞直接接觸，提供細胞無毒化的環境。 3. 透過液ΘΒ^4裝置系統架設’细其投影出不關形之光 源，選擇性激發不同孔洞内的氧偵測碌光粒子，可達到動態偵 測不同孔洞裡的細胞代謝軌，以及即時感測的目的。 Μ木用4光相位1測系統，除了擁有靈敏度高、專—性高、反應 料υυ48〇 速度決、且可以在惡劣的環境中作長期的監測等多項優點。 5·此糸統架設簡單且縣低廉，錄於昂貴之培養_機台更符 合經濟實惠。 本創作之主要目的製作出細胞制㈣統，以光源激發孔洞 裡氧偵_光粒子’利_光相位差之細胞耗氧量量測方法，觀 察、、’田胞在裝置裡的呼吸侧，達成細胞檢測之目的，並達到即時 感測的功能。 【實施方式】 如第一圖所示，此裝置主要以孔盤作為細胞陣列化培養的構件， 其實施之詳細製作步驟依序為： 第一步驟：首先，將含細胞的懸浮培養液、水膠溶液及氧偵測磷 光粒子依比例混和（10)，滴入孔盤（3)内，在滴入固化 劑將孔洞内水膠固化後吸出，此時孔盤(3)達到細胞包 覆的效果。 第二步驟：將壓克力⑵板密封於孔盤(3)上，使孔洞（3)内水膠， 與外在環境隔絕，不會破壞孔洞内環境，細胞可透過 水膠溶液（10)得到養分，在密封孔盤(3)裡生存。 第三步驟：將孔盤⑶放置於檢測系統上，孔洞⑶内水膠包覆氧 偵測磷光粒子，由電腦定義激發光源照射圖形以及位 置，控制液晶顯示器（4)定位激發孔洞陣列，即可觀察 各孔洞（3)内細胞之呼吸情況。 M400480 第四步驟：由電腦程式LabView控制資料擷取卡（7)送出訊號’ 觸發LED Driver(8)，LED Driver(8)依據所給予之訊 號頻率同時觸發LED(5)，LED(5)之光源經液晶顯示器 (4)定義光源圖形後，透過分光鏡(9)，一部分由光強 度偵測器（1)接收訊號作為參考訊號，一部分照射至待 量測孔洞（3)内的磷光粒子激發出磷光，濾光鏡(6)過 • 濾其他雜光，由光強度偵測器（1)接收訊號作為磷光訊 # 號。將兩光強度偵測器（1)之訊號送到資料擷取卡⑺ 後，資料擷取卡（7)記錄兩光強度偵測器（1)所測得的 光訊號，將訊號傳送到LabView程式中。 第五步驟：訊號處理程式分別以快速傅立葉、鎖相兩種方式，將 光強度偵測器（1)所測之訊號，分別計算出相位差，計 算結果平均後輸出得到該秒之相位結果。將所得相位 差結果經由理論，推得裝置各孔洞（3)内氧濃度之 胃 化，分析當下細胞的活性。 【圖式簡單說明】 第一圖為本創作之系統檢測示意圖。 第一圖為本創作之系統檢測平面示意圖。 第二圖為本創作之系統檢測流程示意圖 8 M400480 【主要元件符號說明】 1光強度偵測器 3 96孔盤 5 UV-LED 陣列 7資料擷取卡 9分光鏡 11感測裝置 2壓克力板 4 LCD 6滤光片 8 LED-driver 10細胞加上水膠混合液 12量測裝置V. New description: 【New technology field】 · This work uses traditional cell culture in the hole plate to complete the biomedical sensing device by using the phosphorescence phase difference oxygen consumption detection system. This device can accurately analyze the cell status changes. It can be applied to the development of new drugs and biomedical testing. [Prior Art] 1. At present, many methods of cell localization and oxygen sensing have been proposed, and only for the analysis and comparison of the devices related to this creation, are summarized as follows: 2· 2007 'David A· Chang-Yen et al. The person in the oxygen-consuming county particle Ru is mixed with the polymer material and spin-coated on the surface of the glass to form a layer of nano film to complete the oxygen consumption sensing device. After the device is in standby, the blue LED with a wavelength of 45 () nm is illuminated as a lamp slide, and the light will be totally reflected in the glass, thereby exciting the fluorescence on the surface of the glass, and finally receiving a certain range. The surface of the green number is converted to the oxygen concentration by the Stern-VolmerS program. Passing _ 100% oxygen concentration in the device, respectively sensing the relationship between its fluorescence intensity at different oxygen concentrations, and when the oxygen concentration is below 1%, the fluorescence intensity and oxygen concentration can still be linear. _, so the literature can use this oxygen-consuming sensing device to measure small changes in oxygen concentration, which will be applied in biological metabolism research, environmental detection and cell observation in the future. 3. In 2007, Ylna Kuang et al. will sell 1 (10) (10) diameter polystyrene nanoparticles to produce positive-positive roots, and then coat the fluorescent particles mtheniumdi) on the surface of the navel. Positive-electrode nanoparticles are coated on the cell surface of the beer, and the surface of the cells is attached to the cell surface for continuous oxygen consumption glory intensity measurement. This gentamic polystyrene nanoparticle is non-invasive 'will not cause cell death due to cell swallowing. It is reversible. 'If necessary, the oxygen-consuming camping light particles attached to the cell can be excluded. Because of the close proximity of the patch, the oxygen consumption of the cells can be accurately sensed instantly. 4. In 2009, Timothy W. Molter et al. fabricated a microwell array on a glass substrate and then distributed the fluorescent particles into a solution on the microwell array device. When the device is covered, it is willing to 'exclude excess solution. After leaving the solution in the microwell array, the fluorescent particles will sink in the microwell array. After the cover is opened, the solution is baked and the glory particles automatically form a ring structure at the bottom of the microwell, and then sintered and fixed. In the aspect of oxygen-consuming fluorescence detection, this paper chooses to measure the life of the dish. When the excitation light source is turned off, the fluorescent particles will have a short-time light emission time, which is shot in the disc. Two fluorescent images with different exposure times, then divide the two fluorescent surfaces into a dimensionless ratio under a certain oxygen concentration. When the oxygen concentration is different, the two photo intensity-independent ratios will correspond. change. The scale light can not be changed because of the amount of fluorescent particles, but only because of the oxygen concentration of the environment. Therefore, when this method is used to overcome the traditional measurement fluorescence intensity, the concentration of the camping light particles will affect the intensity of the camp light. Problem, and reduce the fluorescence quenching effect (Ph〇t〇bleach) 'extend the use time of this oxygen consumption sensor. [New content] M400480 The sensing device of this design, the method of cultivating the cells in the orifice plate, adding oxygen to detect the phosphorescent particles and sealing the orifice plate, and measuring the pores in the orifice plate in combination with the phosphorescence phase difference oxygen consumption detection system The amount of dissolved oxygen. This system is mainly composed of liquid crystal display. Through the optical erection of this patent, it can control different excitation light source patterns to achieve single hole phosphorescence excitation or phosphorescence excitation between different holes, and then compare the difference of cell activity between different holes, and increase the observation. The degree of freedom. The principle of oxygen consumption measurement is based on the phosphorescence phase measurement method, and the principle is based on the generation of phosphorescence lifetime. Generally, when the excitation light illuminates the light-filling material, the _disc material is excited to emit the light, and when the excitation light stops, the light is kept for a period of time, which is called the light-breaking life. Recording the waveform to form 'the phosphorescence peak will delay for a period of time to form a phase difference. The phase difference obtained by comparing the two signals will meet tan( 5 ) = 2; τ I» r, from which the ball can also be derived. Changes in light life. It is the phase difference, u is the excitation light frequency, and r is the phosphorescence lifetime. This phase difference can be used in conjunction with the excitation light frequency to calculate the phosphorescence lifetime using the formula. In Equation 1 by Stern-Volmer. /1= td/t =1 + Lu kq:r〇[P〇2] ’-style towel 1〇, r. Small r indicates the intensity and lifetime of fluorescence (phosphorescence) in the absence of oxygen and oxygen, kq is the bimolecular quenching constant, and [p〇2] is the oxygen concentration. The dissolved oxygen concentration 〇mg/L and the filling life of the analyte are measured and substituted into r. / ^ can get the dissolved oxygen concentration of the analyte. Therefore, the larger the phase difference is, the longer the fluorescence lifetime is, and the lower the oxygen concentration is, thereby judging the change in the oxygen consumption of the cells. The measurement system can be disassembled into two parts. 'Firstly, the trigger and receive parts are controlled by the computer program LabView to retrieve the signal. The LED driver triggers the LED driver according to the given signal frequency. The LED light source passes through the liquid crystal. The display defines the excitation light m4〇〇48〇 source pattern. A part of the light source passing through the beam splitter receives the signal as a reference signal by the light intensity detector, and the other part is irradiated to the phosphorescent particles to be measured to excite the phosphorescence, and the light intensity is detected by the light intensity detector. The detector receives the signal as a phosphor signal. The signal recording, calculation, and turn-out part sends the signal of the two light intensity detectors to the data capture card, and the data capture card records the record speed of 500k data per second, and records the two light intensity detectors. The measured optical signal 'signal is transmitted to the program written in this patent, and the first-order Butter w〇rth bandpass filter is used to effectively remove the external light source and the noise generated by the current noise. After filtering and removing noise, the program calculates the phase difference in two ways: fast Fourier and phase-locked. The phase calculation is performed once every second, and the average of one-time calculation results is output. Fruit health. This patent proposes an optical disc optical phase difference 1 measurement system device, and it is expected that by analyzing the oxygen consumption in the cell, the survival of the cell, the intracellular mitochondrial disorder, the poisoning impact of the drug or the chemical on the cell, and the Changes in cellular metabolism caused by viral infection or disease...etc. The characteristics of this creation are as follows: 1. The method of cultivating cells in a well plate can be used for the purpose of freshening. This method does not require any process and the material is easy to handle and easy to operate. 2. Use the secreted (10) remuneration, shame the lake oxygen concentration to judge the cell's breathing' without direct contact with the cells, providing an environment in which the cells are non-toxic. 3. Through the liquid helium ^4 device system to set up a 'light-projecting light source that can not be turned off, selectively exciting the oxygen detecting light particles in different holes, can dynamically detect the cellular metabolic track in different holes, and instantly The purpose of sensing. The 4-Phase Phase 1 measurement system of eucalyptus has many advantages such as high sensitivity, high specificity, speed of reaction material υυ48〇, and long-term monitoring in harsh environments. 5. This raft is simple to set up and the county is low. It is recorded in the expensive training _ machine is more economical. The main purpose of this creation is to produce a cell system (four) system, which uses a light source to illuminate the oxygen consumption of the oxygen detectors in the holes, and observes the cell's oxygen consumption in the device. Achieve the purpose of cell detection and achieve the function of instant sensing. [Embodiment] As shown in the first figure, the device mainly uses a well plate as a member for cell array culture, and the detailed production steps of the device are as follows: First step: First, the cell-containing suspension culture solution and water are used. The glue solution and the oxygen detecting phosphorescent particles are mixed in proportion (10), dropped into the orifice plate (3), and the water gel in the hole is solidified by dropping the curing agent, and then sucked out, and the orifice plate (3) reaches the cell coating. effect. The second step: sealing the acrylic (2) plate on the orifice plate (3), so that the water glue in the hole (3) is isolated from the external environment, does not damage the environment inside the hole, and the cell can penetrate the water glue solution (10) Get nutrients and survive in the sealed hole plate (3). The third step: placing the orifice plate (3) on the detection system, and the water gel in the hole (3) is coated with oxygen to detect the phosphorescent particles, and the computer defines the excitation light source to illuminate the pattern and the position, and controls the liquid crystal display (4) to locate the excitation hole array. Observe the respiration of cells in each hole (3). M400480 Step 4: Control the data capture card by the computer program LabView (7) Send the signal ' Trigger LED Driver (8), LED Driver (8) trigger LED (5) according to the given signal frequency, LED (5) After the light source is defined by the liquid crystal display (4), the light source pattern is transmitted through the beam splitter (9), and a part of the light intensity detector (1) receives the signal as a reference signal, and a part of the light source is irradiated to the phosphor particles in the hole (3) to be excited. Phosphorescence, filter (6) over • Filter other stray light, the light intensity detector (1) receives the signal as the phosphor photo #. After the signals of the two light intensity detectors (1) are sent to the data capture card (7), the data capture card (7) records the optical signals measured by the two light intensity detectors (1), and transmits the signals to the LabView. In the program. The fifth step: the signal processing program calculates the phase difference by the signals measured by the light intensity detector (1) in two ways, namely, fast Fourier and phase-locked, and the calculated result is averaged to obtain the phase result of the second. The resulting phase difference results were theoretically derived to obtain gastricization of the oxygen concentration in each well (3) of the device, and the activity of the current cells was analyzed. [Simple description of the diagram] The first diagram is a schematic diagram of the system detection of the creation. The first picture is a schematic diagram of the system detection plane of the creation. The second picture is the schematic diagram of the system detection process of the creation. 8 M400480 [Main component symbol description] 1 light intensity detector 3 96 hole disk 5 UV-LED array 7 data capture card 9 beam splitter 11 sensing device 2 acrylic Board 4 LCD 6 filter 8 LED-driver 10 cells plus water glue mixture 12 measuring device