CN103529011A - Method for detecting DNA (deoxyribonucleic acid) through high sensitivity Raman spectrum - Google Patents
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Abstract
本发明涉及一种高灵敏拉曼光谱检测DNA的方法。该方法利用磁珠表面上的链替代聚合和银增强作用实现信号的双重放大。磁珠用于分子信标的固定和链替代产物的分离。在目标DNA与分子信标杂交后，信标环被打开，引物修饰的拉曼染料-银纳米复合物通过引物与信标茎端的杂交与磁珠连接，诱导DNA链聚合反应而释放目标分子，被释放的DNA分子与另一磁珠上的分子信标杂交而产生另一聚合循环。在循环链替代聚合完成后，分离链替代产物，并在纳米复合物表面沉积银纳米壳层，增强染料的拉曼信号。利用拉曼信号与目标DNA浓度与序列的相关性进行DNA分析。这一方法检测DNA的浓度范围是10-13-10-8mol L-1，并可区分完全配对的目标DNA和单碱基错配的DNA，具有一定的应用价值。
The invention relates to a method for detecting DNA with high-sensitivity Raman spectrum. The method utilizes chain displacement polymerization on the surface of magnetic beads and silver reinforcement to achieve double amplification of signal. Magnetic beads are used for the immobilization of molecular beacons and the isolation of strand displacement products. After the target DNA is hybridized with the molecular beacon, the beacon ring is opened, and the primer-modified Raman dye-silver nanocomplex is connected to the magnetic bead through the hybridization of the primer and the stem end of the beacon, which induces DNA chain polymerization to release the target molecule and is activated. The released DNA molecule hybridizes to a molecular beacon on another magnetic bead to create another cycle of polymerization. After the cyclic chain substitution polymerization is completed, the chain substitution products are isolated and silver nanoshells are deposited on the surface of the nanocomposite to enhance the Raman signal of the dye. DNA analysis using the correlation of Raman signal with target DNA concentration and sequence. The concentration range of this method for detecting DNA is 10 -13 -10 -8 mol L -1 , and it can distinguish perfectly paired target DNA from single base mismatched DNA, which has certain application value.
Description
一、技术领域 1. Technical field
本发明为一种高灵敏拉曼光谱检测DNA的方法。它通过磁珠表面上的链替代聚合和银增强作用进行双重信号放大，利用外磁场分离磁珠上的链替代产物，用拉曼光谱进行高灵敏DNA检测。The invention is a method for detecting DNA with high-sensitivity Raman spectrum. It performs dual signal amplification through chain substitution polymerization on the surface of magnetic beads and silver enhancement, uses an external magnetic field to separate chain substitution products on magnetic beads, and uses Raman spectroscopy for highly sensitive DNA detection.
二、背景技术 2. Background technology
DNA是遗传信息的承担者，DNA分子中碱基序列的变异与人类许多遗传疾病有关。因此，对特定序列的DNA进行分析并进行碱基突变的检测在基因筛选、遗传疾病的早期诊断等方面具有深远的意义。DNA杂交是核酸浓度检测和结构分析的重要手段。DNA杂交检测技术包括荧光、化学发光、电化学和表面等离子共振等，其中荧光分析技术可以进行多种DNA的同时检测。但自身的光猝灭、复杂的操作和不同信号的光谱重叠限制了荧光分析技术的应用。虽然半导体量子点有相对窄的光谱带，其自身的毒性也是一个广泛关注的问题。因此需要发展新颖的分析技术。DNA is the bearer of genetic information, and the variation of base sequences in DNA molecules is related to many genetic diseases of human beings. Therefore, analyzing the DNA of a specific sequence and detecting the base mutation has far-reaching significance in gene screening, early diagnosis of genetic diseases, and the like. DNA hybridization is an important method for nucleic acid concentration detection and structural analysis. DNA hybridization detection techniques include fluorescence, chemiluminescence, electrochemistry, and surface plasmon resonance, among which fluorescence analysis techniques can simultaneously detect multiple DNAs. However, its own light quenching, complex operation and spectral overlap of different signals limit the application of fluorescence analysis technology. Although semiconductor quantum dots have relatively narrow spectral bands, their own toxicity is also a widespread concern. Therefore, novel analytical techniques need to be developed.
表面增强拉曼光谱检测技术可以弥补荧光检测技术的缺陷。由于其很窄的光谱带、低的光猝灭和自身猝灭，拉曼光谱检测DNA的方法已得到发展。其中之一是利用众所周知的金纳米颗粒进行固定。这一过程需要很长的温育时间、重复的洗涤步骤，并要克服干扰物的非特性吸附。因此本发明建立了一种简便、灵敏的DNA拉曼光谱检测方法。Surface-enhanced Raman spectroscopy detection technology can make up for the defects of fluorescence detection technology. Due to its narrow spectral band, low photoquenching and self-quenching, methods for the detection of DNA by Raman spectroscopy have been developed. One of them is immobilization with the well-known gold nanoparticles. This process requires long incubation times, repeated washing steps, and overcoming non-specific adsorption of interfering substances. Therefore, the present invention establishes a simple and sensitive DNA Raman spectrum detection method.
三、发明内容 3. Contents of the invention
本发明的目的是：以磁性纳米颗粒为基底固定识别探针分子信标，通过分子信标探针与目标DNA分子，并随后与引物修饰的拉曼染料-银纳米复合物的杂交将纳米复合物连到磁性纳米颗粒上，利用链替代聚合和银增强作用实现双信号放大，建立一种不需要清洗步骤的检测DNA的灵敏方法。The purpose of the present invention is: use magnetic nanoparticles as the substrate to immobilize the recognition probe molecular beacon, and through the hybridization of the molecular beacon probe with the target DNA molecule, and subsequently with the primer-modified Raman dye-silver nanocomposite, the nanocomposite The material is linked to magnetic nanoparticles, and the dual signal amplification is achieved by strand substitution polymerization and silver enhancement, and a sensitive method for detecting DNA without washing steps is established.
本发明通过以下技术方案来实现：The present invention is realized through the following technical solutions:
将目标DNA分子与固定在磁珠表面的分子信标探针进行杂交，打开分子信标环，加入引物修饰的拉曼染料-银纳米复合物，通过引物与分子信标茎端的杂交将复合物连接到磁珠上，在聚合酶与dNTP存在下诱导DNA链发生聚合反应而释放目标DNA分子，被释放的目标DNA分子与固定在另一个磁珠上的分子信标杂交，产生另一个聚合循环。在循环链替代聚合完成后，利用磁珠分离链替代产物，并在产物连接的纳米复合物表面沉积一层银纳米壳层，增强染料的拉曼信号。检测标准DNA溶液与样品获得的拉曼信号，进行DNA浓度检测与单碱基错配的判别。The target DNA molecule is hybridized with the molecular beacon probe immobilized on the surface of the magnetic bead, the molecular beacon ring is opened, and the primer-modified Raman dye-silver nanocomposite is added. Attached to magnetic beads, in the presence of polymerase and dNTPs, the DNA strands are induced to polymerize to release target DNA molecules, and the released target DNA molecules hybridize with molecular beacons immobilized on another magnetic bead, resulting in another cycle of polymerization . After the cyclic chain substitution polymerization is completed, the chain substitution products are separated by magnetic beads, and a silver nanoshell is deposited on the surface of the product-linked nanocomposite to enhance the Raman signal of the dye. Detect the Raman signal obtained from the standard DNA solution and the sample, and perform DNA concentration detection and discrimination of single base mismatches.
上述的识别探针通过羧基与氨基的共价键合将一端带有氨基的分子信标固定在羧基功能化的磁性纳米颗粒上而获得；引物修饰的拉曼染料-银纳米复合物可由带有巯基的引物与拉曼染料在银纳米颗粒上的组装制得。The above-mentioned recognition probes are obtained by immobilizing molecular beacons with amino groups at one end on carboxyl-functionalized magnetic nanoparticles through covalent bonding between carboxyl groups and amino groups; primer-modified Raman dye-silver nanocomposites can be obtained by The thiol-based primers were prepared by the assembly of Raman dyes on silver nanoparticles.
DNA的高灵敏拉曼光谱检测涉及磁珠表面上的链替代聚合和银增强作用进行双重信号放大(图1)，其具体步骤如下：The highly sensitive Raman spectroscopic detection of DNA involves double signal amplification by strand substitution polymerization and silver enhancement on the surface of magnetic beads (Figure 1), and the specific steps are as follows:
1)通过羧基与氨基的共价键将一端带有氨基的分子信标固定在羧基功能化的磁性纳米颗粒上，获得识别探针。同时将带有巯基的引物组装在银纳米颗粒上，进一步组装带巯基的拉曼染料，制得引物修饰的拉曼染料-银纳米复合物。1) A molecular beacon with an amino group at one end is immobilized on a carboxyl-functionalized magnetic nanoparticle through a covalent bond between a carboxyl group and an amino group to obtain a recognition probe. At the same time, the primers with sulfhydryl groups are assembled on the silver nanoparticles, and the Raman dyes with sulfhydryl groups are further assembled to prepare the primer-modified Raman dye-silver nanocomposites.
2)将目标DNA分子与识别探针混合，在磁珠表面进行杂交后，打开分子信标环，引物修饰的拉曼染料-银纳米复合物与分子信标茎端进行杂交，在聚合酶与dNTP存在下，在磁珠表面诱导DNA链聚合反应，并释放目标DNA分子，发生循环链替代聚合，实现第一次信号放大。2) Mix the target DNA molecule with the recognition probe, and after hybridization on the surface of the magnetic bead, the molecular beacon ring is opened, and the primer-modified Raman dye-silver nanocomposite hybridizes with the stem end of the molecular beacon, after the polymerase and In the presence of dNTP, the DNA chain polymerization reaction is induced on the surface of the magnetic beads, and the target DNA molecule is released, and the cyclic chain replacement polymerization occurs, realizing the first signal amplification.
3)在外加磁场的作用下将链替代聚合的产物从混合物中分离出来，与银增强溶液混合，在银纳米颗粒外围沉积一层银壳，以增加拉曼染料的信号强度，获得第二次信号放大。3) Under the action of an external magnetic field, the product of the chain substitution polymerization is separated from the mixture, mixed with the silver enhancement solution, and a layer of silver shell is deposited on the periphery of the silver nanoparticles to increase the signal intensity of the Raman dye, and obtain a second Signal amplification.
4)检测被增强的拉曼染料信号，利用DNA的标准溶液获得检测的工作曲线，从工作曲线进行DNA的分析。4) Detecting the enhanced Raman dye signal, using the DNA standard solution to obtain a detection working curve, and performing DNA analysis from the working curve.
本方法检测DNA的原理：The principle of this method to detect DNA:
通过目标DNA与固定在磁性纳米颗粒表面的分子信标杂交，打开分子信标，让分子信标的茎端与组装在拉曼染料-银纳米复合物上的引物结合，引发DNA链发生聚合反应而释放目标DNA分子，产生循环链替代聚合信号放大。在分离链替代产物后，在纳米复合物表面沉积一层银纳米壳层，进一步放大染料的拉曼信号，利用被增强的拉曼信号进行DNA浓度检测与单碱基错配的判别。By hybridizing the target DNA with the molecular beacon immobilized on the surface of the magnetic nanoparticle, the molecular beacon is opened, and the stem end of the molecular beacon binds to the primer assembled on the Raman dye-silver nanocomposite, triggering the polymerization reaction of the DNA chain and The target DNA molecule is released to generate circular strands instead of polymerized signal amplification. After separating the strand replacement products, a layer of silver nanoshell is deposited on the surface of the nanocomposite to further amplify the Raman signal of the dye, and use the enhanced Raman signal to detect DNA concentration and distinguish single base mismatches.
本发明与现有技术相比，具有以下特点：Compared with the prior art, the present invention has the following characteristics:
本发明用制备的拉曼染料-银纳米复合物为示踪探针，利用双重信号放大，通过拉曼光谱检测，建立了一种高灵敏的拉曼光谱检测DNA的方法。相对于现有的检测方法，有以下特点：The invention uses the prepared Raman dye-silver nanocomposite as a tracer probe, utilizes double signal amplification, and establishes a highly sensitive Raman spectrum detection method for DNA through Raman spectrum detection. Compared with existing detection methods, it has the following characteristics:
(1)通过拉曼光谱检测，操作简单。(1) It is detected by Raman spectroscopy, and the operation is simple.
(2)拉曼光谱检测在磁性纳米颗粒上进行，不需要重复的洗涤步骤，并且磁性纳米粒子价格低廉。(2) Raman spectroscopy detection is performed on magnetic nanoparticles without repeated washing steps, and the magnetic nanoparticles are cheap.
(3)设计了一种拉曼染料-银纳米复合物为示踪标记探针，可通过银增强作用进行拉曼信号放大。(3) A Raman dye-silver nanocomposite was designed as a tracer label probe, which can amplify Raman signal through silver enhancement.
(4)利用链替代聚合反应使目标DNA可以循环使用，实现放大信号。(4) The target DNA can be recycled by using strand substitution polymerization reaction to realize signal amplification.
(5)双重信号放大提高了检测灵敏度。(5) Double signal amplification improves detection sensitivity.
四、附图说明 4. Description of drawings
图1.拉曼光谱检测DNA方法的示意图Figure 1. Schematic diagram of the method for detecting DNA by Raman spectroscopy
五、具体实施方式 5. Specific implementation
实施例1：引物修饰的拉曼染料-银纳米复合物的制备Embodiment 1: the preparation of the Raman dye-silver nanocomposite of primer modification
将200mL 1.0mM硝酸银溶液煮沸后，在搅拌下加入5mL 35mM柠檬酸钠溶液，混合液继续搅拌加热1小时，得到银纳米粒子的胶体溶液，避光4℃保存。进一步在1mL银纳米粒子的胶体溶液中加入100μL巯基修饰的引物(10μM)，搅拌18个小时后，在8000转/分钟下离心15分钟纯化三次，将得到的引物修饰银纳米粒子重新分散在1mL 10mM PBS溶液中。然后，在此溶液中加入4μL 1mM 4-巯基苯甲酸溶液，搅拌12小时后离心纯化三次，得到引物修饰的拉曼染料-银纳米复合物，重新在1mL 10mM PBS溶液中待用。After boiling 200mL of 1.0mM silver nitrate solution, 5mL of 35mM sodium citrate solution was added under stirring, and the mixture was stirred and heated for 1 hour to obtain a colloidal solution of silver nanoparticles, which was stored in the dark at 4°C. Further, 100 μL of sulfhydryl-modified primers (10 μM) were added to 1 mL of colloidal solution of silver nanoparticles, stirred for 18 hours, and purified by centrifugation at 8000 rpm for 15 minutes for three times, and the obtained primer-modified silver nanoparticles were redispersed in 1 mL 10mM PBS solution. Then, 4 μL of 1 mM 4-mercaptobenzoic acid solution was added to this solution, stirred for 12 hours, and centrifuged and purified three times to obtain the primer-modified Raman dye-silver nanocomposite, which was re-introduced in 1 mL of 10 mM PBS solution for use.
实施例2：磁珠表面分子信标的固定Example 2: Immobilization of molecular beacons on the surface of magnetic beads
将50μL羧基化的磁性纳米颗粒分散到1mL PBS(0.01M，pH 7.4)中，滴入200μL0.1M 1-乙基-3-(3-二甲基氨丙基)-碳化二亚胺盐酸盐溶液，400μL 0.05M N-羟基琥珀酰亚胺溶液和150μL 5.4μM分子信标，室温下搅拌4小时，利用磁场分离出磁性纳米颗粒，用水清洗几次，得到分子信标修饰的磁性纳米颗粒，重新分散在1mL PBS(0.01M，pH 7.4)中4℃保存待用。Disperse 50 μL of carboxylated magnetic nanoparticles into 1 mL of PBS (0.01M, pH 7.4) and drop into 200 μL of 0.1M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride Saline solution, 400 μL 0.05M N-hydroxysuccinimide solution and 150 μL 5.4 μM molecular beacon, stirred at room temperature for 4 hours, separated magnetic nanoparticles by using a magnetic field, washed several times with water, and obtained magnetic nanoparticles modified by molecular beacons , redispersed in 1mL PBS (0.01M, pH 7.4) and stored at 4°C until use.
实施例3：拉曼光谱检测DNAEmbodiment 3: Raman spectroscopy detects DNA
(1)在10μL分子信标修饰的磁性纳米颗粒分散液中滴入10μL不同浓度的DNA溶液、5μL引物修饰的拉曼染料-银纳米复合物溶液、2μL聚合酶(4U)和dNTPs(每个核苷酸均为12μM)，在37℃温育100分钟，然后用磁场分离固定在磁性纳米颗粒表面的链替代产物；(1) In 10 μL molecular beacon-modified magnetic nanoparticle dispersion, 10 μL DNA solution of different concentrations, 5 μL primer-modified Raman dye-silver nanocomposite solution, 2 μL polymerase (4 U) and dNTPs (each Nucleotides are all 12 μM), incubated at 37°C for 100 minutes, and then separated by a magnetic field to replace the strands immobilized on the surface of the magnetic nanoparticles;
(2)将链替代产物与10μL含1∶1银增强溶液A与B混合物混合，银增强反应2分钟后进行拉曼检测。(2) The chain substitution product was mixed with 10 μL of the mixture containing 1:1 silver enhancement solutions A and B, and Raman detection was performed after 2 minutes of silver enhancement reaction.
(3)记录不同浓度DNA溶液与样品的拉曼信号，获得DNA检测的工作曲线，并求出样品中的DNA浓度。(3) Record the Raman signals of different concentrations of DNA solutions and samples, obtain the working curve of DNA detection, and calculate the concentration of DNA in the sample.
(4)通过比较相同DNA浓度下的拉曼信号可进行单碱基错配或不配对DNA序列的判别。(4) By comparing the Raman signals at the same DNA concentration, the single base mismatch or unpaired DNA sequence can be discriminated.
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