TWI816007B - Method of detecting biological sample - Google Patents
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Abstract
Description
本發明是有關於一種檢測方法，且特別是有關於一種生物樣品的檢測方法。The present invention relates to a detection method, and in particular, to a detection method of biological samples.
目前，生物樣品的檢測一般是使用傳統光學檢測技術來進行。然而，傳統光學檢測技術對於低濃度生物樣本的光學辨識困難，且在進行光譜辨識時容易受到背景基質干擾，因而導致檢測靈敏度降低。因此，發展一不受光學辨識限制的檢測技術及方法，為目前本領域重要的課題。Currently, the detection of biological samples is generally performed using traditional optical detection techniques. However, traditional optical detection technology has difficulty in optical identification of low-concentration biological samples, and is prone to background matrix interference during spectral identification, resulting in reduced detection sensitivity. Therefore, developing a detection technology and method that is not limited by optical identification is currently an important issue in this field.
本發明提供一種生物樣品的檢測方法，其可具有較佳的檢測靈敏度(sensitivity)。The present invention provides a method for detecting biological samples, which can have better detection sensitivity.
本發明提出一種生物樣品的檢測方法，包括以下步驟。提供磁感晶片，其中磁感晶片包括基板與位在基板上的磁感測層。在磁感晶片上接上多個探針。將包含標記有第一標記物的多個生物樣品的樣品液提供至磁感晶片上，以使標記有第一標記物的生物樣品與探針進行雜交反應(hybridization)。將標記有第二標記物的多個磁珠提供至磁感晶片上，以使標記有第二標記物的磁珠接合至標記有第一標記物的生物樣品上。以磁感測器檢測磁感測層所感測到的訊號。The invention proposes a method for detecting biological samples, which includes the following steps. A magnetic sensing chip is provided, wherein the magnetic sensing chip includes a substrate and a magnetic sensing layer located on the substrate. Connect multiple probes to the magnetic sensor chip. The sample liquid containing a plurality of biological samples labeled with the first marker is provided onto the magnetic sensing chip, so that the biological samples labeled with the first marker and the probe undergo hybridization reaction (hybridization). A plurality of magnetic beads labeled with the second marker are provided on the magnetic sensitive chip, so that the magnetic beads labeled with the second marker are bonded to the biological sample labeled with the first marker. A magnetic sensor is used to detect the signal sensed by the magnetic sensing layer.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，磁感測器例如是穿隧磁阻(Tunnel Magnetoresistance, TMR)感測器或巨磁阻(Giant Magnetoresistance, GMR)感測器。According to an embodiment of the present invention, in the above biological sample detection method, the magnetic sensor is, for example, a tunnel magnetoresistance (TMR) sensor or a giant magnetoresistance (GMR) sensor. device.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，探針例如是核酸探針。According to an embodiment of the present invention, in the above biological sample detection method, the probe is, for example, a nucleic acid probe.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，核酸探針的長度例如是20單體單元至40單體單元(monomeric unit, mer)。According to an embodiment of the present invention, in the above biological sample detection method, the length of the nucleic acid probe is, for example, 20 to 40 monomeric units (monomeric units, mer).
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，探針可位在磁感晶片的感測區中。According to an embodiment of the present invention, in the above biological sample detection method, the probe may be located in the sensing area of the magnetic sensing chip.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，生物樣品例如是待測單股核酸。According to an embodiment of the present invention, in the above method for detecting biological samples, the biological sample is, for example, a single-stranded nucleic acid to be detected.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，待測單股核酸的長度例如是80單體單元至120單體單元。According to an embodiment of the present invention, in the above method for detecting biological samples, the length of the single-stranded nucleic acid to be detected is, for example, 80 monomer units to 120 monomer units.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，標記有第一標記物的生物樣品的製備方法可包括以下步驟。提供待測雙股核酸。使用標記有第一標記物的引子對待測雙股核酸進行增幅處理(amplication)，而獲得標記有第一標記物的多個待測雙股核酸。對標記有第一標記物的待測雙股核酸進行變性處理，而獲得標記有第一標記物的待測單股核酸的樣品液。According to an embodiment of the present invention, in the above biological sample detection method, the preparation method of the biological sample labeled with the first marker may include the following steps. Provide double-stranded nucleic acid to be tested. The primer labeled with the first label is used to amplify the double-stranded nucleic acid to be tested, thereby obtaining a plurality of double-stranded nucleic acids to be tested labeled with the first label. The double-stranded nucleic acid to be tested labeled with the first label is denatured to obtain a sample solution of the single-stranded nucleic acid to be tested labeled with the first label.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，待測雙股核酸的增幅處理例如是進行聚合酶鏈鎖反應(polymerase chain reaction, PCR)。變性處理例如是高溫裂解處理(thermal decomposition)。According to an embodiment of the present invention, in the above method for detecting biological samples, the amplification process of the double-stranded nucleic acid to be detected is, for example, polymerase chain reaction (PCR). Denaturation treatment is, for example, thermal decomposition.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，生物樣品的來源可為待測生物檢體。待測生物檢體例如是尿液、唾液、血清或血漿。According to an embodiment of the present invention, in the above biological sample detection method, the source of the biological sample may be the biological sample to be tested. The biological sample to be tested is, for example, urine, saliva, serum or plasma.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，第一標記物可為生物素(biotin)，且第二標記物可為鏈霉親和素(Streptavidin)。According to an embodiment of the present invention, in the above method for detecting biological samples, the first label may be biotin, and the second label may be streptavidin.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，磁珠的粒徑例如是0.2μm至2μm。According to an embodiment of the present invention, in the above biological sample detection method, the particle size of the magnetic beads is, for example, 0.2 μm to 2 μm.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，磁珠的材料例如是四氧化三鐵(Fe3 O4 )。According to an embodiment of the present invention, in the above biological sample detection method, the material of the magnetic beads is, for example, ferric iron oxide (Fe 3 O 4 ).
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，以磁感測器所進行的檢測更包括定量分析。定量分析可包括以下步驟。在進行雜交反應之後，且在將磁珠接合至生物樣品上之前，使用磁感測器進行量測而獲得第一訊號。在將磁珠接合至生物樣品上之後，使用磁感測器進行量測而獲得第二訊號。藉由第二訊號與第一訊號的差值計算出接合至生物樣品上的磁珠的數量，以對與探針進行雜交的生物樣品進行定量。According to an embodiment of the present invention, in the above method for detecting biological samples, the detection using a magnetic sensor further includes quantitative analysis. Quantitative analysis may include the following steps. After performing the hybridization reaction and before coupling the magnetic beads to the biological sample, a magnetic sensor is used for measurement to obtain a first signal. After the magnetic beads are coupled to the biological sample, a magnetic sensor is used to measure and obtain a second signal. The number of magnetic beads bound to the biological sample is calculated based on the difference between the second signal and the first signal, so as to quantify the biological sample hybridized with the probe.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，第一訊號與第二訊號例如是電壓訊號。第二訊號的電壓值可高於第一訊號的電壓值。According to an embodiment of the present invention, in the above biological sample detection method, the first signal and the second signal are, for example, voltage signals. The voltage value of the second signal may be higher than the voltage value of the first signal.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，第一訊號與第二訊號可在室溫下進行量測。According to an embodiment of the present invention, in the above biological sample detection method, the first signal and the second signal can be measured at room temperature.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，更可包括以下步驟。在磁感晶片上接上多個探針之前，對磁感晶片進行表面改質處理。According to an embodiment of the present invention, the above method for detecting biological samples may further include the following steps. Before connecting multiple probes to the magnetic sensor chip, the surface of the magnetic sensor chip is modified.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，表面改質處理例如是在磁感晶片上形成二氧化矽介電層。According to an embodiment of the present invention, in the above biological sample detection method, the surface modification treatment is, for example, forming a silicon dioxide dielectric layer on the magnetic sensor wafer.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，更可包括以下步驟。在進行雜交反應之後，進行清洗處理，以移除未與探針雜交的生物樣品。According to an embodiment of the present invention, the above method for detecting biological samples may further include the following steps. After the hybridization reaction, a cleaning process is performed to remove the biological sample that has not hybridized with the probe.
依照本發明的一實施例所述，在上述生物樣品的檢測方法中，更可包括以下步驟。在將磁珠接合至生物樣品上之後，進行清洗處理，以移除未接合在生物樣品上的磁珠。According to an embodiment of the present invention, the above method for detecting biological samples may further include the following steps. After the magnetic beads are bound to the biological sample, a cleaning process is performed to remove the magnetic beads that are not bound to the biological sample.
基於上述，在本發明所提出的生物樣品的檢測方法中，在以磁感晶片中的磁感測器進行檢測時，由於生物樣本中幾乎沒有磁性物質，因此不會受到基質干擾。如此一來，在利用磁感晶片中的磁感測器進行生物樣品的檢測時，可具有較佳的檢測靈敏度。Based on the above, in the biological sample detection method proposed by the present invention, when the magnetic sensor in the magnetic sensing chip is used for detection, since there is almost no magnetic material in the biological sample, there will be no matrix interference. In this way, when the magnetic sensor in the magnetic sensor chip is used to detect biological samples, better detection sensitivity can be achieved.
為讓本發明的上述特徵和優點能更明顯易懂，下文特舉實施例，並配合所附圖式作詳細說明如下。In order to make the above-mentioned features and advantages of the present invention more obvious and easy to understand, embodiments are given below and described in detail with reference to the accompanying drawings.
圖1為本發明一實施例的生物樣品的檢測方法的流程圖。圖2A至圖2F為本發明一實施例的生物樣品的檢測流程的示意圖。圖3為本發明一實施例的生物樣品的製備方法的示意圖。圖4為本發明一實施例的定量分析的流程圖。Figure 1 is a flow chart of a biological sample detection method according to an embodiment of the present invention. 2A to 2F are schematic diagrams of a biological sample detection process according to an embodiment of the present invention. Figure 3 is a schematic diagram of a biological sample preparation method according to an embodiment of the present invention. Figure 4 is a flow chart of quantitative analysis according to an embodiment of the present invention.
請參照圖1與圖2A，進行步驟S100，提供磁感晶片100。磁感晶片100是指含有磁感測器的晶片。在本實施例中，磁感晶片100包括基板102與位在基板102上的磁感測層104。基板102例如是半導體基板，如矽基板。磁感測層104可為磁性穿遂接面(magnetic tunnel junctions, MTJ)結構。舉例來說，磁性穿遂接面結構可為包含上下兩層鐵磁性金屬(如，NiFe或CoFe)與中間的絕緣層(如，氧化鋁或氧化鎂)的三明治結構。磁感測層104可藉由半導體製程進行製作。在一些實施例中，磁感晶片100可為微流道晶片，但本發明並不以此為限。Referring to FIG. 1 and FIG. 2A , step S100 is performed to provide the magnetic sensor chip 100 . The magnetic sensing chip 100 refers to a chip containing a magnetic sensor. In this embodiment, the magnetic sensing chip 100 includes a substrate 102 and a magnetic sensing layer 104 located on the substrate 102 . The substrate 102 is, for example, a semiconductor substrate, such as a silicon substrate. The magnetic sensing layer 104 may be a magnetic tunnel junctions (MTJ) structure. For example, the magnetic tunnel junction structure may be a sandwich structure including two upper and lower layers of ferromagnetic metal (eg, NiFe or CoFe) and an intermediate insulating layer (eg, aluminum oxide or magnesium oxide). The magnetic sensing layer 104 can be fabricated through a semiconductor process. In some embodiments, the magnetic sensor wafer 100 may be a microfluidic wafer, but the invention is not limited thereto.
此外，可進行步驟S102，對磁感晶片100進行表面改質處理，以利於在後續製程中將探針接到磁感晶片100上。表面改質處理例如是在磁感晶片100上形成二氧化矽介電層106，但本發明並不以此為限。舉例來說，二氧化矽介電層106可形成在磁感測層104上。在其他實施例中，表面改質處理可為在磁感晶片100上形成其他合適材料的介電層。In addition, step S102 can be performed to perform surface modification treatment on the magnetic sensor wafer 100 to facilitate connecting the probe to the magnetic sensor wafer 100 in subsequent processes. The surface modification treatment includes, for example, forming a silicon dioxide dielectric layer 106 on the magnetic sensor wafer 100, but the invention is not limited thereto. For example, a silicon dioxide dielectric layer 106 may be formed on the magnetic sensing layer 104 . In other embodiments, the surface modification treatment may be to form a dielectric layer of other suitable materials on the magnetic sensor wafer 100 .
請參照圖1與圖2B，進行步驟S104，在磁感晶片100上接上多個探針108。舉例來說，探針108可接在二氧化矽介電層106上。此外，探針108可位在磁感晶片100的感測區R中。磁感晶片100的感測區R可為磁感晶片100中的孔洞(well)或流道。在本實施例中，探針108是以核酸探針為例，但本發明並不以此為限。此外，核酸探針的長度例如是20單體單元至40單體單元，例如可為約20至25單體單元、約25至30單體單元、約30至35單體單元或約35至40單體單元，但不限於此。在一實施例中，核酸探針的長度可為20單體單元至30單體單元。在磁感晶片100上接上探針108的方法例如是化學交聯法。Referring to FIG. 1 and FIG. 2B , step S104 is performed to connect a plurality of probes 108 to the magnetic sensor chip 100 . For example, probe 108 may be connected to silicon dioxide dielectric layer 106 . In addition, the probe 108 may be located in the sensing region R of the magnetic sensing chip 100 . The sensing area R of the magnetic sensor chip 100 may be a well or a flow channel in the magnetic sensor chip 100 . In this embodiment, the probe 108 is a nucleic acid probe, but the invention is not limited thereto. In addition, the length of the nucleic acid probe is, for example, 20 to 40 monomer units, for example, it can be about 20 to 25 monomer units, about 25 to 30 monomer units, about 30 to 35 monomer units, or about 35 to 40 monomer units. Single unit, but not limited to this. In one embodiment, the length of the nucleic acid probe may be 20 to 30 monomeric units. The method of connecting the probe 108 to the magnetic sensor wafer 100 is, for example, a chemical cross-linking method.
請參照圖1與圖2C，進行步驟S106，將包含標記有第一標記物112的多個生物樣品的樣品液S1提供至磁感晶片100上，以使標記有第一標記物112的生物樣品與探針108進行雜交反應。生物樣品的來源可為待測生物檢體。待測生物檢體例如是尿液、唾液、血清或血漿。在生物樣品的來源為待測生物檢體的情況下，可對待測生物檢體進行前處理，如萃取處理或純化處理。雜交反應的溫度範圍例如是45℃至65℃，但本發明並不以此為限。例如，雜交反應的溫度範圍可為約45℃至50℃、約50℃至55℃、約55℃至60℃或約60℃至65℃等，但不限於此。在一實施例中，雜交反應的溫度範圍可為約55℃至62℃，藉此可縮短反應時間。在一些實施例中，雜交反應的溫度範圍可為約50℃、約55℃或約60℃。Referring to FIGS. 1 and 2C , step S106 is performed to provide the sample liquid S1 containing a plurality of biological samples labeled with the first marker 112 onto the magnetic sensing chip 100 , so that the biological samples labeled with the first marker 112 Perform hybridization reaction with probe 108. The source of the biological sample may be the biological specimen to be tested. The biological sample to be tested is, for example, urine, saliva, serum or plasma. When the source of the biological sample is the biological sample to be tested, the biological sample to be tested can be pre-processed, such as extraction or purification. The temperature range of the hybridization reaction is, for example, 45°C to 65°C, but the present invention is not limited thereto. For example, the temperature range of the hybridization reaction may be about 45°C to 50°C, about 50°C to 55°C, about 55°C to 60°C, or about 60°C to 65°C, but is not limited thereto. In one embodiment, the temperature range of the hybridization reaction can be about 55°C to 62°C, thereby shortening the reaction time. In some embodiments, the temperature range of the hybridization reaction may be about 50°C, about 55°C, or about 60°C.
在本實施例中，生物樣品是以待測單股核酸110a為例，但本發明並不以此為限。待測單股核酸110a的長度例如是80單體單元至120單體單元，例如可為約80至90單體單元、約90至100單體單元、約100至110單體單元或約110至120單體單元，但不限於此。在一實施例中，待測單股核酸110a的長度可為90單體單元至110單體單元。第一標記物112可為生物素，但本發明並不以為為限。In this embodiment, the biological sample is the single-stranded nucleic acid 110a to be tested as an example, but the invention is not limited thereto. The length of the single-stranded nucleic acid 110a to be tested is, for example, 80 to 120 monomer units, for example, it can be about 80 to 90 monomer units, about 90 to 100 monomer units, about 100 to 110 monomer units, or about 110 to 110 monomer units. 120 single units, but not limited to this. In one embodiment, the length of the single-stranded nucleic acid 110a to be tested may range from 90 monomer units to 110 monomer units. The first marker 112 may be biotin, but the present invention is not limited thereto.
請參照圖3，在生物樣品為待測單股核酸110a的情況下，標記有第一標記物112的生物樣品的製備方法可包括以下步驟，但本發明並不以此為限。提供待測雙股核酸110。舉例來說，待測雙股核酸110可藉由對待測生物檢體進行前處理而獲得。待測雙股核酸110可包括待測單股核酸110a與單股核酸110b。接著，使用標記有第一標記物112的引子114a對待測雙股核酸110進行增幅處理116，而獲得標記有第一標記物112的多個待測雙股核酸110。待測雙股核酸的增幅處理例如是進行聚合酶鏈鎖反應(PCR)。在增幅處理116中，可使用成對的引子114a與引子114b來進行增幅。然後，對標記有第一標記物112的待測雙股核酸110進行變性處理118，而獲得標記有第一標記物112的待測單股核酸110a的樣品液S1(圖2C)。此外，樣品液S1除了包括待測單股核酸110a之外，更可包括單股核酸110b(圖2C中未示出)。亦即，待測雙股核酸110可經由變性處理118而形成標記有第一標記物112的待測單股核酸110a與單股核酸110b。變性處理118例如是高溫裂解處理。Referring to FIG. 3 , when the biological sample is a single-stranded nucleic acid 110a to be tested, the method of preparing the biological sample labeled with the first marker 112 may include the following steps, but the invention is not limited thereto. A double-stranded nucleic acid to be tested 110 is provided. For example, the double-stranded nucleic acid 110 to be tested can be obtained by preprocessing the biological sample to be tested. The double-stranded nucleic acid 110 to be detected may include the single-stranded nucleic acid 110a and the single-stranded nucleic acid 110b to be detected. Next, the primer 114a labeled with the first marker 112 is used to perform an amplification process 116 on the double-stranded nucleic acid to be tested 110, and a plurality of double-stranded nucleic acids to be tested 110 labeled with the first marker 112 are obtained. The amplification process of the double-stranded nucleic acid to be detected is, for example, polymerase chain reaction (PCR). In the amplification process 116, amplification may be performed using a pair of primers 114a and 114b. Then, the double-stranded nucleic acid to be tested 110 labeled with the first marker 112 is subjected to a denaturation process 118 to obtain a sample solution S1 of the single-stranded nucleic acid to be tested 110a labeled with the first marker 112 (Fig. 2C). In addition, the sample liquid S1 may further include a single-stranded nucleic acid 110b (not shown in Figure 2C) in addition to the single-stranded nucleic acid 110a to be tested. That is, the double-stranded nucleic acid to be tested 110 can undergo a denaturation process 118 to form single-stranded nucleic acid 110a and single-stranded nucleic acid 110b that are labeled with the first marker 112 . The denaturation treatment 118 is, for example, high temperature lysis treatment.
請參照圖1與圖2D，可進行步驟S108，在進行雜交反應之後，進行清洗處理，以移除未與探針108雜交的生物樣品(如，待測單股核酸110a)。舉例來說，可利用緩衝液S2進行上述清洗處理。緩衝液S2例如是Tris(產品名)緩衝液，但本發明並不以此為限。Tris緩衝液可含有三羥甲基氨基甲烷(Tris(hydroxymethyl)aminomethane，Tris)、氯化鈉(NaCl)與Tween 20(產品名，辛格瑪艾瑞契(Sigma Aldrich)公司製)。在一實施例中，Tris緩衝液的pH值可為7.6，且可包括濃度為0.05M的Tris、濃度為0.15M的NaCl與0.02%的Tween 20。在其他實施例中，可省略步驟S108的清洗處理。Referring to Figure 1 and Figure 2D, step S108 can be performed. After performing the hybridization reaction, a cleaning process is performed to remove biological samples that are not hybridized with the probe 108 (eg, the single-stranded nucleic acid 110a to be tested). For example, buffer S2 can be used to perform the above cleaning process. Buffer S2 is, for example, Tris (product name) buffer, but the present invention is not limited thereto. The Tris buffer may contain Tris(hydroxymethyl)aminomethane (Tris), sodium chloride (NaCl), and Tween 20 (product name, manufactured by Sigma Aldrich). In one embodiment, the pH value of the Tris buffer may be 7.6, and may include Tris at a concentration of 0.05M, NaCl at a concentration of 0.15M, and Tween 20 at a concentration of 0.02%. In other embodiments, the cleaning process of step S108 may be omitted.
請參照圖1與圖2E，進行步驟S110，將標記有第二標記物120的多個磁珠122提供至磁感晶片100上，以使標記有第二標記物120的磁珠122接合至標記有第一標記物112的生物樣品(如，待測單股核酸110a)上。舉例來說，標記有第二標記物120的磁珠122可預先製備成磁珠溶液S3，再將磁珠溶液S3提供至磁感晶片100上。此外，磁珠122可藉由第二標記物120與第一標記物112之間的親和力接合至生物樣品(如，待測單股核酸110a)上。在本實施例中，在第一標記物112為生物素的情況下，第二標記物120可為鏈霉親和素，但本發明並不以此為限。標記有第二標記物120的磁珠122的製備方法例如是化學還原法。磁珠122可為空心磁珠或實心磁珠。磁珠122的粒徑例如是0.2μm至2μm。磁珠122的材料例如是四氧化三鐵(Fe3 O4 )。Referring to FIG. 1 and FIG. 2E, step S110 is performed to provide a plurality of magnetic beads 122 marked with the second marker 120 to the magnetic sensor chip 100, so that the magnetic beads 122 marked with the second marker 120 are bonded to the marker. On the biological sample (eg, the single-stranded nucleic acid to be detected 110a) with the first marker 112. For example, the magnetic beads 122 labeled with the second marker 120 can be prepared in advance as a magnetic bead solution S3, and then the magnetic bead solution S3 is provided on the magnetic sensor wafer 100. In addition, the magnetic beads 122 can be bound to the biological sample (eg, the single-stranded nucleic acid 110a to be tested) through the affinity between the second label 120 and the first label 112 . In this embodiment, when the first label 112 is biotin, the second label 120 may be streptavidin, but the invention is not limited thereto. The preparation method of the magnetic beads 122 labeled with the second marker 120 is, for example, a chemical reduction method. The magnetic beads 122 may be hollow magnetic beads or solid magnetic beads. The particle size of the magnetic beads 122 is, for example, 0.2 μm to 2 μm. The material of the magnetic beads 122 is, for example, ferroferric oxide (Fe 3 O 4 ).
請參照圖1與圖2F，可進行步驟S112，在將磁珠122接合至生物樣品(如，待測單股核酸110a)上之後，進行清洗處理，以移除未接合在生物樣品(如，待測單股核酸110a)上的磁珠122。舉例來說，可利用緩衝液S4進行上述清洗處理。緩衝液S4例如是Tris緩衝液，但本發明並不以此為限。Tris緩衝液可含有Tris、NaCl與Tween 20(產品名，Sigma Aldrich公司製)。在一實施例中，Tris緩衝液的pH值可為7.6，且可包括濃度為0.05 M的Tris、濃度為0.15 M的NaCl與0.02%的Tween 20。在其他實施例中，可省略步驟S112的清洗處理。Referring to FIG. 1 and FIG. 2F, step S112 can be performed. After the magnetic beads 122 are coupled to the biological sample (eg, the single-stranded nucleic acid 110a to be tested), a cleaning process is performed to remove unbound particles in the biological sample (eg, Magnetic beads 122 on the single-stranded nucleic acid to be detected 110a). For example, buffer S4 can be used to perform the above cleaning process. Buffer S4 is, for example, Tris buffer, but the present invention is not limited thereto. The Tris buffer may contain Tris, NaCl, and Tween 20 (product name, manufactured by Sigma Aldrich). In one embodiment, the pH value of the Tris buffer may be 7.6, and may include Tris at a concentration of 0.05 M, NaCl at a concentration of 0.15 M, and Tween 20 at a concentration of 0.02%. In other embodiments, the cleaning process of step S112 may be omitted.
請參照圖1、圖2F與圖4，進行步驟S114，以磁感測器檢測磁感測層104所感測到的訊號。磁感測器例如是穿隧磁阻(TMR)感測器或巨磁阻(GMR)感測器。以磁感測器所進行的檢測更可包括定量分析或定性分析。亦即，本實施例的生物樣品的檢測方法可用於各種疾病的定性檢測與定量檢測。在本實施例中，以磁感測器所進行的檢測是以定量分析為例來進行說明。Referring to FIG. 1 , FIG. 2F and FIG. 4 , step S114 is performed to detect the signal sensed by the magnetic sensing layer 104 with the magnetic sensor. The magnetic sensor is, for example, a tunneling magnetoresistance (TMR) sensor or a giant magnetoresistance (GMR) sensor. The detection performed by the magnetic sensor may further include quantitative analysis or qualitative analysis. That is to say, the biological sample detection method of this embodiment can be used for qualitative detection and quantitative detection of various diseases. In this embodiment, the detection performed by the magnetic sensor is quantitative analysis as an example for explanation.
舉例來說，定量分析可包括以下步驟。首先，進行步驟S1140，在進行雜交反應之後，且在將磁珠122接合至生物樣品(如，待測單股核酸110a)上之前，使用磁感測器進行量測而獲得第一訊號。接著，進行步驟S1142，在將磁珠122接合至生物樣品(如，待測單股核酸110a)上之後，使用磁感測器進行量測而獲得第二訊號。然後，進行步驟S1144，藉由第二訊號與第一訊號的差值計算出接合至生物樣品(如，待測單股核酸110a)上的磁珠122的數量，以對與探針108進行雜交的生物樣品(如，待測單股核酸110a)進行定量。第一訊號與第二訊號可在室溫下進行量測，以減少訊號受到溫度變動的影響。第一訊號與第二訊號例如是電壓訊號。在第一訊號與第二訊號為電壓訊號的情況下，在磁珠122接合至生物樣品(如，待測單股核酸110a)上時的第二訊號的電壓值可高於在磁珠122未接合至生物樣品(如，待測單股核酸110a)上時的第一訊號的電壓值。磁感測器的量測模式可為即時(real time)量測模式。舉例來說，即時量測模式可在步驟S106至步驟步驟S114的期間持續進行量測。For example, quantitative analysis may include the following steps. First, step S1140 is performed. After performing the hybridization reaction and before joining the magnetic beads 122 to the biological sample (eg, the single-stranded nucleic acid to be tested 110a), a magnetic sensor is used to measure and obtain a first signal. Next, step S1142 is performed. After the magnetic beads 122 are bonded to the biological sample (eg, the single-stranded nucleic acid 110a to be tested), a magnetic sensor is used for measurement to obtain a second signal. Then, step S1144 is performed to calculate the number of magnetic beads 122 bound to the biological sample (such as the single-stranded nucleic acid 110a to be tested) based on the difference between the second signal and the first signal, so as to perform hybridization with the probe 108 The biological sample (for example, the single-stranded nucleic acid 110a to be tested) is quantified. The first signal and the second signal can be measured at room temperature to reduce the influence of temperature changes on the signals. The first signal and the second signal are, for example, voltage signals. In the case where the first signal and the second signal are voltage signals, the voltage value of the second signal when the magnetic beads 122 are bonded to the biological sample (eg, the single-stranded nucleic acid 110a to be tested) may be higher than when the magnetic beads 122 are not. The voltage value of the first signal when bonded to a biological sample (eg, single-stranded nucleic acid 110a to be detected). The measurement mode of the magnetic sensor may be a real time measurement mode. For example, the real-time measurement mode may continue to perform measurement during the period from step S106 to step S114.
在其他實施例中，在以磁感測器進行定性分析的情況下，只要上述第二訊號與上述第一訊號具有顯著差異，即可判定為生物樣品(如，待測單股核酸110a)與探針108進行雜交。In other embodiments, in the case of qualitative analysis using a magnetic sensor, as long as there is a significant difference between the second signal and the first signal, it can be determined that the biological sample (for example, the single-stranded nucleic acid 110a to be tested) and Probe 108 hybridizes.
基於上述實施例可知，在上述生物樣品的檢測方法中，在以磁感晶片100中的磁感測器進行檢測時，由於生物樣本中幾乎沒有磁性物質，因此不會受到基質干擾。如此一來，在利用磁感晶片100中的磁感測器進行生物樣品的檢測時，可具有較佳的檢測靈敏度。Based on the above embodiments, it can be known that in the above biological sample detection method, when the magnetic sensor in the magnetic sensing chip 100 is used for detection, since there is almost no magnetic material in the biological sample, there will be no matrix interference. In this way, when the magnetic sensor in the magnetic sensor chip 100 is used to detect biological samples, better detection sensitivity can be achieved.
>實驗例>>Experimental Examples>
圖5為本發明一實驗例的讀取訊號與時間的關係圖。FIG. 5 is a diagram showing the relationship between read signal and time in an experimental example of the present invention.
請參照圖5，在藉由上述實施例的生物樣品的檢測方法對標記有生物素的待測單股核酸(生物樣品)進行檢測後，所獲得的檢測結果說明如下。Please refer to Figure 5. After the single-stranded nucleic acid to be tested (biological sample) labeled with biotin is detected by the biological sample detection method of the above embodiment, the detection results obtained are explained as follows.
在約400秒時，加入標記有生物素的待測單股核酸至磁感晶片的感測區，用以與感測區內的核酸探針進行雜交反應。在約700秒至約1300秒時，升溫至56℃至62℃，進行10分鐘的雜交反應(區段A)。藉由升溫至56℃至62℃，可加快雜交反應。在階段A中，訊號會受溫度升高的影響而下降。At about 400 seconds, the single-stranded nucleic acid to be detected labeled with biotin is added to the sensing area of the magnetic sensor chip to perform a hybridization reaction with the nucleic acid probe in the sensing area. At about 700 seconds to about 1300 seconds, the temperature is raised to 56°C to 62°C, and a hybridization reaction is performed for 10 minutes (section A). By raising the temperature to 56°C to 62°C, the hybridization reaction can be accelerated. In phase A, the signal will decrease due to the increase in temperature.
在約1300秒時，開始降溫至室溫，此時訊號會受溫度降低的影響而上升。At about 1300 seconds, it begins to cool down to room temperature. At this time, the signal will increase due to the influence of the temperature decrease.
在約1700秒時，利用Tris緩衝液清洗感測區，將未與核酸探針雜交的待測單股核酸沖離感測區(區段B)。Tris緩衝液的pH值為7.6，且包括濃度為0.05M的Tris、濃度為0.15M的NaCl與0.02%的Tween 20。At about 1700 seconds, the sensing area is washed with Tris buffer, and the single-stranded nucleic acid to be detected that has not hybridized with the nucleic acid probe is washed away from the sensing area (section B). The pH value of the Tris buffer is 7.6 and includes Tris at a concentration of 0.05M, NaCl at a concentration of 0.15M and Tween 20 at a concentration of 0.02%.
在約2000秒時，回復到室溫(如，28℃)，並使用磁感晶片的穿隧磁阻(TMR)感測器量測訊號，且此時的訊號可作為量測的初始訊號(區段C)。At about 2000 seconds, return to room temperature (e.g., 28°C), and use the tunneling magnetoresistance (TMR) sensor of the magnetic sensing chip to measure the signal, and the signal at this time can be used as the initial signal for measurement ( Section C).
在約2200秒時，加入標記有鏈霉親和素的磁珠，在室溫下反應10分鐘，以使標記有鏈霉親和素的磁珠接合至標記有生物素的待測單股核酸上，且藉由磁珠產生磁訊號反應(區段D)。At about 2200 seconds, add magnetic beads labeled with streptavidin and react at room temperature for 10 minutes to bind the magnetic beads labeled with streptavidin to the single-stranded nucleic acid to be tested labeled with biotin. And generate a magnetic signal reaction through magnetic beads (section D).
在約2800秒時，利用Tris緩衝液清洗感測區，將未接合在待測單股核酸上的磁珠沖離感測區。Tris緩衝液的pH值為7.6，且包括濃度為0.05 M的Tris、濃度為0.15 M的NaCl與0.02%的Tween 20。此外，將穿隧磁阻(TMR)感測器在此區段(區段E)所量測的訊號減去磁珠加入前的初始訊號(區段C)，可得到電壓差ΔV(約12.5 μV)，且藉由此電壓差可計算出在感測區中與核酸探針雜交的待測單股核酸的數量。At about 2800 seconds, the sensing area is washed with Tris buffer, and the magnetic beads that are not bound to the single-stranded nucleic acid to be detected are washed away from the sensing area. The pH value of Tris buffer is 7.6 and includes Tris at a concentration of 0.05 M, NaCl at a concentration of 0.15 M, and Tween 20 at a concentration of 0.02%. In addition, by subtracting the initial signal before the magnetic beads are added (section C) from the signal measured by the tunneling magnetoresistance (TMR) sensor in this section (section E), the voltage difference ΔV (about 12.5 μV), and by using this voltage difference, the number of single-stranded nucleic acids to be detected that hybridizes with the nucleic acid probe in the sensing area can be calculated.
綜上所述，在上述實施例的生物樣品的檢測方法中，在以磁感晶片中的磁感測器進行檢測時，由於生物樣本中幾乎沒有磁性物質，因此不會受到基質干擾。如此一來，在利用磁感晶片中的磁感測器進行生物樣品的檢測時，可具有較佳的檢測靈敏度。To sum up, in the biological sample detection method of the above embodiment, when the magnetic sensor in the magnetic sensor chip is used for detection, since there is almost no magnetic material in the biological sample, there will be no matrix interference. In this way, when the magnetic sensor in the magnetic sensor chip is used to detect biological samples, better detection sensitivity can be achieved.
雖然本發明已以實施例揭露如上，然其並非用以限定本發明，任何所屬技術領域中具有通常知識者，在不脫離本發明的精神和範圍內，當可作些許的更動與潤飾，故本發明的保護範圍當視後附的申請專利範圍所界定者為準。Although the present invention has been disclosed above through embodiments, they are not intended to limit the present invention. Anyone with ordinary knowledge in the technical field may make some modifications and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention shall be determined by the appended patent application scope.
100:磁感晶片 102:基板 104:磁感測層 106:二氧化矽介電層 108:探針 110:待測雙股核酸 110a:待測單股核酸 110b:單股核酸 112:第一標記物 114a、114b:引子 116:增幅處理 118:變性處理 120:第二標記物 122:磁珠 R:感測區 S1:樣品液 S2、S4:緩衝液 S3:磁珠溶液 S100、S102、S104、S106、S108、S110、S112、S114、S1140、S1142、S1144:步驟 ΔV:電壓差100:Magnetic chip 102:Substrate 104: Magnetic sensing layer 106:SiO2 dielectric layer 108:Probe 110: Double-stranded nucleic acid to be tested 110a: Single-stranded nucleic acid to be tested 110b: Single-stranded nucleic acid 112:First marker 114a, 114b: Introduction 116: Amplification processing 118: Denaturation treatment 120:Second marker 122:Magnetic beads R: Sensing area S1: sample liquid S2, S4: buffer S3: Magnetic bead solution S100, S102, S104, S106, S108, S110, S112, S114, S1140, S1142, S1144: Steps ΔV: voltage difference
圖1為本發明一實施例的生物樣品的檢測方法的流程圖。 圖2A至圖2F為本發明一實施例的生物樣品的檢測流程的示意圖。 圖3為本發明一實施例的生物樣品的製備方法的示意圖。 圖4為本發明一實施例的定量分析的流程圖。 圖5為本發明一實驗例的讀取訊號與時間的關係圖。Figure 1 is a flow chart of a biological sample detection method according to an embodiment of the present invention. 2A to 2F are schematic diagrams of a biological sample detection process according to an embodiment of the present invention. Figure 3 is a schematic diagram of a biological sample preparation method according to an embodiment of the present invention. Figure 4 is a flow chart of quantitative analysis according to an embodiment of the present invention. FIG. 5 is a diagram showing the relationship between read signal and time in an experimental example of the present invention.
S100、S102、S104、S106、S108、S110、S112、S114:步驟S100, S102, S104, S106, S108, S110, S112, S114: Steps
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| US20080309323A1 (en) * | 2007-06-12 | 2008-12-18 | Canon Kabushiki Kaisha | Method for biochemical analysis |
| CN103529011A (en) * | 2012-07-06 | 2014-01-22 | 南京大学 | Method for detecting DNA (deoxyribonucleic acid) through high sensitivity Raman spectrum |
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