技术领域technical field
本发明属于电化学检测领域，具体地涉及一种基于多腺嘌呤的DNA捕获探针、生物传感器及其检测方法。The invention belongs to the field of electrochemical detection, and in particular relates to a polyadenine-based DNA capture probe, a biosensor and a detection method thereof.
背景技术Background technique
电化学的发展历史悠久，与尖端科学技术和学科的发展紧密相关，电化学生物传感器具有特异性分子识别功能，在疾病基因诊断、抗癌药物筛选等方面显示了广阔的应用前景，是当今生物学、医学领域的前沿的研究手段。The development of electrochemistry has a long history and is closely related to the development of cutting-edge science and technology and disciplines. Electrochemical biosensors have specific molecular recognition functions and have shown broad application prospects in disease gene diagnosis and anticancer drug screening. Cutting-edge research methods in the fields of science and medicine.
电化学生物传感器测定DNA的大致步骤包括：一、DNA捕获探针的固定，即将某一特定序列的单链DNA(ssDNA)固定在电极的表面；二、杂交，将固定有DNA捕获探针的电极放入到含有与之匹配的待测DNA的被测溶液中，使待测DNA与DNA捕获探针在电极表面发生杂交反应形成双链DNA(dsDNA)；三、将杂交反应的信号转化为可测定的电化学信号；四、将电化学信号进行放大检测。The general steps for the electrochemical biosensor to measure DNA include: 1. Immobilization of the DNA capture probe, that is, immobilizing a single-stranded DNA (ssDNA) of a specific sequence on the surface of the electrode; 2. Hybridization, immobilizing the DNA capture probe The electrode is put into the measured solution containing the matched DNA to be tested, so that the DNA to be tested and the DNA capture probe hybridize on the surface of the electrode to form double-stranded DNA (dsDNA); 3. Convert the signal of the hybridization reaction into Measurable electrochemical signals; 4. Amplify and detect electrochemical signals.
DNA捕获探针的固定是DNA电化学传感器制作中的首要问题，固定的量和活性直接影响传感器的灵敏度。目前常见的DNA固定方法主要有共价键合法、自组装法和亲和素-生物素方法。共价键和法是通过形成共价键，如酰胺键、酯键、醚键等使DNA固定到电极表面，需要对电极表面进行活化预处理，引入所需的活性基团，通过双官能团试剂或偶联活化剂将衍生后的DNA分子与电极联结。但是该方法由于形成共价键的化学反应效率不大的问题，导致组装在电极表面的DNA捕获探针的密度不高。自组装法(SAM法)主要用于DNA捕获探针在金电极表面的固定，具体方法是将DNA捕获探针的末端巯基化，通过金硫键形成自组装层达到固定DNA的目的。但是该方法需要巯基修饰，成本较高，并且在实验操作中容易形成双硫键而影响检测结果的准确性。亲和素-生物素法是将亲和素(avidin)通过静电作用或共价偶联作用连接于电极表面，利用生物素和亲和素之间极强的生物识别亲和作用，将生物素修饰的DNA固定到电极上。以上三种方法均需要对DNA捕获探针进行修饰，合成成本较高，步骤繁琐。The immobilization of DNA capture probes is the primary problem in the fabrication of DNA electrochemical sensors, and the amount and activity of the immobilization directly affect the sensitivity of the sensor. At present, the common DNA immobilization methods mainly include covalent bonding method, self-assembly method and avidin-biotin method. The covalent bonding method is to fix DNA to the electrode surface by forming covalent bonds, such as amide bonds, ester bonds, ether bonds, etc., which requires activation and pretreatment of the electrode surface to introduce the required active groups. Or coupling activator to link the derivatized DNA molecule with the electrode. However, due to the low efficiency of the chemical reaction for forming covalent bonds in this method, the density of DNA capture probes assembled on the electrode surface is not high. The self-assembly method (SAM method) is mainly used for the immobilization of DNA capture probes on the surface of gold electrodes. The specific method is to thiolate the ends of DNA capture probes, and form a self-assembly layer through gold-sulfur bonds to achieve the purpose of immobilizing DNA. However, this method requires sulfhydryl modification, which is expensive, and it is easy to form disulfide bonds in the experimental operation, which will affect the accuracy of the detection results. The avidin-biotin method is to connect avidin to the electrode surface through electrostatic interaction or covalent coupling, and use the strong biological recognition affinity between biotin and avidin to modify biotin The DNA is immobilized on the electrodes. The above three methods all need to modify the DNA capture probe, the synthesis cost is high, and the steps are cumbersome.
然而，目前本领域技术人员难以明确如何调控电化学生物传感器电极表面DNA捕获探针的组装密度，这直接制约了电化学生物传感器的进一步发展，因此亟待获得一种全新的DNA捕获探针固定方法及效果优异的电化学生物传感器。However, at present, it is difficult for those skilled in the art to know how to regulate the assembly density of DNA capture probes on the electrode surface of electrochemical biosensors, which directly restricts the further development of electrochemical biosensors. Therefore, it is urgent to obtain a new immobilization method for DNA capture probes And the electrochemical biosensor with excellent effect.
发明内容Contents of the invention
本发明要解决的技术问题是解决了调控电化学生物传感器电极表面DNA捕获探针的组装密度的难题，从而提供了一种基于多腺嘌呤的DNA捕获探针、生物传感器及其检测方法。该DNA捕获探针和生物传感器能够灵敏度高、特异性佳地检测待测DNA，操作简便，应用前景广阔。The technical problem to be solved in the present invention is to solve the difficult problem of regulating the assembly density of the DNA capture probe on the electrode surface of the electrochemical biosensor, thereby providing a polyadenine-based DNA capture probe, a biosensor and a detection method thereof. The DNA capture probe and the biosensor can detect DNA to be tested with high sensitivity and good specificity, are easy to operate and have broad application prospects.
本发明的发明人经过深入的研究和反复的试验，利用腺嘌呤与金电极的相互作用，设计了包含多个连续腺嘌呤(poly A)的DNA捕获探针，并且将这样的DNA捕获探针固定在金电极表面，通过“夹心式”的杂交方法，将待测DNA和信号探针引入金电极表面，再依据检测杂交前后催化氧化还原电流变化指示待测DNA的浓度，从而完成了本发明。The inventors of the present invention have designed a DNA capture probe comprising a plurality of continuous adenines (poly A) by utilizing the interaction between adenine and gold electrodes through in-depth research and repeated experiments, and made such DNA capture probes It is fixed on the surface of the gold electrode, and the DNA to be tested and the signal probe are introduced into the surface of the gold electrode through a "sandwich" hybridization method, and then the concentration of the DNA to be tested is indicated according to the change of the catalytic redox current before and after detection of hybridization, thus completing the present invention .
本发明的技术方案之一是：一种基于多腺嘌呤的DNA捕获探针，其核苷酸序列如序列表中SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5所示。One of the technical solutions of the present invention is: a DNA capture probe based on polyadenine, its nucleotide sequence is as shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID Shown in No.4 or SEQ ID No.5.
本发明中，所述的多腺嘌呤的DNA捕获探针含有不同个数(5个、10个、20个、30个和40个)的腺嘌呤，分别称为poly A5、poly A10、poly A20、poly A30和poly A40。In the present invention, the polyadenine DNA capture probes contain different numbers of adenines (5, 10, 20, 30 and 40), which are called poly A5, poly A10, and poly A20 respectively. , poly A30 and poly A40.
本发明中，较佳地，所述DNA捕获探针为poly A30，其核苷酸序列如序列表中SEQID No.4所示。In the present invention, preferably, the DNA capture probe is poly A30, the nucleotide sequence of which is shown in SEQID No.4 in the sequence listing.
本发明的技术方案之二是：一种电化学生物传感器，其由包括以下步骤的方法制备而成：The second technical solution of the present invention is: an electrochemical biosensor prepared by a method comprising the following steps:
(1)制备基于多腺嘌呤的DNA捕获探针，所述DNA捕获探针的核苷酸序列如序列表中SEQ ID No.1、SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5所示；(1) Prepare a DNA capture probe based on polyadenine, the nucleotide sequence of the DNA capture probe is as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No in the sequence listing .4 or shown in SEQ ID No.5;
(2)将步骤(1)所得的基于多腺嘌呤的DNA捕获探针滴加到电极上，反应，得组装电极；(2) adding the polyadenine-based DNA capture probe obtained in step (1) dropwise onto the electrode, and reacting to obtain an assembled electrode;
(3)用巯基己醇(MCH)对步骤(2)所得的组装电极的空位封闭，得封闭的电极。(3) Sealing the vacancies of the assembled electrode obtained in step (2) with mercaptohexanol (MCH) to obtain a sealed electrode.
本发明中，步骤(2)为：将步骤(1)所得的基于多腺嘌呤的DNA捕获探针滴加到电极上，反应，得组装电极。其中，所述DNA捕获探针滴加到电极上时的组装密度较佳地为0.01～3μM，更佳地为0.1μM。所述反应的温度为20～40℃，较佳地为25℃。所述组装的时间为本领域常规的时间，较佳地为过夜，即大于12小时。In the present invention, the step (2) is: drop the polyadenine-based DNA capture probe obtained in the step (1) onto the electrode, react, and assemble the electrode. Wherein, when the DNA capture probe is dropped onto the electrode, the assembly density is preferably 0.01-3 μM, more preferably 0.1 μM. The reaction temperature is 20-40°C, preferably 25°C. The assembly time is conventional in the art, preferably overnight, that is, more than 12 hours.
步骤(2)中，所述的电极为常规，较佳地，所述的电极为处理干净的电极；更佳地，所述处理干净的电极由包括以下步骤的方法制得：经物理打磨、硫酸电化学清洗，再用超纯水彻底冲洗电极表面，然后用N2吹干。所述的电极较佳的是金电极。In step (2), the electrode is conventional, preferably, the electrode is a cleaned electrode; more preferably, the cleaned electrode is made by a method comprising the following steps: physical grinding, Electrochemical cleaning with sulfuric acid, and then thoroughly rinse the electrode surface with ultrapure water, and then blow dry withN2 . The electrodes are preferably gold electrodes.
本发明中，步骤(3)为：用巯基己醇(MCH)对步骤(2)所得的组装电极的空位封闭，得封闭的电极。其中，所述MCH的浓度较佳地为0.01～1mM，更佳地为0.1mM。所述封闭的时间为5～60分钟，较佳地为30分钟。所述封闭的温度为本领域常规的温度，较佳地为25℃。In the present invention, step (3) is: using mercaptohexanol (MCH) to seal the vacancies of the assembled electrode obtained in step (2) to obtain a sealed electrode. Wherein, the concentration of MCH is preferably 0.01-1 mM, more preferably 0.1 mM. The blocking time is 5-60 minutes, preferably 30 minutes. The sealing temperature is a conventional temperature in the art, preferably 25°C.
本发明中，经研究发现证实，最佳地，上述电化学生物传感器采用所述的polyA30作为DNA捕获探针；polyA30的组装密度为0.1μM；且步骤(3)中采用0.1mM MCH封闭30分钟。In the present invention, it has been confirmed through research that, optimally, the above-mentioned electrochemical biosensor uses the polyA30 as a DNA capture probe; the assembly density of polyA30 is 0.1 μM; and in step (3), 0.1 mM MCH is used to block for 30 minutes .
本发明的技术方案之三是：一种利用上述的电化学生物传感器检测待测DNA的方法，其包括以下的步骤：The third technical solution of the present invention is: a method for detecting DNA to be tested using the above-mentioned electrochemical biosensor, which includes the following steps:
(Ⅰ)将待测DNA和核苷酸序列如序列表中SEQ ID No.7所示且在3’端连接生物素的信号探针预杂交，得预杂交物A；(1) pre-hybridize the DNA and nucleotide sequence to be tested as shown in SEQ ID No.7 in the sequence listing and connect the signal probe with biotin at the 3' end to obtain pre-hybrid A;
(Ⅱ)将步骤(Ⅰ)所得的预杂交物A与如权利要求2～5任一项所述的电化学生物传感器的电极上的DNA捕获探针杂交，得杂交的电极；(II) Hybridizing the prehybrid A obtained in step (I) with the DNA capture probe on the electrode of the electrochemical biosensor according to any one of claims 2 to 5 to obtain a hybridized electrode;
(Ⅲ)向步骤(Ⅱ)所得的杂交的电极上滴加亲和素-辣根过氧化酶后孵化；加入底物TMB，进行检测分析。(III) Add avidin-horseradish peroxidase dropwise to the hybridized electrode obtained in step (II) and incubate; add substrate TMB for detection and analysis.
本发明中，步骤(Ⅰ)为：将待测DNA和核苷酸序列如序列表中SEQ ID No.7所示且在3’端连接生物素的信号探针预杂交，得预杂交物A。In the present invention, step (I) is: pre-hybridize the signal probe with the DNA and nucleotide sequence to be tested as shown in SEQ ID No.7 in the sequence listing and connect biotin at the 3' end to obtain pre-hybrid A .
其中，较佳地，所述待测DNA的浓度为0～1000pM，所述信号探针的浓度为100nM。较佳地，所述预杂交在60～100℃保持1～10分钟，然后20～40℃放置5～60分钟。更佳地，所述预杂交的温度为80℃。更佳地，所述预杂交的时间为5分钟。较佳地，所述放置的时间为20分钟。较佳地，所述放置的温度为25℃。较佳地，所述预杂交在缓冲液下进行。所述缓冲液为本领域常规的缓冲液，较佳地，所述缓冲液为包含1M NaCl的10mM TE缓冲溶液，所述TE缓冲液包括10mM Tris-HCl和1mM EDTA，pH7.0。Wherein, preferably, the concentration of the DNA to be detected is 0-1000 pM, and the concentration of the signal probe is 100 nM. Preferably, the pre-hybridization is maintained at 60-100°C for 1-10 minutes, and then placed at 20-40°C for 5-60 minutes. More preferably, the temperature of the pre-hybridization is 80°C. More preferably, the pre-hybridization time is 5 minutes. Preferably, the standing time is 20 minutes. Preferably, the storage temperature is 25°C. Preferably, the pre-hybridization is performed under buffer. The buffer is a conventional buffer in the art, preferably, the buffer is 10 mM TE buffer solution containing 1M NaCl, the TE buffer includes 10 mM Tris-HCl and 1 mM EDTA, pH 7.0.
本发明中，步骤(Ⅱ)为：将步骤(Ⅰ)所得的预杂交物A与所述的电化学生物传感器的电极上的DNA捕获探针杂交，得杂交的电极。其中，较佳地，步骤(Ⅱ)所述的杂交在25～45℃杂交30～120分钟；更佳地，所述杂交的温度为37℃。较佳地，所述杂交的时间为60分钟。较佳地，所述杂交完成后还包括清洗电极的步骤。所述清洗的清洗液是本领域常规的清洗液，较佳地，为含有1M NaCl的10mM TE缓冲溶液，所述TE缓冲液包括10mM Tris-HCl和1mMEDTA，pH7.0。In the present invention, the step (II) is: hybridizing the prehybrid product A obtained in the step (I) with the DNA capture probe on the electrode of the electrochemical biosensor to obtain a hybridized electrode. Wherein, preferably, the hybridization in step (II) is performed at 25-45°C for 30-120 minutes; more preferably, the hybridization temperature is 37°C. Preferably, the hybridization time is 60 minutes. Preferably, after the hybridization is completed, a step of washing the electrodes is also included. The cleaning solution for cleaning is a conventional cleaning solution in the field, preferably, 10 mM TE buffer solution containing 1M NaCl, the TE buffer solution includes 10 mM Tris-HCl and 1 mM EDTA, pH 7.0.
本发明中，步骤(Ⅲ)为：向步骤(Ⅱ)所得的杂交的电极上滴加亲和素-辣根过氧化酶(avidin-HRP酶)后孵化；加入底物TMB，进行检测分析。其中，较佳地，所述的亲和素-辣根过氧化酶为1000倍稀释的亲和素-辣根过氧化酶；较佳地，所述1000倍稀释的亲和素-辣根过氧化酶的添加量为1～5μL，更佳地为3μL。所述孵化的温度为本领域常规的温度，较佳地为室温，优选25℃。所述孵化的时间为本领域常规的时间，较佳地为15分钟。步骤(III)中加入底物TMB，进行检测分析的方法为本领域常规。本发明中，采用所述的方法进行电化学检测后，根据测得的电流的绝对值的大小，可以根据电流曲线图，拟合出曲线相应的待测DNA数量与电流绝对值之间的数学公式，一般而言，待测样品中待测DNA数量越大，相应的，测得的电流的绝对值越大。In the present invention, the step (III) is: adding avidin-horseradish peroxidase (avidin-HRP enzyme) to the hybridized electrode obtained in the step (II) and then incubating; adding the substrate TMB for detection and analysis. Wherein, preferably, described avidin-horseradish peroxidase is 1000-fold diluted avidin-horseradish peroxidase; preferably, described 1000-fold diluted avidin-horseradish peroxidase The amount of oxidase added is 1-5 μL, more preferably 3 μL. The incubation temperature is a conventional temperature in the art, preferably room temperature, preferably 25°C. The incubation time is a conventional time in the art, preferably 15 minutes. The method of adding the substrate TMB in step (III) for detection and analysis is routine in the art. In the present invention, after adopting the method for electrochemical detection, according to the magnitude of the absolute value of the measured current, the mathematical relationship between the amount of DNA to be tested and the absolute value of the current corresponding to the curve can be fitted according to the current curve. Generally speaking, the greater the amount of DNA to be tested in the sample to be tested, the correspondingly, the greater the absolute value of the measured current.
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于：本发明将包含多个连续腺嘌呤的DNA探针固定在金电极表面，首次构建了基于多腺嘌呤DNA的电化学生物传感器，可以达到基于巯基修饰DNA组装金电极同样的效果，将开创此类新型电化学生物传感器的新纪元。该固定方法不仅实现DNA探针无需巯基修饰步骤，降低成本的同时可以避免实验操作中双硫键的形成，而且可以通过改变DNA探针中连续腺嘌呤的个数就可以调控金电极表面DNA探针的组装密度。DNA捕获探针固定后，一方面其他碱基(G、C、T)也会非特异性地吸附在金电极表面，影响DNA捕获探针与待测DNA形成双链；另一方面，形成双链后在金电极表面又出现了空位，会吸附酶(如avidin-HRP酶(亲和素-辣根过氧化酶))，而使后续电化学检测产生很高背景信号，从而不利于获得最优的信噪比。然而本发明进一步地在将DNA捕获探针固定于金电极表面后，采用低浓度的巯基己醇对电极表面的空位点进行一段时间的封闭，有利于电极表面的DNA探针有序直立起来，利于后续的杂交反应，信噪比明显提高。本发明的检测方法能够广泛地应用于DNA检测中。本发明能够高灵敏度、高特异性地检测DNA，灵敏度可高至1pM，操作简便，应用前景广阔。The positive progress effect of the present invention is that the present invention immobilizes the DNA probe containing multiple continuous adenines on the surface of the gold electrode, constructs an electrochemical biosensor based on polyadenine DNA for the first time, and can achieve gold electrode assembly based on mercapto-modified DNA The same effect will usher in a new era of such novel electrochemical biosensors. This immobilization method not only realizes that the DNA probe does not need a sulfhydryl modification step, reduces the cost but also avoids the formation of disulfide bonds in the experimental operation, and can regulate the DNA detection on the surface of the gold electrode by changing the number of consecutive adenines in the DNA probe. The packing density of the needles. After the DNA capture probe is immobilized, on the one hand, other bases (G, C, T) will also be non-specifically adsorbed on the surface of the gold electrode, affecting the formation of double strands between the DNA capture probe and the DNA to be tested; on the other hand, the formation of double strands Later, vacancies appear on the surface of the gold electrode, which will adsorb enzymes (such as avidin-HRP enzyme (avidin-horseradish peroxidase)), which will cause a high background signal in the subsequent electrochemical detection, which is not conducive to obtaining the optimal signal-to-noise ratio. However, in the present invention, after immobilizing the DNA capture probe on the surface of the gold electrode, a low concentration of mercaptohexanol is used to seal the empty sites on the electrode surface for a period of time, which is conducive to the orderly erection of the DNA probes on the electrode surface. It is beneficial to the subsequent hybridization reaction, and the signal-to-noise ratio is significantly improved. The detection method of the present invention can be widely used in DNA detection. The invention can detect DNA with high sensitivity and high specificity, the sensitivity can be as high as 1pM, the operation is simple and the application prospect is wide.
附图说明Description of drawings
图1为采用基于DNA捕获探针进行电化学检测的示意图。Figure 1 is a schematic diagram of electrochemical detection using DNA-based capture probes.
图2A～2C为不同组装方法的电化学检测的结果，其中A为使用0.1mM的MCH封闭；B为使用0.1μM的A5封闭；C为不进行封闭步骤。2A-2C are the results of electrochemical detection of different assembly methods, wherein A is blocked with 0.1 mM MCH; B is blocked with 0.1 μM A5; C is not blocked.
图3为不同组装方法进行电化学检测的信噪比的结果。Figure 3 shows the results of the signal-to-noise ratio of electrochemical detection by different assembly methods.
图4为不同组装方法进行电化学检测的电极交流阻抗的结果。Figure 4 shows the results of the AC impedance of the electrode for electrochemical detection by different assembly methods.
图5A～5D为不同组装方法进行电化学检测的电子转移速率系数的结果。其中，A为裸金电极；B为组装poly A30但是不进行封闭步骤的电极；C为组装poly A30并且用0.1mM的MCH封闭的电极；D为组装poly A30并且用0.1μM的A5封闭的电极。5A-5D are the results of electron transfer rate coefficients for electrochemical detection by different assembly methods. Among them, A is a bare gold electrode; B is an electrode assembled with poly A30 but without sealing step; C is an electrode assembled with poly A30 and sealed with 0.1 mM MCH; D is an electrode assembled with poly A30 and sealed with 0.1 μM A5 .
图6为基于多腺嘌呤的DNA捕获探针中腺嘌呤的数目对信噪比的影响的结果。Figure 6 shows the results of the effect of the number of adenines in the polyadenine-based DNA capture probe on the signal-to-noise ratio.
图7A～7E为含有不同数目腺嘌呤的基于多腺嘌呤的DNA捕获探针对组装密度的影响的结果。其中，A为polyA5，B为polyA10，C为polyA20，D为polyA30，E为polyA40。7A-7E are the results of the effect of polyadenine-based DNA capture probes containing different numbers of adenines on the packing density. Among them, A is polyA5, B is polyA10, C is polyA20, D is polyA30, and E is polyA40.
图8为poly A30的浓度对信噪比的影响的结果。Fig. 8 is the result of the influence of the concentration of poly A30 on the signal-to-noise ratio.
图9为0.1mM的MCH的封闭时间对信噪比的影响的结果。Fig. 9 is the result of the influence of the blocking time of 0.1 mM MCH on the signal-to-noise ratio.
图10为不同浓度的待测DNA进行电化学检测的结果(i-t曲线)。Fig. 10 is the result of electrochemical detection (i-t curve) of different concentrations of DNA to be tested.
图11为不同浓度的待测DNA进行电化学检测的结果(柱状图)。Fig. 11 is the results (bar graph) of electrochemical detection of different concentrations of DNA to be tested.
图12为采用单核苷酸错配序列、完全不互补的不互补DNA进行电化学检测的结果。Figure 12 shows the results of electrochemical detection using single nucleotide mismatch sequences and completely non-complementary non-complementary DNA.
具体实施方式detailed description
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.
如序列表中SEQ ID No.1～12所示的单链DNA均由life technology生物有限公司合成。The single-stranded DNAs shown in SEQ ID No.1-12 in the sequence listing were all synthesized by Life Technology Biological Co., Ltd.
实施例中所述的室温为本领域常规的室温，一般指20～40℃。The room temperature described in the examples is a conventional room temperature in the art, generally referring to 20-40°C.
实施例1多腺嘌呤的DNA捕获探针的构建The construction of the DNA capture probe of embodiment 1 polyadenine
设计并合成含有不同个数(5个、10个、20个、30个和40个)的腺嘌呤的DNA捕获探针(分别称为poly A5、poly A10、poly A20、poly A30和poly A40)，其核苷酸序列分别如序列表中SEQ ID No.1～5所示，人工合成(life technology生物有限公司)后即获得多腺嘌呤的DNA捕获探针。Design and synthesis of DNA capture probes containing different numbers (5, 10, 20, 30 and 40) of adenines (referred to as poly A5, poly A10, poly A20, poly A30 and poly A40) , the nucleotide sequences of which are respectively shown in SEQ ID No. 1-5 in the sequence listing, and the polyadenine DNA capture probes are obtained after artificial synthesis (Life Technology Biological Co., Ltd.).
同时合成有且仅有5个腺嘌呤的单链DNA(即A5，其核苷酸序列如序列表中SEQ IDNo.6所示)，以便作为后续试验的对照。Simultaneously, a single-stranded DNA with and only 5 adenines (namely A5, whose nucleotide sequence is shown in SEQ ID No. 6 in the sequence listing) was synthesized to serve as a control for subsequent experiments.
实施例2含有多腺嘌呤的DNA捕获探针的生物传感器Example 2 Biosensors Containing Polyadenine-Based DNA Capture Probes
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)将5μL 0.1μM实施例1所得的多腺嘌呤的DNA捕获探针poly A30滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) Add 5 μL of 0.1 μM polyadenine DNA capture probe poly A30 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25° C., and assemble overnight to obtain an assembled electrode.
(3)使用150μL0.1mM的MCH(购自sigma)对步骤(2)所得的组装电极的空位封闭30分钟，得封闭的电极，即为可用于检测的生物传感器。(3) Use 150 μL of 0.1 mM MCH (purchased from sigma) to seal the vacancy of the assembled electrode obtained in step (2) for 30 minutes, and the sealed electrode is a biosensor that can be used for detection.
(4)将1nM的待测DNA(其核苷酸序列如序列表中SEQ ID No.8所示)和100nM信号探针(其核苷酸序列如序列表中SEQ ID No.7所示，并且在3’端连接生物素)在含有1M NaCl的10mM TE缓冲溶液(包括10mM Tris-HCl和1mM EDTA，pH7.0)中预杂交，80℃保持5分钟，然后25℃放置20分钟，得预杂交物A。(4) 1nM of the DNA to be tested (its nucleotide sequence is as shown in SEQ ID No.8 in the sequence listing) and 100nM signal probe (its nucleotide sequence is as shown in SEQ ID No.7 in the sequence listing, and biotin was attached at the 3' end) in 10mM TE buffer solution containing 1M NaCl (including 10mM Tris-HCl and 1mM EDTA, pH 7.0), pre-hybridized at 80°C for 5 minutes, and then placed at 25°C for 20 minutes to obtain Prehybrid A.
(5)将步骤(4)所得的预杂交物A与步骤(3)所得的封闭的电极上的DNA捕获探针37℃杂交60分钟，再用含有1M NaCl的10mM TE缓冲溶液(包括10mM Tris-HCl和1mM EDTA，pH7.0)彻底冲洗电极。(5) Hybridize the prehybrid A obtained in step (4) with the DNA capture probe on the closed electrode obtained in step (3) at 37°C for 60 minutes, and then use 10mM TE buffer solution containing 1M NaCl (including 10mM Tris -HCl and 1 mM EDTA, pH 7.0) rinse the electrode thoroughly.
(6)向步骤(5)所得的可检测的电极上滴加3μL能催化氧化还原反应的avidin-HRP酶(购自eBioscience，1000倍稀释)，室温孵化15min，使其与该电极上的DNA捕获探针连接；然后加入能催化avidin-HRP酶的反应底物TMB(购自Neogen)进行电化学检测分析。(6) Add 3 μL of avidin-HRP enzyme (purchased from eBioscience, 1000-fold dilution) that can catalyze the redox reaction onto the detectable electrode obtained in step (5), and incubate at room temperature for 15 min to allow it to bind to the DNA on the electrode The capture probe is connected; then the reaction substrate TMB (purchased from Neogen) which can catalyze the avidin-HRP enzyme is added for electrochemical detection and analysis.
进行电化学检测分析的详细原理参见图1，具体地，首先利用腺嘌呤与金电极之间的强相互作用，将一段富含多腺嘌呤(poly A)的DNA捕获探针连接到金电极表面，在待测DNA(其属于大肠杆菌基因组的一段核苷酸序列)存在的情况下，待测DNA和DNA捕获探针、生物素修饰的信号探针采用“夹心法”进行杂交。连接到电极表面的信号探针进而通过生物素-链霉素之间牢固的结合力将HRP酶连接到金电极表面，通过电极表面捕获的HRP酶与底物发生反应，从而灵敏地检测到催化反应所产生的催化电流。The detailed principle of electrochemical detection and analysis is shown in Figure 1. Specifically, firstly, a DNA capture probe rich in polyadenine (poly A) is connected to the surface of the gold electrode by using the strong interaction between adenine and the gold electrode. , in the presence of the test DNA (which belongs to a nucleotide sequence of the Escherichia coli genome), the test DNA, the DNA capture probe, and the biotin-modified signal probe are hybridized using a "sandwich method". The signal probe connected to the electrode surface then connects the HRP enzyme to the surface of the gold electrode through the strong binding force between biotin and streptomycin, and the HRP enzyme captured on the electrode surface reacts with the substrate, thereby sensitively detecting the catalytic The catalytic current generated by the reaction.
实施例3不同组装方法的电化学检测Electrochemical detection of different assembly methods of embodiment 3
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)将5μL0.1μM实施例1所得的poly A30滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) Add 5 μL of 0.1 μM poly A30 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25° C., and assemble overnight to obtain an assembled electrode.
(3)分别使用0.1mM的MCH、0.1μM的实施例1制备的A5对步骤(2)所得的组装电极的空位封闭30分钟，得封闭的电极A、B。(3) Use 0.1 mM MCH and 0.1 μM A5 prepared in Example 1 to seal the gaps of the assembled electrodes obtained in step (2) for 30 minutes to obtain sealed electrodes A and B.
此外，按步骤(2)操作后直接进行步骤(4)得滴加poly A30组装然而不经任何物质进行封闭的电极C。In addition, step (4) is directly performed after step (2) to obtain an electrode C assembled by adding poly A30 dropwise but not sealed by any substance.
其余所有步骤与实施例2完全相同。All the other steps are identical to Example 2.
结果如图2A～2C、图3和表1所示。结果说明，不使用物质进行封闭即不进行任何占位时(即电极C)，电化学检测出的本底信号很高，且检测1nM的信号也不高，信噪比很小，因此不能检测到低浓度的待测DNA。当使用A5进行封闭金电极表面的空位后(即电极B)，情况不能改善，本底信号依旧很高，而检测1nM的信号略有下降，CV峰的催化峰也更加不明显。原因可能是采用0.1μM捕获探针poly A30组装金电极后用0.1μM A5进行封闭，相当于0.2μMA30组装的金电极，更加阻碍电子传递，导致信号降低。而使用MCH进行封闭金电极表面的空位后(即电极A)，本底信号明显降低，这可能因为用巯基己醇将金电极表面封闭，阻碍了对酶的吸附；检测1nM的信号明显提高，原因可能因为采用MCH占位后，组装在金电极表面的DNA变得有序，有利于杂交；信噪比也明显提高，CV峰也趋于正常的催化峰。因此，选择使用MCH封闭的多腺嘧啶捕获探针组装的金电极进行电化学生物传感器的实验。The results are shown in Figures 2A-2C, Figure 3 and Table 1. The results show that when no substance is used for sealing, that is, when no place is occupied (i.e., electrode C), the background signal detected by electrochemical detection is very high, and the signal detected at 1nM is not high, and the signal-to-noise ratio is very small, so it cannot be detected to low concentrations of the DNA to be tested. After using A5 to seal the vacancies on the surface of the gold electrode (i.e., electrode B), the situation cannot be improved, the background signal is still high, but the signal detected at 1nM drops slightly, and the catalytic peak of the CV peak is even less obvious. The reason may be that the gold electrode was assembled with 0.1 μM capture probe poly A30 and then blocked with 0.1 μM A5, which is equivalent to the gold electrode assembled with 0.2 μM A30, which further hindered electron transfer and resulted in a decrease in signal. After using MCH to seal the vacancy on the surface of the gold electrode (i.e., electrode A), the background signal is significantly reduced, which may be due to the use of mercaptohexanol to seal the surface of the gold electrode, which hinders the adsorption of the enzyme; the signal detected at 1nM is significantly improved, The reason may be that the DNA assembled on the surface of the gold electrode becomes orderly after the use of MCH, which is conducive to hybridization; the signal-to-noise ratio is also significantly improved, and the CV peak also tends to the normal catalytic peak. Therefore, gold electrodes assembled with MCH-blocked polyadenylation capture probes were chosen for electrochemical biosensor experiments.
表1电化学检测Table 1 Electrochemical detection
实施例4不同组装方法的电极交联阻抗表征Embodiment 4 Electrode cross-linking impedance characterization of different assembly methods
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)将5μL 0.1μM实施例1所得的poly A30滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) Add 5 μL of 0.1 μM poly A30 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25° C., and assemble overnight to obtain an assembled electrode.
(3)分别使用0.1mM的MCH、0.1μM的实施例1制备的A5对步骤(2)所得的组装电极的空位封闭30分钟，得封闭的电极A、B。(3) Use 0.1 mM MCH and 0.1 μM A5 prepared in Example 1 to seal the gaps of the assembled electrodes obtained in step (2) for 30 minutes to obtain sealed electrodes A and B.
此外，按步骤(2)操作后直接进行步骤(4)得滴加poly A30组装然而不经任何物质进行封闭的电极C。In addition, step (4) is directly performed after step (2) to obtain an electrode C assembled by adding poly A30 dropwise but not sealed by any substance.
此外，将步骤(1)所得的电极称为裸金电极。In addition, the electrodes obtained in step (1) are called bare gold electrodes.
(4)将上述得到的四种情况的电极(裸金电极、A、B和C)在含有0.1M KCl的10.0mMK3Fe(CN)6/K4Fe(CN)6溶液中进行交流阻抗表征。(4) Conduct AC impedance in 10.0mM K3 Fe(CN)6 /K4 Fe(CN)6 solution containing 0.1M KCl for the electrodes (bare gold electrodes, A, B and C) obtained above characterization.
结果如图4和表2所示。结果说明，裸金电极的阻抗非常小，只有0.479kΩ，组装了polyA30捕获探针后，阻抗大大增加为9.20kΩ，说明polyA30捕获探针已经组装在金电极表面，从而阻碍了金电极表面的电子传递，引起阻抗增大；当使用0.1mM的MCH占位30分钟后，阻抗又变小为6.48kΩ，这是因为使用MCH占位后，电极表面的捕获探针变得有序了，有利于电子传递。对比组装polyA30捕获探针、polyA30组装后MCH处理、polyA30与polyA5共组装的金电极的阻抗可以看出来，使用polyA5占位，并不能达到MCH的效果，反而阻抗更大，这可能是因为：由于polyA5的存在，更多的腺嘌呤组装在电极表面，更加阻碍电子的传递。The results are shown in Figure 4 and Table 2. The results show that the impedance of the bare gold electrode is very small, only 0.479kΩ. After the polyA30 capture probe is assembled, the impedance is greatly increased to 9.20kΩ, indicating that the polyA30 capture probe has been assembled on the surface of the gold electrode, thus hindering the electrons on the surface of the gold electrode. transfer, causing the impedance to increase; when using 0.1mM MCH to occupy the place for 30 minutes, the impedance becomes smaller to 6.48kΩ, this is because the capture probes on the electrode surface become orderly after using MCH Electron delivery. Comparing the impedance of assembled polyA30 capture probes, MCH treatment after polyA30 assembly, and gold electrodes co-assembled with polyA30 and polyA5, it can be seen that the use of polyA5 occupying space cannot achieve the effect of MCH, but the impedance is greater. This may be because: With the presence of polyA5, more adenine is assembled on the electrode surface, which hinders the transfer of electrons even more.
表2阻抗检测Table 2 Impedance detection
实施例5不同组装方法的电极表观电子转移速率系数Electrode apparent electron transfer rate coefficient of different assembly methods in embodiment 5
(1)～(3)步骤同实施例4。(1)～(3) steps are the same as embodiment 4.
(4)将上述得到的四种情况的电极(裸金电极、A、B和C)在含有0.1M KCl的10.0mMK3Fe(CN)6液中进行循环伏安检测，扫速分别设置为0.1V/s、0.2V/s、0.3V/s、0.4V/s、0.5V/s、0.6V/s。(4) The electrodes (bare gold electrodes, A, B and C) of the above four conditions obtained above were subjected to cyclic voltammetry detection in 10.0mM K3 Fe(CN)6 solution containing 0.1M KCl, and the scan speed was set to 0.1V/s, 0.2V/s, 0.3V/s, 0.4V/s, 0.5V/s, 0.6V/s.
根据Laviron方程[参见E.Laviron,J.Electroanal.Chem.101(1979)19-28]可以估算出各电极的表观电子转移速率系数(Ks)。The apparent electron transfer rate coefficient (Ks) of each electrode can be estimated according to the Laviron equation [see E. Laviron, J. Electroanal. Chem. 101 (1979) 19-28].
结果如图5A～5D和表3所示。结果说明，在(0.1-0.6)mV/s扫描速度范围内，可以看到明显的且几乎对称的氧化还原峰。随着扫速的增加，氧化和还原的峰电流同时增加并且在(0.1-0.6)mV/s扫描速度范围内呈线性增加，表明电极的氧化还原反应是扩散控制的电化学过程。裸金电极由于导电性好，有利于电子传递，Ks＝0.65s-1；当金电极表面组装了多腺嘧啶捕获探针，Ks＝0.01s-1，这是由于腺嘧啶固定在金电极表面，阻碍了电子传递，所以表观电子转移速率常数会变小很多；然而当用0.1mM MCH对金电极空位进行封闭后，使得非特异性吸附在金电极表面的DNA离开金电极表面，而且使得固定在金电极表面的捕获探针变得有序，所以表观电子转移速率常数较未用MCH封闭的大了6倍之多，即Ks达到0.07s-1；当使用捕获探针与polyA5共组装方法封闭金电极表面的位点时，表观电子转移速率常数仍是0.01s-1，未能改变多腺嘧啶捕获探针组装金电极的情况。所以，选择采用MCH封闭的多腺嘧啶捕获探针组装的金电极。The results are shown in FIGS. 5A to 5D and Table 3. The results show that in the range of (0.1-0.6) mV/s scanning speed, obvious and almost symmetrical redox peaks can be seen. With the increase of scan rate, the peak currents of oxidation and reduction increased simultaneously and linearly in the range of (0.1-0.6) mV/s scan rate, indicating that the redox reaction of the electrode is a diffusion-controlled electrochemical process. The bare gold electrode is conducive to electron transfer due to its good conductivity, Ks=0.65s-1 ; when the surface of the gold electrode is assembled with a polyadenylation capture probe, Ks=0.01s-1 , which is due to the fact that adenine is fixed on the surface of the gold electrode , which hinders the electron transfer, so the apparent electron transfer rate constant will be much smaller; however, when the gold electrode vacancies are blocked with 0.1mM MCH, the DNA non-specifically adsorbed on the gold electrode surface leaves the gold electrode surface, and the immobilized The capture probe on the surface of the gold electrode becomes ordered, so the apparent electron transfer rate constant is 6 times larger than that of the unblocked MCH, that is, Ks reaches 0.07s-1 ; when the capture probe is co-assembled with polyA5 When the method seals the sites on the gold electrode surface, the apparent electron transfer rate constant is still 0.01s-1 , which fails to change the situation of polyadenylation capture probes assembling gold electrodes. Therefore, a gold electrode assembled with an MCH-blocked polyadenylation capture probe was selected.
表3电子转移速率系数检测Table 3 Electron transfer rate coefficient detection
综上，确定采用低浓度MCH对多腺嘧啶捕获探针组装的金电极进行封闭的效果最佳。In summary, it is determined that low concentration of MCH has the best sealing effect on gold electrodes assembled with polyadenylation capture probes.
实施例6多腺嘌呤的DNA捕获探针中腺嘌呤的数目对信噪比的影响Influence of the number of adenines in the DNA capture probe of embodiment 6 polyadenines on the signal-to-noise ratio
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)分别将5μL 0.1μM实施例1所得的poly A5、poly A10、poly A20、poly A30和poly A40滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) Add 5 μL 0.1 μM of poly A5, poly A10, poly A20, poly A30 and poly A40 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25°C, and assemble overnight to obtain an assembly electrode.
其余步骤与实施例2相同。All the other steps are the same as in Example 2.
结果如图6和表4所示。结果说明，随着捕获探针中腺嘌呤(A)数目的增加，检测1nM待测DNA的信号逐渐增加，直到腺嘌呤数目为30时达到饱和，而且信号也越来越稳定，这是因为组装在金电极表面的捕获探针密度越小，越有利于杂交，致使信号逐渐增加；随着捕获探针中腺嘌呤(A)数目的增加，组装在金电极表面的捕获探针也越稳定，致使信号越来稳定。随着DNA捕获探针中A数目的增加，本底信号逐渐降低。这可能是因为，A的数目越多，组装在金电极表面的捕获探针也越稳定，吸附在电极表面的碱基A有一定阻碍酶吸附的作用。随着A数目的增加，信噪比逐渐增加，直到腺嘌呤数目为30时信噪比最大，所以选择polyA30作为最佳的DNA捕获探针进行后续实验。The results are shown in Figure 6 and Table 4. The results show that with the increase of the number of adenine (A) in the capture probe, the signal of detecting 1nM DNA to be tested gradually increases until the number of adenine reaches 30, and the signal becomes more and more stable. The smaller the capture probe density on the surface of the gold electrode, the more conducive to hybridization, resulting in a gradual increase in signal; as the number of adenine (A) in the capture probe increases, the capture probe assembled on the surface of the gold electrode is also more stable. causing the signal to become more stable. As the number of A in the DNA capture probe increases, the background signal gradually decreases. This may be because the greater the number of A, the more stable the capture probe assembled on the surface of the gold electrode, and the base A adsorbed on the surface of the electrode can hinder the adsorption of the enzyme to a certain extent. As the number of A increases, the signal-to-noise ratio increases gradually, until the number of adenine is 30, the signal-to-noise ratio is the largest, so polyA30 is selected as the best DNA capture probe for subsequent experiments.
表4电化学检测Table 4 electrochemical detection
实施例7不同多腺嘌呤的DNA捕获探针与组装密度Example 7 DNA Capture Probes and Assembly Densities of Different Polyadenines
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)分别将5μL 0.1μM实施例1所得的poly A5、poly A10、poly A20、poly A30和poly A40滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) Add 5 μL 0.1 μM of poly A5, poly A10, poly A20, poly A30 and poly A40 obtained in Example 1 dropwise to the cleaned electrode obtained in step (1), react at 25°C, and assemble overnight to obtain an assembly electrode.
(3)使用150μL 0.1mM的MCH(购自sigma)，对步骤(2)所得的组装电极的空位封闭30分钟，得封闭的电极，即为可用于检测的生物传感器。(3) Use 150 μL of 0.1 mM MCH (purchased from sigma) to seal the vacancy of the assembled electrode obtained in step (2) for 30 minutes, and the sealed electrode is a biosensor that can be used for detection.
(4)本实验用到的电解液为包含1M NaCl的10mM TE缓冲溶液(10mM Tris-HCl,1mMEDTA，pH 7.0)，电解液需要使用时新鲜配制。实验前均向电解液中通入15分钟的N2以彻底除去氧气，将上述得到的电极分别首先放置在除氧后的电解液中进行计时电量法检测；然后在电解液中加入50μM RuHex(购自Sigma)，吸附5分钟后再次进行计时电量法检测。(4) The electrolyte used in this experiment is 10mM TE buffer solution (10mM Tris-HCl, 1mMEDTA, pH 7.0) containing 1M NaCl, and the electrolyte is freshly prepared when needed. Before the experiment,15 minutes of N was passed into the electrolyte to completely remove oxygen, and the electrodes obtained above were first placed in the electrolyte after deoxygenation for chronocoulometric detection; then 50 μM RuHex was added to the electrolyte ( Purchased from Sigma), after 5 minutes of adsorption, the chronocoulometry detection was performed again.
通过计时电量法测定RuHex配合物的氧化还原电量推算出poly A捕获探针的表面密度，结果如图7A～7E和表5所示。结果说明，随着A的个数增加，组装在金电极表面的DNA的表面密度逐渐减小。这是因为金电极的表面积是一定的，而理论上组装在金电极表面上A的总数是不变的，因此随着A的个数增加，表面密度逐渐减小。因此根据上述结论，可以通过改变DNA探针中连续腺嘌呤的个数来调控金电极表面DNA探针的组装密度。The surface density of the poly A capture probe was calculated by measuring the redox charge of the RuHex complex by chronocoulometry, and the results are shown in Figures 7A-7E and Table 5. The results showed that as the number of A increased, the surface density of DNA assembled on the gold electrode surface gradually decreased. This is because the surface area of the gold electrode is constant, and the total number of A assembled on the surface of the gold electrode is theoretically constant, so as the number of A increases, the surface density gradually decreases. Therefore, according to the above conclusions, the assembly density of DNA probes on the gold electrode surface can be regulated by changing the number of continuous adenines in the DNA probes.
表5表面密度Table 5 surface density
实施例8poly A30组装浓度对信噪比的影响Example 8 Effect of poly A30 assembly concentration on signal-to-noise ratio
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)分别将实施例1所得的poly A30以0.02μM、0.1μM和0.5μM的浓度5μL的体积滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) The poly A30 obtained in Example 1 was added dropwise at a volume of 5 μL at a concentration of 0.02 μM, 0.1 μM and 0.5 μM to the cleaned electrode obtained in step (1), reacted at 25 ° C, and assembled overnight to obtain an assembled electrode .
其余步骤与实施例2相同。All the other steps are the same as in Example 2.
结果如图8和表6所示。结果说明，当组装0.02μM的polyA30捕获探针时，检测100pM目标时信号比较大，说明密度越小越有利于DNA的杂交，但是本底信号很大。当组装0.5μM的polyA30时，检测100pM目标时信号较小，可能是因为电极表面的组装密度大了，不利于DNA的杂交。从实验结果看，组装0.1μM的poly A30时，信噪比最大。The results are shown in Figure 8 and Table 6. The results show that when 0.02μM polyA30 capture probe is assembled, the signal is relatively large when detecting 100pM target, indicating that the smaller the density is, the more conducive to DNA hybridization, but the background signal is very large. When 0.5μM polyA30 is assembled, the signal is small when detecting 100pM target, which may be because the assembly density on the electrode surface is high, which is not conducive to DNA hybridization. From the experimental results, the signal-to-noise ratio is the largest when 0.1 μM poly A30 is assembled.
表6电化学检测Table 6 Electrochemical detection
实施例9封闭时间的优化The optimization of embodiment 9 sealing time
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)将实施例1所得的poly A30以0.1μM浓度5μL的体积滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) The poly A30 obtained in Example 1 was added dropwise at a volume of 5 μL at a concentration of 0.1 μM to the cleaned electrode obtained in step (1), reacted at 25° C., and assembled overnight to obtain an assembled electrode.
(3)使用150μL的1mM MCH(购自sigma)分别对步骤(2)所得的组装电极的空位封闭0、5、10、20、30和60分钟，得封闭的电极。(3) Use 150 μL of 1 mM MCH (purchased from sigma) to block the vacancies of the assembled electrodes obtained in step (2) for 0, 5, 10, 20, 30, and 60 minutes, respectively, to obtain blocked electrodes.
其余步骤与实施例2相同。All the other steps are the same as in Example 2.
结果如图9和表7所示。结果说明，使用巯基己醇占位后，检测1nM目标的信号明显增加。随着封闭时间的延长，检测1nM目标的信号逐渐降低，背景信号也逐渐降低，但是信噪比是逐渐增大的，当封闭时间为30分钟时，电化学检测的信噪比最大。因此，选择控制封闭时间为30分钟。The results are shown in Figure 9 and Table 7. The results showed that after using mercaptohexanol to occupy the site, the signal for detecting 1nM target increased significantly. With the prolongation of the blocking time, the detection signal of 1nM target gradually decreased, and the background signal also gradually decreased, but the signal-to-noise ratio gradually increased. When the blocking time was 30 minutes, the signal-to-noise ratio of electrochemical detection was the largest. Therefore, the control closure time was chosen to be 30 minutes.
综上，通过以上条件的优化，采用本发明的生物传感器进行电化学检测的最优条件为：使用polyA30的捕获探针；并且使该捕获探针的组装浓度为0.1μM；用0.1mM MCH封闭30分钟。In summary, through the optimization of the above conditions, the optimal conditions for electrochemical detection using the biosensor of the present invention are: use a capture probe of polyA30; and make the assembly concentration of the capture probe 0.1 μM; block with 0.1 mM MCH 30 minutes.
表7电化学检测Table 7 Electrochemical detection
效果实施例1灵敏度检测Effect Example 1 Sensitivity Detection
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)将实施例1所得的poly A30以0.1μM浓度5μL的体积滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) The poly A30 obtained in Example 1 was added dropwise at a volume of 5 μL at a concentration of 0.1 μM to the cleaned electrode obtained in step (1), reacted at 25° C., and assembled overnight to obtain an assembled electrode.
(3)使用150μL的1mM MCH(购自sigma)分别对步骤(2)所得的组装电极的空位封闭30分钟，得封闭的电极。(3) Use 150 μL of 1 mM MCH (purchased from sigma) to block the vacancies of the assembled electrodes obtained in step (2) for 30 minutes to obtain sealed electrodes.
(4)分别将1pM、10pM、100pM、200pM、500pM和1nM的待测DNA(其核苷酸序列如序列表中SEQ ID No.8所示)和100nM信号探针(其核苷酸序列如序列表中SEQ ID No.7所示，并且在3’端连接生物素)在含有1M NaCl的10mM TE缓冲溶液(包括10mM Tris-HCl和1mMEDTA，pH7.0)中预杂交，80℃保持5分钟，然后25℃放置20分钟，得预杂交物A。(4) DNA to be tested (its nucleotide sequence is shown in SEQ ID No.8 in the sequence listing) and 100nM signal probe (its nucleotide sequence is as shown in SEQ ID No.8) of 1pM, 10pM, 100pM, 200pM, 500pM and 1nM respectively Shown in SEQ ID No.7 in the sequence listing, and biotin is connected at the 3' end) in 10mM TE buffer solution containing 1M NaCl (including 10mM Tris-HCl and 1mMEDTA, pH7.0) pre-hybridization, 80 ℃ for 5 minutes, and then placed at 25°C for 20 minutes to obtain pre-hybrid A.
其余步骤与实施例2相同。All the other steps are the same as in Example 2.
结果如图10～11和表8所示。结果说明，当没有待测DNA存在时，只能观察到很小的背景电流；当加入待测DNA，检测电流明显增大，且随着待测DNA浓度的增加而增加，因此采用本发明所述的含多腺嘌呤的DNA捕获探针及其自组装金电极进行电化学检测，所检测的待测DNA的检出限为1pM。The results are shown in Figures 10-11 and Table 8. The result shows that when there is no DNA to be tested, only a small background current can be observed; when the DNA to be tested is added, the detection current increases significantly, and increases with the concentration of the DNA to be tested. The above polyadenine-containing DNA capture probe and its self-assembled gold electrode are used for electrochemical detection, and the detection limit of the detected DNA is 1pM.
表8电化学检测Table 8 Electrochemical detection
效果实施例2特异性检测Effect Example 2 Specificity Detection
(1)取经过物理打磨和硫酸电化学清洗的金电极(2mm直径，上海辰华仪器有限公司)，用超纯水Milli-Q彻底冲洗电极表面，然后用N2吹干，得处理干净的电极。(1) Take the gold electrode (2mm diameter, Shanghai Chenhua Instrument Co., Ltd.) that has been physically polished and electrochemically cleaned with sulfuric acid, rinse the electrode surface thoroughly with ultrapure water Milli-Q, and then blow dry withN2 to get a clean electrode.
(2)将实施例1所得的poly A30以0.1μM浓度5μL的体积滴加到步骤(1)所得的处理干净的电极，25℃反应，过夜组装，得组装电极。(2) The poly A30 obtained in Example 1 was added dropwise at a volume of 5 μL at a concentration of 0.1 μM to the cleaned electrode obtained in step (1), reacted at 25° C., and assembled overnight to obtain an assembled electrode.
(3)使用150μL的1mM MCH(购自sigma)分别对步骤(2)所得的组装电极的空位封闭30分钟，得封闭的电极。(3) Use 150 μL of 1 mM MCH (purchased from sigma) to block the vacancies of the assembled electrodes obtained in step (2) for 30 minutes to obtain sealed electrodes.
(4)分别将100pM的待测DNA(其核苷酸序列如序列表中SEQ ID No.8所示)、单核苷酸错配序列SNP(A)、SNP(C)和SNP(G)(其核苷酸序列依次如序列表中SEQ ID No.10～12所示)以及将充分过量的与上述待测DNA的核苷酸序列完全非互补DNA(NT)(其核苷酸序列如序列表中SEQ ID No.9所示)和100nM信号探针(其核苷酸序列如序列表中SEQ ID No.7所示，并且在3’端连接生物素)在含有1M NaCl的10mM TE缓冲溶液(包括10mM Tris-HCl和1mMEDTA，pH7.0)中预杂交，80℃保持5分钟，然后25℃放置20分钟，得预杂交物A。(4) 100pM of the DNA to be tested (its nucleotide sequence is shown in SEQ ID No.8 in the sequence listing), single nucleotide mismatch sequences SNP (A), SNP (C) and SNP (G) (its nucleotide sequence is successively as shown in SEQ ID No.10～12 in the sequence list) and fully non-complementary DNA (NT) with the nucleotide sequence of the above-mentioned DNA to be tested in sufficient excess (its nucleotide sequence is as shown in Shown in SEQ ID No.9 in the sequence listing) and 100nM signal probe (its nucleotide sequence is shown in SEQ ID No.7 in the sequence listing, and biotin is connected at the 3' end) in 10mM TE containing 1M NaCl Pre-hybridize in a buffer solution (including 10 mM Tris-HCl and 1 mM EDTA, pH 7.0), keep at 80° C. for 5 minutes, and then stand at 25° C. for 20 minutes to obtain pre-hybrid A.
相应地，有一个对应的不与任何待测DNA杂交的电极作为空白对照。Correspondingly, there is a corresponding electrode that does not hybridize with any DNA to be tested as a blank control.
其余步骤与实施例2相同。All the other steps are the same as in Example 2.
结果如图12和表9所示。结果说明，本发明所述的含有多腺嘌呤的DNA捕获探针及其自组装金电极进行的电化学检测的特异性非常高。The results are shown in Figure 12 and Table 9. The results show that the specificity of the electrochemical detection performed by the polyadenine-containing DNA capture probe and the self-assembled gold electrode of the present invention is very high.
众所周知，对单碱基错配的区分在单核苷多态性检测中具有很重要的应用。通过对仅仅与待测DNA存在单核苷酸差异的单碱基错配DNA链(SNP(A)、SNP(C)和SNP(G))探究特异性，其得到的信号强度大约是背景信号的2倍，而这仅仅是真正的待测DNA所检测得到的信号强度的五分之一左右，可见该自组装电极具有高度的序列特异性。It is well known that the discrimination of single base mismatches has important applications in the detection of single nucleotide polymorphisms. Specificity is explored for single-base mismatched DNA strands (SNP(A), SNP(C) and SNP(G)) that differ only by a single nucleotide from the test DNA, with signal intensities approximately background signal , which is only about one-fifth of the signal intensity detected by the real DNA to be tested. It can be seen that the self-assembled electrode has a high degree of sequence specificity.
表9电化学检测Table 9 electrochemical detection
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。It should be understood that after reading the above content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
<110> 上海市计量测试技术研究院<110> Shanghai Institute of Metrology and Testing Technology
<120> 基于多腺嘌呤的DNA捕获探针、生物传感器及其检测方法<120> DNA capture probe, biosensor and detection method based on polyadenine
<130> P1610939C<130> P1610939C
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610959082.1ACN106338539B (en) | 2016-11-03 | 2016-11-03 | DNA capture probe, biosensor and its detection method based on polyadenous purine |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610959082.1ACN106338539B (en) | 2016-11-03 | 2016-11-03 | DNA capture probe, biosensor and its detection method based on polyadenous purine |
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| CN106338539Atrue CN106338539A (en) | 2017-01-18 |
| CN106338539B CN106338539B (en) | 2019-07-12 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610959082.1AActiveCN106338539B (en) | 2016-11-03 | 2016-11-03 | DNA capture probe, biosensor and its detection method based on polyadenous purine |
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| CN (1) | CN106338539B (en) |
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