υ0301：〇ν 玖、發明說明 【發明所屬之技術領域】 本發明大體上係關於決定核酸被測物的方法及儀器。 尤其，本發明是關於偵測一不含傳統光學可辨識標誌（標 記）物質的被測物。 【先前技術】 使用一實質上爲平面的系統，專家稱之爲生物感應器 或生物晶片，以決定特定核酸被測物之性質與/或含量，例 如DNA，是所熟知的。這些生物晶片的表面含有支持物， 通常會形成許多偵測區域，大多的例子是排列成格子狀， 此處每一個別區域或一群區域對特定待測之被測物具有的 專一性都彼此不同。在待測DNA被測物的例子中，其專一 的核酸探針，例如，寡核苷酸或cDNA，大多是形成單股的 形式，且其對核酸的個別專一性是由序列順序所決定（探 針設計），以直接或間接固定於支持物表面的個別區域。 在本文相對應的偵測方法，將以此種方式作用的晶片表面 與欲測定的DNA被測物接觸，在此狀況下確定（確定先前 可偵測的已標記之標的核酸是存在的結果下）後者與固定探 針分子雜交。稍後對一個或更多專一形成的雜交複合物進 行定性與視需要的定量偵測，大多數例子係利用物理光學 冷光偵測，及分配所得到的數據到個別的偵測區域，因此 可以決定，例如，核酸被測物的存在或序列及視需要的其 含量。 除了這些以冷光爲基礎的方法，在這幾年的努力下， DNA的分析不需要冷光標定且不需要利用偵測及影像。 舉例來說，嘗試利用場效應電晶體以區別單股及雙 時期的狀態（E· Souteyrand等人，藉場效應直接偵測合成 的同形寡聚物DNA序列之雜交（Direct detection of the hybridisation of synthetic homo-oligomer DNA sequences by field effect) 5 J. Phys. Chem. B. ^ 1001，2980，[1997])或利 用阻抗結構（見，例如，P. VanGerwen等人，奈米級交叉 指型電極排列做爲生化感應器（Nanoscaled interdigitai electrode arrays for biochemical sensors) ，Sensors and Actuators，B49，73-80，[1998]) o 該技藝的另一方法是有關於利用沙雷氏桿菌（Airs仏 )細胞外內切酶的酵素活性，其中酵素媒介的 DNA分解導致酸鹼値的改變，可被酸鹼感應器偵測（S. Reher，利用沙雷氏桿菌細胞外內切酶酵素活性以電量法、 電位法及光學法分析DNA及RNA(DNA and RNA analysis by voltammetric，potentiometric and optical methods using the extracellular endonucleases of S err at i a marcescens) 5 ISBN 3-89825-030-X ， 1999) o 此外，一現有的發表中，描述利用特定的標誌（標記 )物質施行DNA的分析，但其偵測不利用光學方法。一方 法是關於利用電子標定標記雜交的DNA，及讓此雜交DNA 吸附到貴金屬電極上，此處的結合事件可利用電極讀取。 (www.microsensor.com/TechnologySystem.html ， Clinical microsensors，2000 )。其他文獻描述偶合一小的順磁體到 2ϋ0301：07 DNA分子上，藉由磁場的變化來讀取。（D.R. Baselt等人 ’以致電阻技術爲基礎的生物感應器（A biosensor based on magnetoresistance technology) ， Biosensors & Bioelectronics 1998 j 13 ( 7-8) : 731-9，[1998])。 雖然上述所提到的文獻示範其他不同於以冷光爲基礎 的核酸分析，卻遭遇雜交反應需花數小時時間的困難，加 上用來測量的感應器測量上的偏差，即專家所知的偏移 (ddft)。此偏移導致訊號隨時間改變，其通常無法從實際的 訊號中被辨識或適當的分離，因後者與此偏移位於同一頻 率強度。進而，通常較容易在可能的最短時間內讀到達到 該訊號最大値的訊號。 【發明內容】 因此，本發明的任務是提供一改善的方法，以克服與 偏移問題有關的缺點。 此任務根據本發明中主要申請專利範圍的方法而解決。 根據一項具體實例，本發明是有關決定核酸被測物的 方法，其係將被測物雜交至適合的固定在固相的核酸探針 上，其中 (a) 此核酸被測物與核酸探針在適合的雜交狀態下一起 培養，形成雜交複合物，且 (b) 此被測物的決定是利用酵素參與導致雜交複合物質 量增加或減少而測得的物理化學數據， 此處測量的數據，是利用至少一個感應器測量，其是 固相中不可缺少的一部分。 8 由於這種間接的解決方法，本發明成功的大大避免偏 移問題，藉由轉換此偵測至不同的時間視窗，因此產生不 同的頻率，且較佳的只需幾秒鐘或幾分鐘（見圖1)。本方 法較佳的具體實例描述在附屬的申請專利範圍中。 本文中所指的“決定”，是關於分析不只是核酸，尤其 是包含偵測樣品中要檢驗的核酸被測物。進而，包含應用 的形式，例如，決定核酸序列及偵測在特定單核酸多型性 (SNPs)中的變異。因此本方法可確保一廣泛的可能使用 範圍，因其可應用至所有目前及未來以雜交複合物形成爲 基礎的決定及/或偵測技術中。 根據一較佳的具體實例，導致雜交複合物質量增加或 減少的酵素是選自由包含聚合酶、結合酶、核酶、半催化 核酸、DNA酶/RNA酶（外及/或內切核酸酶，包括限制內 切核酸酶）及RNA酶Η所組成的群組中，其中，尤其是具 有5’及/或3’-外切核酸酶活性的聚合酶是更佳的。 除了此DNA依賴的DNA聚合酶外，根據本發明，視 存在的核酸組成（RNA或DNA)而定的質量增加可利用 RNA依賴的DNA聚合酶（反轉錄酶）或RNA依賴的RNA 聚合酶（複製酶）而產生。質量的增加也可利用適當的聚 合酶活化的核酶或半催化的RNAs產生。根據本發明，所 有聚合酶（包括核酶或半催化RNAs)應用的原則係熱穩定 及不耐熱的酵素都可使用。 酵素導致的質量增加亦可利用結合酶完成。文中引人 注意處，是一適當的使用本發明提及之結合酶活化之核酶 2^3〇i：〇y ，或半催化的RNAs。根據本發明，所有結合酶（包括核酶 或半催化的RNAs)應用的原則係熱穩定及不耐熱的酵素都 可使用。 相對於質量增加，質量的減少亦可被偵測到。藉由核 酸酶（RNA酶，DNA酶）切除結合的核酸可導致質量減少 。5’及/或3’-外切及內切核酸酶及RNA酶Η皆可使用。單 股及雙股酶或具有兩者活性的酶皆可使用。關於核酸酶， 原則是具有序列專一性及非序列專一性的酶都可使用。核 酶或具有核酸酶活性的半催化的RNAs亦可。通常，核酶 或半催化的RNAs其作用具有序列專一性，此處的專一性 ，可利用個別的雜交序列的需求調整。 因此，本發明反映了大部分固相結合的核酸被測物所 遭遇的情形，即固定到固相的單股核酸探針的存在（見圖 1A)。在適合的狀況下，如果核酸被測物與存在的探針序 列互補，即在此探針處形成至少部分至少雙股的雜交複合 物（見圖1B)。 根據本發明，啓動酶的步驟（見圖1C)是在複合物形 成之後，酶的作用導致一可測得的複合物質量改變。 舉例來說，如果一雜交複合物包含較短的DNA探針， 及存在一相對較長的核酸被測物，在適合的狀況下且含有 四種核苷酸三磷酸（A;T;G;C)，可以使用聚合酶，其 可塡滿（至少部分）由較長的核酸被測物所造成的單股區 域（見圖1C及1D)。根據假設的平均結合速度，即每分 鐘一仟個鹼基的規模，此連續性的聚合作用在幾分鐘之內 10υ0301: 〇ν 发明 Description of the invention [Technical field to which the invention belongs] The present invention relates generally to a method and an apparatus for determining a nucleic acid test substance. In particular, the present invention relates to detecting a test object that does not contain a conventional optically identifiable mark (marker). [Prior art] The use of a substantially planar system, which experts call biosensors or biochips, to determine the properties and / or content of specific nucleic acid analytes, such as DNA, is well known. The surface of these biochips contains supports and usually forms many detection areas. Most of the examples are arranged in a grid. Here, each individual area or group of areas has different specificities for a particular test object to be tested. . In the case of the DNA to be tested, the specific nucleic acid probes, such as oligonucleotides or cDNA, are mostly in the form of single strands, and their individual specificity for nucleic acids is determined by the sequence ( Probe design) to directly or indirectly fix individual areas of the support surface. The corresponding detection method in this article is to contact the surface of the wafer in this way with the DNA to be measured, and determine under this condition (under the result that the previously detectable labeled target nucleic acid is present). ) The latter hybridizes with immobilized probe molecules. Later, one or more specifically formed hybrid complexes are subjected to qualitative and quantitative detection as needed. Most examples use physical optical cold light detection, and assign the obtained data to individual detection areas, so it can be determined For example, the presence or sequence of a nucleic acid test substance and its content if necessary. In addition to these cold light-based methods, under the efforts of recent years, DNA analysis does not require cold calibration and does not require detection and imaging. For example, try to use field-effect transistors to distinguish between single-strand and dual-phase states (E. Souteyrand et al., Using field effect to directly detect the hybridization of synthetic homologous oligo DNA sequences (Direct detection of the hybridisation of synthetic homo-oligomer DNA sequences by field effect) 5 J. Phys. Chem. B. ^ 1001, 2980, [1997]) or the use of impedance structures (see, for example, P. VanGerwen et al., nanoscale interdigitated electrode arrangements As bioscale sensors (Nanoscaled interdigitai electrode arrays for biochemical sensors), Sensors and Actuators, B49, 73-80, [1998]) o Another method of this technique is related to the use of extracellular cells of Serratia (Airs 仏) Enzyme activity of endonucleases, in which the degradation of the enzyme-mediated DNA causes changes in the pH of the enzyme, can be detected by the acid-base sensor (S. Reher, using Serratia extracellular endonuclease enzyme activity by electrical method, potential DNA and RNA analysis by voltammetric, potentiometric and optical methods using the extra cellular endonucleases of Ser err at ia marcescens 5 ISBN 3-89825-030-X, 1999) In addition, an existing publication describes the use of specific markers to perform DNA analysis, but its detection does not use optical methods. One method is to label the hybridized DNA by electronic calibration and to adsorb the hybridized DNA to the precious metal electrode. The binding event here can be read by the electrode. (www.microsensor.com/TechnologySystem.html, Clinical microsensors, 2000). Other literatures describe coupling a small paramagnet to 2ϋ0301: 07 DNA molecules, which are read by changes in the magnetic field. (D.R.Baselt et al. 'A biosensor based on magnetoresistance technology', Biosensors & Bioelectronics 1998 j 13 (7-8): 731-9, [1998]). Although the above-mentioned literature demonstrates that other nucleic acid analysis based on cold light is different, it encounters the difficulty that the hybridization reaction takes several hours, plus the measurement deviation of the sensor used for the measurement, which is known by experts. Shift (ddft). This offset causes the signal to change over time, which usually cannot be identified or properly separated from the actual signal because the latter is at the same frequency intensity as this offset. Furthermore, it is usually easier to read a signal that reaches the maximum value of the signal in the shortest possible time. SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide an improved method to overcome the disadvantages associated with the offset problem. This task is solved according to the method of the main patent application in the present invention. According to a specific example, the present invention relates to a method for determining a nucleic acid test substance, which involves hybridizing the test substance to a suitable nucleic acid probe immobilized on a solid phase, wherein (a) the nucleic acid test substance and the nucleic acid probe are The needles are cultivated together in a suitable hybridization state to form a hybrid complex, and (b) the determination of this test object is the physical and chemical data measured by using enzymes to cause the mass of the hybrid complex to increase or decrease. , Is measured with at least one sensor, which is an integral part of the solid phase. 8 As a result of this indirect solution, the present invention successfully avoids the offset problem greatly. By converting this detection to different time windows, different frequencies are generated, and preferably only a few seconds or minutes ( see picture 1). Preferred specific examples of this method are described in the scope of the attached patent application. The “decision” referred to in this article refers to the analysis of not only nucleic acids, but especially the nucleic acid analytes to be tested in the detection sample. Furthermore, it includes application forms such as determining nucleic acid sequences and detecting variations in specific single nucleic acid polymorphisms (SNPs). The method therefore ensures a wide range of possible uses, as it can be applied to all current and future decision-making and / or detection techniques based on hybrid complex formation. According to a preferred specific example, the enzyme that causes the mass of the hybridization complex to increase or decrease is selected from the group consisting of a polymerase, a binding enzyme, a ribozyme, a semicatalytic nucleic acid, a DNase / RNase (exo and / or endonuclease, Including the group consisting of restriction endonucleases) and RNases, among them, a polymerase having 5 'and / or 3'-exonuclease activity is more preferable. In addition to this DNA-dependent DNA polymerase, according to the present invention, an increase in mass depending on the nucleic acid composition (RNA or DNA) present can utilize an RNA-dependent DNA polymerase (reverse transcriptase) or an RNA-dependent RNA polymerase ( Replicase). The increase in mass can also be produced using appropriate polymerase-activated ribozymes or semi-catalyzed RNAs. According to the present invention, all polymerases (including ribozymes or semicatalytic RNAs) are applied on the principle that thermostable and thermolabile enzymes can be used. The increase in mass caused by enzymes can also be accomplished with bound enzymes. What draws attention in the text is an appropriate use of the ribozymes activated by the binding enzymes mentioned in the present invention 2 ^ 30i: Oy, or semi-catalyzed RNAs. According to the invention, all binding enzymes (including ribozymes or semi-catalyzed RNAs) are applied on the principle that thermostable and thermolabile enzymes can be used. Relative to an increase in mass, a decrease in mass can also be detected. Removal of bound nucleic acids by ribonucleases (RNases, DNase) can result in reduced mass. 5 'and / or 3'-exonuclease and RNase can be used. Both single-stranded and double-stranded enzymes or enzymes with both activities can be used. Regarding nucleases, the principle is that both sequence-specific and non-sequence-specific enzymes can be used. Ribozymes or semi-catalyzed RNAs with nuclease activity are also possible. In general, ribozymes or semi-catalyzed RNAs have sequence specificity for their effects. The specificity here can be adjusted by the needs of individual hybridization sequences. Therefore, the present invention reflects the situation encountered by most solid-phase-bound nucleic acid analytes, that is, the presence of single-stranded nucleic acid probes fixed to the solid phase (see Figure 1A). Under suitable conditions, if the nucleic acid test object is complementary to the existing probe sequence, at least a part of at least a double-stranded hybrid complex is formed at this probe (see Figure 1B). According to the present invention, the step of initiating the enzyme (see Fig. 1C) is that after the complex is formed, the action of the enzyme causes a measurable change in the mass of the complex. For example, if a hybridization complex contains a shorter DNA probe and a relatively long nucleic acid test object, under appropriate conditions and contains four nucleotide triphosphates (A; T; G; C). A polymerase can be used, which can fill (at least in part) a single-stranded region caused by a longer nucleic acid test substance (see Figures 1C and 1D). Based on the assumed average binding rate, which is the size of one base per minute, this continuous polymerization takes place within minutes 10
2u〇3Gi：〇V 發生。這個例子是假設，此兩個雜交的部分具有不同的長 度，亦可應用在相反的例子中，即被測物的長度較探針爲 短。在這種情況下，有利於將探針設計爲至少有100個核 苷酸的長度，且此雜交複合物的單股區域是可被預期的， 而且其可藉由聚合酶的活性延長而塡滿，使其儘可能靠近 感應器的表面。如果探針是藉由3’端固定的且單股區域塡 滿是在固相發生，亦可達到此優點。這些有益的具體實例 可轉移到不只聚合酶，亦包括一般根據本發明合適的所有 酵素，且容易由習於該項技術的人完成，視其想要應用的 領域而定。 當待測樣品中沒有存在與探針序列互補的被測物時， 則因不同的結合能，故在此時間點中沒有雜交複合物的形 成，因此接下來的酵素反應不會發生，且沒有酵素依賴的 測量數據可被紀錄。 因爲不應用電場的雜交反應通常需耗費數小時，根據 本發明，其偵測的時間相當的減少，導致其可在很短的時 間視窗內發生，更適合感應器的讀取。 前文所提的聚合酶活性，是一個可得到焦磷酸根離子 的例子，在雜交複合物單股區域，進行核苷酸三磷酸之聚 合反應時，被釋放出來，且導致局部酸化，因而使酸鹼値 降低。藉局部排列pH感應器或pH偵測器（例如，酸鹼感 測場效應電晶體元件（pH-ISFET))，必要時可以偵測相 對應欲知的特定位置處酸鹼値的變化（見圖2)。 進一步，本發明亦提供間接偵測聚合反應或結合反應 11 過程中釋放出來的焦磷酸根離子，也就是說，藉由一個二 級酵素系列反應。舉例來說，ATP硫化酶及腺苷-5’-磷酸化 硫（APS)參與此二級反應的第一步。在此例子中，ATP硫 化酶轉換在聚合反應或結合反應過程中合倂核苷酸所釋放 的焦磷酸根離子PPi及APS變爲ATP。此產生的ATP接著 催化進一步的酶的反應，其係可被實際偵測到的。舉例來 說，此形成的ATP可催化螢光酶轉換螢光素的反應，導致 其發光而被本發明之光學感應器掃描到。 本發明方法之修飾例子是有關裝載與磁性球體一同使 用的核苷酸三磷酸（Baselt，/oc· c/i·，1998 )或與金屬粒子 一同使用（臨床微感應器（Clinical Micro-sensors)，/oc· cii. ，2000 )。此裝載的結果，由於與核苷酸三磷酸結合的固 體物之附加性質，使讀取的結果進一步加強。進而，核苷 酸三磷酸上會呈現顏色（染料/色素），且藉由固定的發光 二極體讀取。 因此，根據較佳的具體實例，感應器（至少一個）係選自 由包括電極構造、場效應電晶體、磁感器、光感器及酸鹼 感應器所組成的群組，對應於完全的光譜使用。 更佳的具體例是有關於結合使用前述不同形式的感應 器。舉例來說，其可能可以最適化訊號強度或靈敏度，如 此可得到一個想要偵測的事件的可信度，如果此偵測的儀 器是適合於本發明之方法，其不只有一個探針專一的感應 器（例如場效應電晶體），亦包括其他形式的探針專一感應器 (例如酸鹼感測場效應電晶體元件（pH-ISFET))。必要時， 2υ〇301：〇ν 這些從多參數的測量所得到的數據，可使酵素依賴的訊號 被更正確的評估。 進而，此感應器可裝設一加熱元件。此類元件包括’ 例如，在影像感應（CMOS)過程中應用的傳導體軌跡’後 來被下一層所覆蓋。如此有助於溫度循環的進行’舉例來 說，對文中本發明方法的PCR支援應用是令人滿意的。 因爲有感應器之被測物的加入脈衝，會產生所謂的加 成性波峰，最好是連續的操作本發明之方法（以直通模式）。 根據其他方面，提供一儀器以達成本發明之方法。 此儀器包含至少一固相、至少一核酸探針直接或間接 固定在其上，及至少一個感應器以偵測物理測量數據，此 處的感應器是此固相上不可缺少的一部分，且較佳係選自 於上文所定義之群組，包括電極構造、場效應電晶體、磁 感器、光感器及酸鹼感應器。 根據本發明所提，較適合的儀器是，多數不同的核酸 探針以格子狀排列成微矩陣，此微矩陣更佳係提供至少一 個感應器於每一個固定的核酸探針處或每一個專一的偵測 區域。 由ΕΡ-Α-0 881 490得知的測量儀器，可測量至少一個 待測活細胞的特定物理或型態上的參數，經適當修改後， 可根據本發明使用。此裝備如已述的含有多個感應器，此 感應器是所提供的儀器中不可缺少的部分，所欲檢測的物 質是固定在其上。 根據本發明，此儀器的輔助元件主要包括含嵌入之偵 132u03Gi: 0V occurred. This example assumes that the two hybridized parts have different lengths, and can also be applied to the opposite example, that is, the length of the test object is shorter than that of the probe. In this case, it is advantageous to design the probe to have a length of at least 100 nucleotides, and a single-stranded region of the hybridization complex can be expected, and it can be extended by polymerase activity. Full as close to the surface of the sensor as possible. This advantage can also be achieved if the probe is immobilized by the 3 'end and the single-stranded region is full in the solid phase. These beneficial specific examples can be transferred to not only polymerases, but also all enzymes generally suitable according to the present invention, and can be easily completed by those skilled in the art, depending on the field in which they want to apply. When there is no test substance complementary to the probe sequence in the test sample, there is no hybridization complex formed at this time point due to different binding energies, so the next enzyme reaction will not occur, and no Enzyme-dependent measurement data can be recorded. Because a hybridization reaction without an electric field usually takes several hours, according to the present invention, the detection time is considerably reduced, resulting in that it can occur in a short time window, which is more suitable for reading by sensors. The polymerase activity mentioned above is an example where pyrophosphate ions can be obtained. When a single-stranded region of the hybridization complex undergoes a polymerization reaction of nucleotide triphosphates, it is released and leads to local acidification, thus making the acid Decrease in alkalinity. By locally arranging pH sensors or pH detectors (for example, pH-ISFETs), it is possible to detect changes in pH at specific locations corresponding to the desired position (see figure 2). Furthermore, the present invention also provides indirect detection of pyrophosphate ions released during the polymerization reaction or the binding reaction 11, that is, through a secondary enzyme series reaction. For example, ATP sulfase and adenosine-5'-phosphoryl sulfur (APS) participate in the first step of this secondary reaction. In this example, ATP sulfurylase converts the pyrophosphate ions PPi and APS released by the nucleosides during the polymerization reaction or the binding reaction into ATP. This generated ATP then catalyzes further enzymatic reactions, which are actually detectable. For example, the formed ATP can catalyze the reaction of the luciferase to convert luciferin, causing it to emit light and be scanned by the optical sensor of the present invention. Modification examples of the method of the present invention are related to loading nucleotide triphosphates (Baselt, / oc · c / i ·, 1998) used with magnetic spheres or used with metal particles (Clinical Micro-sensors) , / Oc · cii., 2000). The result of this loading is further enhanced by the additional properties of the solids bound to the nucleotide triphosphate. Furthermore, a color (dye / pigment) appears on the nucleotide triphosphate and is read by a fixed light-emitting diode. Therefore, according to a preferred specific example, the sensor (at least one) is selected from the group consisting of an electrode structure, a field effect transistor, a magnetic sensor, a light sensor, and an acid-base sensor, corresponding to a complete spectrum use. A more specific example is the combination of the aforementioned different types of sensors. For example, it may be able to optimize the signal strength or sensitivity, so as to obtain the credibility of an event to be detected. If the detection instrument is suitable for the method of the present invention, it has more than one probe. Sensors (such as field-effect transistors), but also other types of probe-specific sensors (such as pH-ISFETs). When necessary, 2υ〇301: 〇ν These data obtained from multi-parameter measurements can enable more accurate evaluation of enzyme-dependent signals. Furthermore, the sensor can be equipped with a heating element. Such components include 'for example, conductor traces applied in image sensing (CMOS) processes' and are then covered by the next layer. This facilitates the progress of the temperature cycle. For example, the PCR support application of the method of the present invention is satisfactory. Because the pulse of the measured object with the sensor will generate so-called additive peaks, it is best to continuously operate the method of the present invention (in the through mode). According to other aspects, an apparatus is provided to achieve the method of the invention. The instrument includes at least one solid phase, at least one nucleic acid probe fixed directly or indirectly on the solid phase, and at least one sensor to detect physical measurement data. The sensor here is an indispensable part of the solid phase, and compared with The best line is selected from the group defined above, including electrode structure, field effect transistor, magnetic sensor, light sensor and acid-base sensor. According to the present invention, a more suitable instrument is that most different nucleic acid probes are arranged in a grid into a micromatrix, and the micromatrix is more preferably provided with at least one sensor at each fixed nucleic acid probe or each dedicated Detection area. The measuring instrument known from EP-A-0 881 490 can measure specific physical or type parameters of at least one living cell to be measured, and can be used according to the present invention after being appropriately modified. This equipment, as already mentioned, contains multiple sensors. This sensor is an indispensable part of the instrument provided, and the substance to be detected is fixed on it. According to the present invention, the auxiliary components of this instrument mainly include
2o030i：0V 測層的半導體材料，最好包含數個偵測器，其中至少一個 上述的感應器係倂入作爲偵測器，視需要結合之（見上述 )。進而，此輔助元件中可含有加熱的元件，以在實施過 程中提供不同的溫度（見上述）。在一更佳的具體實例中 ，此訊號的處理至少部份在所使用的感應晶片發生。 根據本發明之一個方面，被掃描輸出的測量數據，舉 例來說，可以利用相似的區域，直接在晶片上估計，例如 ，以每毫秒紀錄一數値，然後與先前測量而得的參考値做 比較，此參考値亦儲存在晶片內。此外，此操作模式使一 些非專一的干擾訊號，例如，分散向內的外部訊號，被計 算出來。 此感應器的表面設計，如微矩陣的排列方式，其上有 多數偵測區域需要被估計，此測量範圍或測量點的訊號可 依序偵測，例如，測量整排或整行的感應器表面，或分部 份測量（多重應用）。 舉例來說，經由類似的數字轉換後，從偵測儀所輸出 的電子訊號，可利用合適的線路，傳送至外部的評估儀器 (見上述）。 爲了使本發明之方法能運用在感應器的這一層，根據 另一個較佳的具體實例，可在其上覆蓋一層可以相結合的 物質。典型的是將感應器晶片表面，例如，這些含有二氧 化矽的組成，浸泡於一含有雙功能分子（所謂的連接物） 的溶液中，其具有鹵砂院（例如，氯砍院）或砂焼氧基， 可與此支持面連接，因此形成自行組合的單一層（SAM) 14 2〇〇3Gi：〇y ，經由感應器表面與受體之間產生共價鍵結。舉例來說， 此覆蓋可使用甘油三矽烷乙氧完成，例如，將其浸泡在含 有1%矽甲烷的甲苯溶液中，緩慢取回，置於120°c中烘烤 ，將其固定。一般利用這種方式覆蓋的厚度約有幾埃。連 接物與受體分子間經由另一個合適的功能基連接，例如， 胺基或環氧基。可連接多個不同的受體分子的適合雙功能 連接物，尤其是源於生物體的受體分子，使其接合到晶片 的表面，熟於此技術的人皆了解，較之於G.T. Hermanson， Academic Press 1996之“生物結合技術”。其可讓此接合的 物質變成一薄的聚合層，而產生具有功能的表面，參見W〇 00/43539 。 根據本發明，前述做爲探針的核酸，可利用一般打印 的設備加以應用且固定。 舉例來說，DNA的雜交可利用本方法在晶片表面上建 立的方法實施。此可藉由，例如，PCR方法製備。在雜交 過程中，DNA被測物與感應器表面探針（如果存在）的配 對股結合。陽性雜交事件可利用本發明之方法偵測到。 特定位置質量增加的測量可用，例如，物理方法。舉 例來說，特定位置折射指數的改變、特定位置電阻或電子 傳導性的改變、特定位置光密度的改變，或甚至特定位置 二向色效應等，皆可被測量。 基本上，本發明普遍的方法適合廣大光譜區域的應用 ，其中在此區域內可區別待測樣品中特定被測物完整診斷 的偵測，與另一方面，利用本方法由複合物衍生的修飾來 15 2υ0301：〇ν 決定序列數據，或是有關基因組功能關係的資訊。然而， 這種區別是供做舉例說明的目的，基本上，本發明公開的 適用性，不限於此。2o030i: 0V layered semiconductor materials, it is best to include several detectors, at least one of which is incorporated as a detector, and combined as needed (see above). Furthermore, this auxiliary element may contain heated elements to provide different temperatures during implementation (see above). In a better specific example, the processing of this signal occurs at least partly in the sensor chip used. According to an aspect of the present invention, the measurement data output by scanning can, for example, use a similar area to estimate directly on the wafer, for example, record a number of milliseconds per millisecond, and then do it with a reference value obtained from previous measurements For comparison, this reference frame is also stored in the chip. In addition, this operating mode allows some non-specific interference signals, such as external signals scattered inward, to be calculated. The surface design of this sensor, such as the arrangement of the micromatrix, has most of the detection areas to be estimated. The signals of this measurement range or measurement point can be detected sequentially, for example, measuring a whole row or a row of sensors Surface, or partial measurement (multiple applications). For example, after similar digital conversion, the electronic signal output from the detector can be transmitted to an external evaluation instrument using a suitable line (see above). In order that the method of the present invention can be applied to this layer of the inductor, according to another preferred embodiment, it can be covered with a layer of a substance that can be combined. Typically, the surface of the sensor chip, for example, these components containing silicon dioxide, is immersed in a solution containing bifunctional molecules (so-called linkers), which has a halide courtyard (for example, chlorin courtyard) or sand The methoxy group can be connected to this support surface, so a self-assembled single layer (SAM) 142003Gi: Oy is formed, and a covalent bond is generated between the surface of the sensor and the receptor. For example, this covering can be done with glycerol trisilane ethoxylate. For example, immerse it in a toluene solution containing 1% silicon dioxide, slowly retrieve it, bake it at 120 ° C, and fix it. Generally, the thickness covered by this method is about several angstroms. The linker is linked to the acceptor molecule via another suitable functional group, such as an amine group or an epoxy group. Suitable bifunctional linkers that can connect multiple different receptor molecules, especially receptor molecules derived from organisms, to make them join to the surface of the wafer. Anyone familiar with this technology knows that, compared to GT Hermanson, Academic Press 1996, "Biocombination Technology." It allows this bonded substance to become a thin polymeric layer, creating a functional surface, see WO 00/43539. According to the present invention, the aforementioned nucleic acid serving as a probe can be applied and fixed using a general printing device. For example, DNA hybridization can be performed using a method established on the wafer surface by this method. This can be prepared, for example, by a PCR method. During hybridization, the DNA analyte is bound to the pair of strands on the sensor surface probe (if present). Positive hybridization events can be detected using the methods of the invention. Measurements of increased mass at specific locations are available, for example, physical methods. For example, changes in the refractive index at specific locations, changes in electrical resistance or electron conductivity at specific locations, changes in optical density at specific locations, or even dichroic effects at specific locations can be measured. Basically, the universal method of the present invention is suitable for the application of a wide spectral region, in which the detection of a complete diagnosis of a specific test object in a test sample can be distinguished, and on the other hand, the modification derived from the complex using this method He 15 2υ0301: 0ν determines sequence data or information about functional relationships in the genome. However, this difference is for the purpose of illustration, and basically, the applicability of the present disclosure is not limited to this.
舉例來說，本發明之方法尤其適用於決定經由巢式 PCR平行放大的DNA的序列，較佳係於一結合液相/固相的 DNA微矩陣系統，因爲其不需使用修飾的核苷酸（例如生物 素或毛地黃素），以及螢光染料和其他傳統使用的標記物質 。在結合液相/固相的DNA微矩陣系統的巢式PCR (見圖4 )具有與傳統PCR相同的敏感度，也就是說，在液相中進 行的反應，可同時具有比傳統雜交分析及引子延展分析更 高的專一性。此優點是由於引子/樣品DNA/聚合酶系統固 有的關於放大反應之專一性被大大的增加，其係藉由固定 在固體支持物的內部PCR引子（因此也做爲探針用）及複製 子（amplkon)間的專一交互作用。整個結果，其專一性是優 於，例如，5’-外切核酸酶分析（例如，當使用TaqManTM聚 合酶時）。 從感應器發出的訊號係利用紀錄器紀錄。紀錄器具有 一快速轉換器，將相似的偵測訊號轉換成數値，儲存在記 憶中。其較佳係用於評估即時產生的訊號，但也可在時間 延遲後評估。普通的微處理器可用來評估此數値。 【實施方式】 本發明舉下列實施例詳述。 根據本發明製備一感應晶片 CMOS感應器係使用1.2微米的CMOS方式裝設在一 5 16 英吋或6英吋的膠紙上。每一個場效應電晶體被置於p-受 質上的η槽。藉由引流器及來源區域的輸入，產生場的氧 化作用。應用一厚度大約10 nm的熱閘門氧化物。此閘門 在接下來的反應過程中，被聚矽化合物保護。利用化學氣 相沉積步驟（CVD)加上一層二氧化矽，鋁也噴灑於其上 。鈍化係藉氮化矽沉積層及氧化矽沉積層達成。此絕緣閘 門暴露在接下來的蝕刻步驟中。 覆蓋CMOS感應器 上述CMOS感應器的製備，是浸泡在含有1% G0PS及 含0.1%三乙胺的甲苯溶液中大約2小時使覆蓋上矽甲烷。 接著從溶液中取出晶片，待一段時間滴乾之後，置於乾燥 箱中，以120°C固定大約2小時。 必要時，以這種方式覆蓋完成的晶片可以儲存在乾燥 的環境中，直到進行生物連接反應爲止。 寡核苷酸探針生物連接反應 上述覆蓋完成的晶片，利用傳統技術，以非接觸的步 驟加上5’-胺基修飾的寡核苷酸探針。爲了達到此目的，此 寡核苷酸保存在濃度爲5 μΜ PBS的緩衝溶液中。待完成 (printing)之後，此連接反應繼續在一 50°C的潮濕容器中進 行。最後，此晶片以蒸餾水沖洗，再利用甲醇沖洗使其乾 燥。任何殘留的溶劑，最後置於排氣櫃中，以蒸發作用移 除。 製備樣品的步驟 來自人類DNA分離物的血色素基因片段藉由pCR放大For example, the method of the present invention is particularly suitable for determining the sequence of DNA amplified in parallel by nested PCR, preferably in a liquid / solid phase DNA micromatrix system, because it does not require the use of modified nucleotides. (Such as biotin or digitonin), as well as fluorescent dyes and other traditionally used labeling substances. The nested PCR (see Figure 4) of the DNA micromatrix system combined with liquid / solid phase has the same sensitivity as traditional PCR, that is, the reaction in the liquid phase can have both Primer extension analysis is more specific. This advantage is due to the increased specificity of the amplification reaction inherent to the primer / sample DNA / polymerase system. (Amplkon) specific interaction. The overall result is more specific than, for example, 5'-exonuclease analysis (for example, when using TaqManTM polymerase). The signal from the sensor is recorded with a recorder. The recorder has a fast converter that converts similar detection signals into data and stores them in memory. It is preferably used to evaluate signals generated in real time, but can also be evaluated after a time delay. Ordinary microprocessors can be used to evaluate this number. [Embodiment] The present invention will be described in detail in the following examples. A sensor chip prepared according to the present invention is a CMOS sensor which is mounted on a 5 16 inch or 6 inch adhesive tape using a 1.2 micron CMOS method. Each field-effect transistor is placed in an n-slot on the p-substrate. With the input of the drain and source area, the field is oxidized. A thermal gate oxide with a thickness of about 10 nm is applied. This gate is protected by polysilicon compounds during the subsequent reaction. A chemical vapor deposition step (CVD) is applied with a layer of silicon dioxide, and aluminum is also sprayed thereon. Passivation is achieved by a silicon nitride deposition layer and a silicon oxide deposition layer. This insulation gate is exposed during the subsequent etching steps. Covering the CMOS sensor The above CMOS sensor was prepared by immersing it in a toluene solution containing 1% GOPS and 0.1% triethylamine for about 2 hours to cover it with silicon methane. Then remove the wafer from the solution. After dripping for a while, place it in a dry box and fix it at 120 ° C for about 2 hours. If necessary, the finished wafers covered in this way can be stored in a dry environment until a bio-ligation reaction takes place. Oligonucleotide probe bio-ligation reaction The above-mentioned covered wafers are conventionally added with 5'-amine-modified oligonucleotide probes in a non-contact step. For this purpose, the oligonucleotides are stored in a buffered solution of 5 μM PBS. After printing, this ligation reaction continues in a humid container at 50 ° C. Finally, the wafer was rinsed with distilled water and then dried with methanol. Any residual solvent is finally placed in the fume hood and removed by evaporation. Steps for sample preparation Hemoglobin gene fragment from human DNA isolate amplified by pCR
2uJ30i：0V 。在放大反應中使用合適的引子序列，例如，美國專利 5,712,098 中所述。 此反應的混合物包括以下的標準試劑（引子：0.5 μΜ ;dATP、dCTP、dGTP ·· 0.1 mM ; dTTP 0·08 mM ; PCR 緩衝 液，MgCl2 ·· 4 mM ; HotStarTaq ( Perkin Elmer) 2 單位（50 μΐ))。原本的核苷酸在PCR反應過程（35個循環，95°C 5 分鐘、95°C 30 秒、60°C 30 秒、72°C 30 秒、72°C 7 分 鐘）中倂入新合成的DNA中。接著，加入T7 Gen6-外切核 酸酶（每一次PCR，100單位/50 μΐ)，以產生單股DNA， 且加熱此反應（37°C 30分鐘，85°C 10分鐘）。 雜交反應 上所述的反應，在5倍SSPE，0.1% SDS ( 12 μΐ)緩衝 溶液中，在晶片上進行雜交，在5 0 °C潮濕的容器中大約2 小時。最後，以2倍SSPE，0.1% SDS沖洗，再以水沖洗 乾淨晶片。 【圖式簡單說明】 (一）圖式部分 本發明及有益的具體實例將以圖輔助詳述： 圖1說明本發明方法之一具體實例的步驟的圖解。（A )核酸探針（2)共價鍵結至表面。（B)加入核酸被測物（1) 之後，形成雜交複合物，通常需要數小時。（C)使用一合 適的酵素，例如，聚合酶，且同時具有四種核苷酸A、T、 G及C (以DNA爲例），此複合物的單股區域，大約在幾 分鐘的短時間內被塡滿，（D)因此很快的產生一個訊號， 182uJ30i: 0V. Suitable primer sequences are used in the amplification reaction, for example, as described in U.S. Patent 5,712,098. This reaction mixture includes the following standard reagents (primers: 0.5 μM; dATP, dCTP, dGTP ················································· Buffer. μΐ)). The original nucleotides were newly synthesized during the PCR reaction (35 cycles, 95 ° C for 5 minutes, 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds, and 72 ° C for 7 minutes). In the DNA. Next, T7 Gen6- exonuclease (100 units / 50 μΐ per PCR) was added to generate single-stranded DNA, and the reaction was heated (37 ° C for 30 minutes, 85 ° C for 10 minutes). Hybridization reaction The reactions described above were performed on a wafer in 5x SSPE, 0.1% SDS (12 μΐ) buffer solution, and placed in a humid container at 50 ° C for approximately 2 hours. Finally, rinse the wafer with 2x SSPE, 0.1% SDS, and rinse with water. [Brief description of the drawings] (I) Schematic part The present invention and useful specific examples will be detailed with the aid of the drawings: FIG. 1 illustrates the steps of a specific example of the method of the present invention. (A) The nucleic acid probe (2) is covalently bonded to the surface. (B) After adding the nucleic acid test object (1), it takes several hours to form a hybrid complex. (C) Use a suitable enzyme, such as polymerase, and have four kinds of nucleotides A, T, G, and C (take DNA as an example). The single-stranded region of the complex is about a few minutes short. Time is full, (D) therefore quickly generates a signal, 18
2uJ3Gi：GV 此訊號會被感應器（4)讀取，其是此固相不可缺的一部分。 圖2說明使用一適合的聚合酶反應的較佳具體實例之 原理，其會釋放出焦磷酸根離子（5)，導致局部酸鹼値改變 。此改變可被固定的感應器（4)偵測到。 圖3說明本文中影像感應（CMOS)過程建構的場效應 電晶體。此場效應電晶體包含，舉例來說，在其η槽中具 有一層ρ-η-ρ(6)的構造，其表面有一層薄的絕緣體（10)(例 如，10 nm的熱氧化物），其上可直接或間接供探針的應用 ，以進行雜交反應。在較佳的具體實例中，場效應電晶體 區域所刻劃的保護（7)是蝕刻成鋒利的或刻度的方式，因此 ，雜交及質量增加（8)的過程可在凹陷處進行。此儀器的表 面藉使用（例如）貴金屬或疏水/親水性物質（9)，可對於核酸 分子的雜交產生主動或被動的影響力，。在測量溶液（11)中 ，例如，1M碳酸氫鈉，在閘門處測量電介質性質的改變， 其係由於雜交複合物的單股區域塡滿。因此造成的高原電 位轉變，可使用位於溶液中的參考電極（12)，藉由場效應電 晶體讀取。舉例來說，引流器與源頭之間的電流，或參考 電極與源頭間的電壓，皆可被紀錄成訊號（見，例如，B. Palan 等人，Fundamental Noise Limits of ISFET-based Microsystems ， Poster contribution 4P26 ， EUROSENSORS XIII ( ISBN 90-76699-02-X)，p. 169 ff.，1999 )。 圖4說明平行放大的步驟，基於所知的“巢式晶片 (Nested〇n Chip)”PCR ( N〇C，見上述），偕同FET掃描電 位變化。在整個N0C過程中，一個探針處的電位變化示於 192uJ3Gi: GV This signal will be read by the sensor (4), which is an integral part of this solid phase. Figure 2 illustrates the principle of using a suitable specific example of a polymerase reaction, which releases pyrophosphate ions (5), resulting in local acid-base changes. This change can be detected by the fixed sensor (4). Figure 3 illustrates the field effect transistor constructed in the image sensing (CMOS) process in this paper. This field-effect transistor includes, for example, a structure with a layer of ρ-η-ρ (6) in its η groove, and a thin layer of insulator (10) on its surface (for example, 10 nm thermal oxide), It can be used directly or indirectly for probe applications to perform hybridization reactions. In a better specific example, the protection (7) of the field effect transistor region is etched into a sharp or graduated manner, so the process of hybridization and mass increase (8) can be performed in the depression. The surface of this instrument, by using, for example, precious metals or hydrophobic / hydrophilic substances (9), can have an active or passive influence on the hybridization of nucleic acid molecules. In the measurement solution (11), for example, 1M sodium bicarbonate, the change in dielectric properties is measured at the gate, which is due to the single-stranded area of the hybridization complex being full. The resulting plateau potential shift can be read using a field effect transistor using a reference electrode (12) in solution. For example, the current between the current collector and the source, or the voltage between the reference electrode and the source, can be recorded as a signal (see, for example, B. Palan et al., Fundamental Noise Limits of ISFET-based Microsystems, Poster contribution 4P26, EUROSENSORS XIII (ISBN 90-76699-02-X), p. 169 ff., 1999). Figure 4 illustrates the steps of parallel magnification, based on the known "Nestedon Chip" PCR (NOC, see above), with different FET scan potential changes. The potential change at one probe throughout the NOC is shown in Figure 19
2uJ30i：0V A。X軸代表循環次數，γ軸代表測量的電壓。引子分子（ 相當於探針分子）連接到探針的位置，標示於X軸下方： 剛開始的循環中，只有一些引子被延長，接著，進入中間 的循環，以指數的形式大量的增加，在後段循環中，逐漸 增加成飽和狀態（大部分的引子已經延長）。此曲線顯示 當探針位置的質量增加時，所測得電壓上升的情形。Β顯 示單一循環（循環次數在中間）的電位圖形。除了引子， 模板及引子從左到右的延長，在此皆有顯示。此說明顯示 ，電壓的上升是引子延長的函數。 (二）元件代表符號 1 :核酸被測物 2 :核酸探針 3 ··四種核苷酸三磷酸（A ; Τ ; G ; C) 4 :感應器 5:焦磷酸根離子 6 _· p-n-p 層 7:場效應電晶體區域所刻劃的保護 8:雜交及質量增加 9 :貴金屬或疏水/親水性物質 10 :絕緣體 11 :待測溶液 12 :參考電極 202uJ30i: 0V A. The X-axis represents the number of cycles and the γ-axis represents the measured voltage. The position where the primer molecule (equivalent to the probe molecule) is connected to the probe is indicated below the X axis: In the initial cycle, only some primers were extended. Then, it entered the intermediate cycle and increased exponentially. Later in the cycle, it gradually increases to saturation (most of the primers have been extended). This curve shows how the measured voltage increases as the mass of the probe position increases. Β shows the potential pattern of a single cycle (the number of cycles is in the middle). Except for primers, templates and primer extensions from left to right are shown here. This description shows that the rise in voltage is a function of primer elongation. (II) Symbols for element 1: Nucleic acid test object 2: Nucleic acid probe 3 · Four nucleotide triphosphates (A; T; G; C) 4: Sensor 5: Pyrophosphate ion 6 _ · pnp Layer 7: Protection delineated by the field effect transistor region 8: Hybridization and mass increase 9: Precious metal or hydrophobic / hydrophilic substance 10: Insulator 11: Test solution 12: Reference electrode 20