CN107435063B - A method for rapid preparation of thiol-modified DNA nano-gold complexes (DNA-AuNPs) - Google Patents

Abstract

Translated fromChinese

本发明涉及一种快速制备巯基修饰DNA纳米金复合物(DNA‑AuNP)的方法。采用具有不同长度的寡乙二醇间隔链(OEG)作为间隔链的巯基修饰DNA来进行DNA‑AuNP的快速制备。可显著提高DNA在纳米金表面的上载速率，而且该上载速率随着盐浓度的提高而增加。该方法是一种制备DNA‑AuNP的通用方法，具有不同巯基修饰位置(5'或者3'端)、长度和序列的DNA均适用。该方法不仅适用于线性单链DNA在纳米金上的上载，而且也适用于具有二级结构的DNA探针在纳米金表面上的上载，可一步直接在纳米金表面上载具有二级结构的DNA探针，比如分子信标。该方法适用于不同尺寸的纳米金，特别是稳定性相对较差的大尺寸的纳米金。

The invention relates to a method for rapidly preparing a thiol-modified DNA nano-gold complex (DNA-AuNP). The rapid preparation of DNA-AuNPs was performed using sulfhydryl-modified DNA with oligoethylene glycol spacers (OEGs) of different lengths as spacers. The uploading rate of DNA on the surface of gold nanoparticles can be significantly improved, and the uploading rate increases with the increase of salt concentration. This method is a general method for the preparation of DNA-AuNPs, and is applicable to DNAs with different sulfhydryl modification positions (5' or 3' end), lengths and sequences. This method is not only suitable for the uploading of linear single-stranded DNA on gold nanoparticles, but also for the uploading of DNA probes with secondary structure on the surface of gold nanoparticles. The DNA with secondary structure can be directly loaded on the surface of gold nanoparticles in one step. Probes, such as molecular beacons. This method is suitable for gold nanoparticles of different sizes, especially large-sized gold nanoparticles with relatively poor stability.

Description

Translated fromChinese

一种快速制备巯基修饰DNA纳米金复合物(DNA-AuNP)的方法A method for rapid preparation of thiol-modified DNA nano-gold complexes (DNA-AuNPs)

技术领域technical field

本发明涉及一种利用寡乙二醇间隔链(OEG)修饰的巯基DNA，快速制备巯基修饰DNA纳米金复合物(DNA-AuNP)的方法，属于生物技术领域。The invention relates to a method for rapidly preparing a sulfhydryl-modified DNA nano-gold complex (DNA-AuNP) by utilizing sulfhydryl DNA modified by an oligoethylene glycol spacer chain (OEG), and belongs to the field of biotechnology.

背景技术Background technique

巯基修饰DNA纳米金复合物(DNA-AuNP)在纳米自组装结构构建、生物传感技术和药物运输等领域都拥有十分重要的应用。纳米金界面上DNA的有效的分子识别是这些应用的基础。广泛的研究表明DNA-AuNP上DNA的数量、密度、构象、保护层的组成、复合探针的形状和尺寸等众多因素都显著影响分子识别的效率。解决基于DNA-AuNP的应用重复性差的技术难题，关键是DNA-AuNP的制备，AuNP界面上的DNA的数量和构象必须得到准确控制。Thiol-modified DNA nano-gold complexes (DNA-AuNPs) have important applications in the construction of nano-self-assembled structures, biosensing technology and drug delivery. Efficient molecular recognition of DNA at nanogold interfaces is the basis for these applications. Extensive studies have shown that numerous factors, such as the number, density, conformation of DNA on DNA-AuNPs, the composition of the protective layer, and the shape and size of the composite probe, all significantly affect the efficiency of molecular recognition. To solve the technical problem of poor repeatability of DNA-AuNP-based applications, the key is the preparation of DNA-AuNPs. The quantity and conformation of DNA on the AuNP interface must be accurately controlled.

然而，现有的DNA-AuNP制备方法即使在理想化的实验室条件下也不能够准确控制AuNP界面上的DNA的数量和构象。而且现有的DNA-AuNP制备技术存在各种各样的问题与不足。DNA-AuNP的现有制备方法主要利用表面活性剂或者对纳米金有亲和力的小分子来提高DNA-AuNP的盐耐受能力，然后在高盐条件下进行巯基DNA在纳米金上的上载。1996年美国西北大学的Mirkin课题组最早制备了DNA-AuNP，他们采用了“老化-加盐”的制备方法，通过不断增加缓冲溶液的NaCl浓度来消弱DNA与柠檬酸根包被的带负电的纳米金之间的静电排斥，从而实现DNA在纳米金上的高密度组装(Nature 1996,382(6592),607-609)。由于柠檬酸根包被的纳米金的稳定性较差，加盐过程中经常会出现聚集的现象，需要小心控制加盐的速度。另外，DNA-AuNP的稳定性比柠檬酸根包被的纳米金提高不少，但还不能承受较高的盐浓度，限制了它在一些场合的应用。1999年加州大学伯克利分校的Alivisatos课题组提出了利用双(对磺酰苯基)苯基膦(BSPP)来先替换包被在纳米金表面上的柠檬酸根，再进行巯基修饰DNA在高盐浓度下组装的方法，大幅提高了DNA-AuNP制备方法的稳定性和对盐离子和不同pH的耐受能力(Angew.Chem.Int.Ed.1999,38(12),1808-1812)。但是用这种方法制备的DNA-AuNP的DNA探针上载量比“老化-加盐”的方法低很多。类似的，在2009年和2012年分别报道了利用非离子含氟表面活性剂(Anal.Chem.2009,81(20),8523-8528)、三磷酸腺嘌呤脱氧核苷酸(dATP)(Bioconjugate Chem.2009,20(6),1218-1222)和大分子量聚乙二醇(PEG)(J.Am.Chem.Soc.2012,134(24),9910-9913)来分别替换包被在纳米金表面上的柠檬酸根的方法，也显著提高了纳米金对盐离子的耐受能力，DNA探针的上载量也都达到与“老化-加盐”的方法相当的水平。另外，经典的“老化-加盐”法需要1-2天。2009年报道了利用非离子含氟表面活性剂或者dATP来分别替换包被在纳米金表面上的柠檬酸根，将制备时间缩短到几个小时。2012年加拿大滑铁卢大学Juewen Liu课题组报道的基于pH调控的简单方法，通过将缓冲溶液的pH降低到3来有效消弱DNA与纳米金之间的静电排斥，仅需3分钟孵育就实现巯基修饰DNA在纳米金上的快速定量包被(J.Am.Chem.Soc.2012,134(17),7266-7269)。However, existing DNA-AuNP preparation methods cannot accurately control the quantity and conformation of DNA at the AuNP interface even under idealized laboratory conditions. Moreover, the existing DNA-AuNP preparation technology has various problems and deficiencies. The existing preparation methods of DNA-AuNP mainly use surfactants or small molecules with affinity for nano-gold to improve the salt tolerance of DNA-AuNP, and then carry out the uploading of sulfhydryl DNA on nano-gold under high-salt conditions. In 1996, Mirkin's research group from Northwestern University first prepared DNA-AuNPs. They used the "aging-salt" preparation method to weaken the negatively charged DNA and citrate-coated DNA by continuously increasing the NaCl concentration of the buffer solution. Electrostatic repulsion between gold nanoparticles to achieve high-density assembly of DNA on gold nanoparticles (Nature 1996, 382(6592), 607-609). Due to the poor stability of citrate-coated gold nanoparticles, aggregation often occurs during salt addition, and the speed of salt addition needs to be carefully controlled. In addition, the stability of DNA-AuNP is much higher than that of citrate-coated gold nanoparticles, but it cannot withstand higher salt concentration, which limits its application in some occasions. In 1999, the Alivisatos research group at the University of California, Berkeley proposed the use of bis(p-sulfonylphenyl) phenylphosphine (BSPP) to replace the citrate group coated on the surface of gold nanoparticles, and then thiol-modified DNA at high salt concentration. The method of lower assembly greatly improves the stability of the DNA-AuNP preparation method and the tolerance to salt ions and different pH (Angew. Chem. Int. Ed. 1999, 38(12), 1808-1812). However, the DNA probe loading of DNA-AuNPs prepared by this method is much lower than that of the "aging-salting" method. Similarly, in 2009 and 2012, the use of nonionic fluorosurfactants (Anal.Chem.2009, 81(20), 8523-8528), adenine triphosphate deoxynucleotide (dATP) (Bioconjugate Chem. 2009, 20(6), 1218-1222) and high molecular weight polyethylene glycol (PEG) (J.Am.Chem.Soc.2012,134(24), 9910-9913) to replace the coating on nanometers, respectively The method of citrate on the gold surface also significantly improved the tolerance of gold nanoparticles to salt ions, and the loading capacity of DNA probes also reached a level comparable to that of the "aging-salt" method. Also, the classic "aging-salt" method takes 1-2 days. In 2009, the use of nonionic fluorosurfactants or dATP to replace citrate coated on the surface of gold nanoparticles, respectively, shortened the preparation time to several hours. In 2012, a simple method based on pH regulation reported by Juewen Liu's group at the University of Waterloo, Canada, effectively weakened the electrostatic repulsion between DNA and gold nanoparticles by reducing the pH of the buffer solution to 3, and achieved thiol modification in only 3 minutes of incubation. Rapid quantitative coating of DNA on gold nanoparticles (J. Am. Chem. Soc. 2012, 134(17), 7266-7269).

但是上述方法分别存在可能影响下游应用(比如细胞毒性)、DNA探针上载量误差大、大分子PEG的空间位阻可能影响DNA探针分子识别的问题。而且应用最广泛的“老化-加盐”方法和最近报道的低pH值条件下快速制备DNA-AuNP的方法不适用于能够在分子内和分子间形成稳定二级结构的DNA的上载。比如在生物传感技术中备受青睐的分子信标。分子信标具有分子内部分互补的结构特点，在按照“老化-加盐”方法制备DNA-AuNP时经常遇到纳米金发生聚集的现象。而低pH值条件下快速制备DNA-AuNP的方法由于低pH条件下氢键不能形成，仅适合线性DNA在纳米金上的上载。2009年樊春海课题组利用与短链辅助探针混合包被的方法实现了对分子信标-AuNP复合物的成功制备，但是DNA探针的上载量比较低(Angew.Chem.Int.Ed.2009,48(46),8670-8674)。因此非常需要寻找一种可以快速、稳定、高效、定量和不干扰下游应用的制备方法来解决具有二级结构的DNA在纳米金表面的上载问题。However, the above methods have problems that may affect downstream applications (such as cytotoxicity), large errors in DNA probe loading, and steric hindrance of macromolecular PEG may affect DNA probe molecular recognition. Moreover, the most widely used "aging-salting" method and the recently reported method for rapid preparation of DNA-AuNPs under low pH conditions are not suitable for the uploading of DNA that can form stable secondary structures within and between molecules. For example, molecular beacons are favored in biosensing technology. Molecular beacons have the structural characteristics of partial complementation within the molecule, and the aggregation of gold nanoparticles is often encountered when DNA-AuNPs are prepared according to the "aging-salting" method. However, the method for rapid preparation of DNA-AuNPs under low pH conditions is only suitable for the uploading of linear DNA on gold nanoparticles because hydrogen bonds cannot be formed under low pH conditions. In 2009, Fan Chunhai's research group achieved the successful preparation of molecular beacon-AuNP complexes by mixed coating with short-chain auxiliary probes, but the uploading capacity of DNA probes was relatively low (Angew.Chem.Int.Ed. 2009, 48(46), 8670-8674). Therefore, it is very necessary to find a preparation method that can be fast, stable, efficient, quantitative and does not interfere with downstream applications to solve the problem of uploading DNA with secondary structure on the surface of gold nanoparticles.

发明内容SUMMARY OF THE INVENTION

本发明目的是提供一种能一步快速制备DNA-AuNP的新方法，该方法可以同时适用于线性DNA和具有二级结构的DNA，比如分子信标。该方法将要进行上载的DNA在5'或者3'的末端修饰巯基，与巯基相连位置修饰上具有不同长度的寡乙二醇间隔链(OEG)，然后，不需要长时间的老化过程，直接在所需要的盐浓度下将该DNA加入柠檬酸根包被的纳米金进行孵育，通过调整DNA与纳米金的计量比调控AuNP上DNA的上载量。The purpose of the present invention is to provide a new method for rapidly preparing DNA-AuNP in one step, which can be applied to both linear DNA and DNA with secondary structure, such as molecular beacons. In this method, sulfhydryl groups are modified at the 5' or 3' end of the DNA to be uploaded, and oligoethylene glycol spacers (OEG) with different lengths are modified at the positions connected to the sulfhydryl groups. The DNA was added to the gold nanoparticles coated with citrate under the required salt concentration for incubation, and the amount of DNA loaded on the AuNP was regulated by adjusting the ratio of DNA to gold nanoparticles.

本发明提供一种快速制备巯基修饰DNA纳米金复合物(DNA-AuNP)的方法，该方法可以同时适用于线性DNA和具有二级结构的DNA，该方法包括如下步骤：步骤一：将要进行上载的DNA在5'或者3'的末端修饰巯基，与巯基相连位置修饰上具有不同长度的寡乙二醇间隔链(OEG)；步骤二：不需要老化过程，直接将该DNA在所需要的盐浓度下与柠檬酸根包被的纳米金进行孵育；步骤三：通过调整DNA与纳米金的计量比调控AuNP上DNA的上载量。The present invention provides a method for rapidly preparing thiol-modified DNA nano-gold complex (DNA-AuNP), which can be applied to both linear DNA and DNA with secondary structure. The method includes the following steps: Step 1: uploading to be performed The DNA is modified with sulfhydryl groups at the 5' or 3' end, and oligoethylene glycol spacers (OEG) with different lengths are modified at the positions connected to the sulfhydryl groups; Step 2: No aging process is required, the DNA is directly in the required salt Incubate with citrate-coated gold nanoparticles at a concentration; step 3: adjust the amount of DNA loaded on AuNPs by adjusting the ratio of DNA to gold nanoparticles.

上述方法的特征在于，该方法适用于具有二级结构的DNA的一步直接组装。The above-mentioned method is characterized in that the method is suitable for one-step direct assembly of DNA with secondary structure.

上述方法的特征在于，上述具有二级结构的DNA为分子信标。The above-mentioned method is characterized in that the above-mentioned DNA with secondary structure is a molecular beacon.

上述方法的特征在于，步骤一如下：将要进行上载的DNA在5'或者3'的末端修饰巯基，与巯基相连位置修饰上具有不同长度的寡乙二醇间隔链(OEG)。The above method is characterized in that the first step is as follows: the DNA to be uploaded is modified with a sulfhydryl group at the 5' or 3' end, and an oligoethylene glycol spacer (OEG) with different lengths is modified at the position connected to the sulfhydryl group.

上述方法的特征在于，寡乙二醇间隔链(OEG)可以含有6,12或18个乙二醇单元。The above method is characterized in that the oligoethylene glycol spacer (OEG) may contain 6, 12 or 18 ethylene glycol units.

上述方法的特征在于，步骤二如下：不需要老化过程，纯化后的DNA与纳米金(AuNP)按照一定物质的量比例直接混合于含有所需盐浓度的磷酸盐缓冲液(10mM PB，pH7.4)中，室温(25℃)下孵育一段时间(根据需要2分钟到2小时)。Aforesaid method is characterized in that,step 2 is as follows: without aging process, purified DNA and nano gold (AuNP) are directly mixed in the phosphate buffer (10mM PB, pH7. 4), incubate at room temperature (25°C) for a period of time (2 minutes to 2 hours as needed).

上述方法的特征在于，所需要的盐浓度为NaCl浓度在0至300mM。The above method is characterized in that the required salt concentration is a NaCl concentration of 0 to 300 mM.

上述方法的特征在于，该方法适用于具有不同巯基修饰位置(5'或者3'端)、长度和各种标记的DNA和大尺寸纳米金(50和100nm)。The above method is characterized in that it is applicable to DNA and large-sized gold nanoparticles (50 and 100 nm) with different thiol modified positions (5' or 3' end), length and various labels.

上述方法的特征在于，该方法在一定的盐浓度条件下可以实现DNA的定量上载。The above-mentioned method is characterized in that the method can realize quantitative uploading of DNA under the condition of a certain salt concentration.

上述方法的特征在于，该方法可以在中性pH值的缓冲溶液中实现DNA的快速上载。The above method is characterized in that the method can achieve rapid DNA uploading in a buffer solution with neutral pH value.

上述方法的特征在于，该方法不需要表面活性剂或其它试剂来预先包被纳米金。The above method is characterized in that the method does not require surfactants or other reagents to pre-coat gold nanoparticles.

上述方法的特征在于，该方法可以利用调节OEG的长度、孵育时间、盐浓度和DNA与纳米金的摩尔比来获得不同的DNA上载量。The above method is characterized in that the method can obtain different DNA loading amounts by adjusting the length of OEG, incubation time, salt concentration and molar ratio of DNA to gold nanoparticles.

该方法具有如下优势：1)OEG为电中性基团，在DNA在纳米金表面上载过程中，可以有效屏蔽携带负电荷的纳米金与同样携带负电荷的DNA之间的排斥作用，从而极大加快上载速率；将传统“老化-加盐”法1-2天的组装时间缩减至2分钟以内；2)具有OEG间隔链的DNA的快速上载过程可以在具有较高盐浓度的中性缓冲溶液中进行，该条件是二级结构形成所需要的，这使得该方法适用于具有二级结构的DNA在纳米金表面上的一步直接组装；3)具有OEG间隔链的DNA在相同组装条件下，在纳米金表面的上载量远高于只有巯基修饰的DNA；4)在投入比例小于150/1时，具有OEG间隔链的DNA可以定量上载到纳米金上，上载结束时溶液中游离态DNA残余量小于10％，极大降低了DNA消耗，而且无需再进行表面上DNA上载量的定量。5)该方法适用于不同尺寸的纳米金，特别是稳定性相对较差的大尺寸的纳米金。该发明为纳米金-巯基修饰DNA复合物的可控制备提供一种显著优于现有技术的新方法。This method has the following advantages: 1) OEG is an electrically neutral group, which can effectively shield the repulsion between the negatively charged nanogold and the same negatively charged DNA during the loading process of DNA on the surface of gold nanoparticles. Greatly accelerates the upload rate; reduces the 1-2 day assembly time of the traditional "aging-salt" method to less than 2 minutes; 2) The rapid upload process of DNA with OEG spacer strands can be performed in neutral buffers with higher salt concentrations carried out in solution, the conditions required for secondary structure formation, which makes this method suitable for one-step direct assembly of DNA with secondary structure on the surface of gold nanoparticles; 3) DNA with OEG spacer strands under the same assembly conditions , the loading amount on the surface of gold nanoparticles is much higher than that of DNA modified only by thiol groups; 4) When the input ratio is less than 150/1, DNA with OEG spacer chains can be quantitatively uploaded to gold nanoparticles, and free DNA remains in the solution at the end of uploading. The amount is less than 10%, the DNA consumption is greatly reduced, and the quantification of the DNA load on the surface is no longer necessary. 5) The method is applicable to gold nanoparticles of different sizes, especially large-sized gold nanoparticles with relatively poor stability. The invention provides a new method significantly superior to the prior art for the controllable preparation of nano-gold-thiol-modified DNA complexes.

本发明的具体实验步骤：Concrete experimental steps of the present invention:

(1)将要进行上载的DNA在5'或者3'的末端修饰巯基，与巯基相连位置修饰上具有不同长度的寡乙二醇间隔链(OEG)；OEG可以含有6,12或18个乙二醇单元；(1) The DNA to be uploaded is modified with a sulfhydryl group at the 5' or 3' end, and an oligoethylene glycol spacer (OEG) with different lengths is modified at the position connected to the sulfhydryl group; OEG can contain 6, 12 or 18 ethylene glycols alcohol unit;

(2)将上述具有OEG间隔链的巯基修饰的DNA进行还原和纯化；该DNA在含有二硫苏糖醇(DTT)和2％(V/V)的三乙胺(TEA)溶液中进行还原，然后利用NAP-5柱进行两次纯化；(2) Reduction and purification of the above-mentioned sulfhydryl-modified DNA with an OEG spacer chain; the DNA was reduced in a triethylamine (TEA) solution containing dithiothreitol (DTT) and 2% (V/V) , followed by two purifications using a NAP-5 column;

(3)不需要老化过程，纯化后的DNA与纳米金(AuNP)按照一定物质的量比例直接混合于含有所需盐浓度的磷酸盐缓冲液(10mM PB，pH 7.4)中，室温(25℃)下孵育一段时间(根据需要2分钟到2小时)；(3) No aging process is required, the purified DNA and gold nanoparticles (AuNP) are directly mixed in a phosphate buffer (10 mM PB, pH 7.4) containing the required salt concentration according to a certain amount of substances at room temperature (25°C). ) for a period of time (2 minutes to 2 hours as needed);

(4)测定DNA-AuNP的浓度：将(3)的混合物进行离心，离心管底部的红色物质重新分散在所需缓冲溶液中，并用紫外可见吸收测定进行纳米金浓度的定量；(4) Determination of the concentration of DNA-AuNP: centrifuge the mixture of (3), the red substance at the bottom of the centrifuge tube is re-dispersed in the required buffer solution, and the concentration of gold nanoparticles is quantified by UV-Vis absorption measurement;

(5)测定DNA在纳米金上的上载量N(条/AuNP)：完成包被过程的DNA-AuNP经过离心，取上清，利用紫外测定其浓度，根据包被时投入的DNA、纳米金和离心后上清的浓度和体积计算包被量。相对应公式为：

(5) Determination of DNA loading amount N on gold nanoparticles (bar/AuNP): The DNA-AuNP after the coating process was centrifuged, the supernatant was taken, and its concentration was measured by ultraviolet light. The amount of coating was calculated from the concentration and volume of the supernatant after centrifugation. The corresponding formula is:

附图说明Description of drawings

图1是本发明与现有技术的方法、效果及原理比较图，其中图1A是现有技术中“老化-加盐”法的方法、效果和不足；图1B是现有技术中“使用酸性缓冲溶液法”的方法、效果和不足；图1C是本发明的方法及其效果；图1D是上述三种方法的原理比较。Fig. 1 is the method, effect and principle comparison diagram of the present invention and the prior art, wherein Fig. 1A is the method, effect and deficiency of the "aging-salting" method in the prior art; Fig. 1B is the prior art "use acid The method, effect and deficiency of "buffer solution method"; Fig. 1C is the method of the present invention and its effect; Fig. 1D is the principle comparison of the above three methods.

图2是本发明中，利用动态光散射实时测定具有相同长度和不同组成的间隔链的巯基修饰DNA按照“老化-加盐”法进行DNA-AuNP制备时的DNA-AuNP的粒径变化图。“老化-加盐”法包括以下三步：老化，加盐，温育。老化过程即开始包被的前18个小时，该过程不另外添加盐；加盐过程即老化过程完成后，逐渐向13nm纳米金与DNA的混合溶液中滴加浓度为1M氯化钠(NaCl)的磷酸盐缓冲溶液(pH 7.4)使得盐浓度缓慢增至0.3M；温育过程即加盐过程完成之后继续孵育一段时间使DNA更加完全地包被于纳米金表面。以上三步均选取数个时间点测定DNA-AuNP粒径。15-A₁₀-SH，15-T₁₀-SH，15-EG₁₈-SH的序列见表1。各DNA与纳米金的摩尔比均为500:1。2 is a graph of the particle size change of DNA-AuNPs when DNA-AuNPs are prepared by the "aging-salting" method using dynamic light scattering to measure thiol-modified DNAs with spacer chains of the same length and different compositions in real time in the present invention. The "aging-salting" method includes the following three steps: aging, salting, and incubation. The aging process is the first 18 hours of coating, and no additional salt is added in this process; the salting process, that is, after the aging process is completed, gradually add 1M sodium chloride (NaCl) dropwise to the mixed solution of 13nm nano-gold and DNA. The phosphate buffer solution (pH 7.4) of phosphate buffer solution (pH 7.4) slowly increased the salt concentration to 0.3M; the incubation process, that is, after the completion of the salt addition process, continued to incubate for a period of time so that the DNA was more completely coated on the surface of the gold nanoparticles. In the above three steps, several time points were selected to determine the particle size of DNA-AuNPs. The sequences of 15-A₁₀ -SH, 15-T₁₀ -SH, 15-EG₁₈ -SH are shown in Table 1. The molar ratio of each DNA to gold nanoparticles was 500:1.

图3示出本发明中，不同长度OEG作为间隔链的巯基修饰DNA与13nm纳米金按照“老化-加盐”法进行DNA-AuNP制备时的DNA-AuNP的粒径变化图(图3中A)；在不同盐浓度下直接将具有不同长度OEG作为间隔链的巯基修饰DNA与13nm纳米金进行2小时孵育过程中DNA-AuNP的粒径变化图(图3中B,C,D)；具有不同长度OEG作为间隔链的巯基修饰DNA与13nm纳米金分别在含有和不含有0.3M NaCl的10mM磷酸盐缓冲溶液(pH7.4)中孵育2小时后的上载量(图3中E)；具有不同间隔链的巯基修饰DNA与13nm纳米金在含有0.3M NaCl的10mM磷酸盐缓冲溶液(pH7.4)中孵育2小时后的照片(图3中F)；15-EG₁₈-SH与13nm纳米金在含有0.3M NaCl的10mM磷酸盐缓冲溶液(pH7.4)中孵育2小时前后的紫外可见光谱(图3中G)。DNA与纳米金的摩尔比为500:1。15-EG₆-SH，15-EG₁₂-SH,15-EG₁₈-SH，15-A₁₀-SH,15-T₁₀-SH的序列见表1。Figure 3 shows the change in particle size of DNA-AuNPs when DNA-AuNPs are prepared by the "aging-salting" method when sulfhydryl-modified DNAs with different lengths of OEG are used as spacer chains and 13nm gold nanoparticles (A in Figure 3 ). ); particle size changes of DNA-AuNPs during 2-hour incubation of sulfhydryl-modified DNA with different lengths of OEG as spacer chains with 13 nm gold nanoparticles under different salt concentrations (B, C, D in Figure 3); with The loading of thiol-modified DNA with different lengths of OEG as spacer chains and 13 nm gold nanoparticles in 10 mM phosphate buffer solution (pH 7.4) with and without 0.3 M NaCl, respectively, after incubation for 2 hours (Fig. 3, E); with Photographs of sulfhydryl-modified DNA with different spacer strands incubated with 13 nm gold nanoparticles in 10 mM phosphate buffer solution (pH 7.4) containing 0.3 M NaCl for 2 h (F in Figure 3); 15-EG₁₈ -SH with 13 nm nanometers UV-Vis spectra of gold before and after incubation in 10 mM phosphate buffer (pH 7.4) containing 0.3 M NaCl for 2 hours (Figure 3, G). The molar ratio of DNA to gold nanoparticles is 500:1. The sequences of 15-EG₆ -SH, 15-EG₁₂ -SH, 15-EG₁₈ -SH, 15-A₁₀ -SH, 15-T₁₀ -SH are shown in the table 1.

图4示出本发明中，在不同物质的量的比例下，15-EG₁₈-SH与13nm纳米金老化孵育2小时过程中DNA-AuNP粒径随时间变化曲线(图4中A)及孵育2小时后的上载量(图4中B)。15-EG₁₈-SH与纳米金的摩尔比为100:1、200:1、300:1、400:1和500:1。孵育溶液为10mM磷酸盐缓冲溶液(pH7.4)。15-EG18-SH的序列见表1。Fig. 4 shows the variation curve of DNA-AuNP particle size with time in the process of 15-EG₁₈ -SH and 13 nm nano-gold aging and incubating for 2 hours under the ratio of the amount of different substances in the present invention (A in Fig. 4 ) and incubation The amount of upload after 2 hours (B in Figure 4). The molar ratios of 15-EG₁₈ -SH to gold nanoparticles were 100:1, 200:1, 300:1, 400:1 and 500:1. The incubation solution was 10 mM phosphate buffer (pH 7.4). The sequence of 15-EG18-SH is shown in Table 1.

图5示出本发明中，15-EG₁₈-SH直接与13nm纳米金在含有不同NaCl浓度的10mM磷酸盐(pH＝7.4)的缓冲溶液中孵育2小时之后的琼脂糖凝胶电泳和DNA的上载量。Figure 5 shows the agarose gel electrophoresis and DNA electrophoresis of 15-EG₁₈ -SH directly incubated with 13 nm gold nanoparticles in buffer solutions containing 10 mM phosphate (pH=7.4) with different NaCl concentrations for 2 hours in the present invention. upload volume.

图6示出本发明中，具有相同长度和不同组成的间隔链的巯基修饰和荧光标记的DNA(FAM-15-A₁₀-SH，FAM-15-T₁₀-SH，FAM-15-EG₁₈-SH，表1)在纳米金表面2小时老化过程中荧光强度随时间变化的曲线(图6中A，DNA与纳米金的摩尔比为75：1)及不同摩尔比例(DNA：AuNP)条件下，所上载的FAM-15-EG₁₈-SH占总投入量的比例(图6中B)。FAM-15-EG₁₈-SH与AuNP的摩尔比为25：1,50:1，75:1，100:1,125:1，150:1和200:1时的NaCl的浓度分别是10,20,30,40,60,80和100mM。Figure 6 shows the thiol-modified and fluorescently labeled DNAs (FAM-15-A₁₀ -SH, FAM-15-T₁₀ -SH, FAM-15-EG₁₈ of the present invention with spacer chains of the same length and different compositions) -SH, Table 1) The curve of the fluorescence intensity with time during the 2-hour aging of the gold nanoparticles surface (A in Figure 6, the molar ratio of DNA to gold nanoparticles is 75:1) and different molar ratio (DNA:AuNP) conditions Below, the ratio of the uploaded FAM-15-EG₁₈ -SH to the total input amount (B in Figure 6). The molar ratios of FAM-15-EG₁₈ -SH to AuNPs were 25:1, 50:1, 75:1, 100:1, 125:1, 150:1 and 200:1, and the NaCl concentrations were 10, 20, 30, 40, 60, 80 and 100mM.

图7,是本发明中，展示本发明的方法适用于具有不同巯基修饰位置(5'或者3'端)、长度的DNA和大尺寸纳米金(50nm)的照片和上载DNA前后纳米金粒径的变化，其中图7中A为5'端巯基修饰的DNA，B为30个碱基长度的巯基修饰的DNA，C为3'端巯基修饰的DNA，D为本发明应用于50nm纳米金。Fig. 7, in the present invention, shows that the method of the present invention is suitable for DNA with different thiol modified positions (5' or 3' end), lengths and large-sized gold nanoparticles (50nm) and the photos of the gold nanoparticles before and after uploading DNA In Figure 7, A is the sulfhydryl-modified DNA at the 5' end, B is the sulfhydryl-modified DNA with a length of 30 bases, C is the sulfhydryl-modified DNA at the 3' end, and D is the application of the present invention to 50nm gold nanoparticles.

图8,是本发明中，通过各步骤的粒径变化证实荧光标记分子信标(MB1-EG₁₈-SH，表1)在13nm纳米金上的成功组装。在10mM磷酸盐缓冲溶液中(1×PB)MB1-EG₁₈-SH以线性结构组装到纳米金上(○)，因此在15mM NaOH中孵育后粒径没有变化。在含有0.15M NaCl的10mM磷酸盐缓冲溶液中(0.15M NaCl，PBS)MB1-EG₁₈-SH以分子信标结构一步组装到纳米金上(■)，因此在15mM NaOH中孵育后粒径显著增加。线性结构DNA在纳米金表面上由于碱基吸附造成的非特异性吸附大于具有分子信标结构的DNA，因此洗涤后前者的粒径下降明显大于后者。Figure 8, in the present invention, confirmed the successful assembly of the fluorescently labeled molecular beacon (MB1-EG₁₈ -SH, Table 1) on 13 nm gold nanoparticles by the particle size change of each step. In 10 mM phosphate buffer (1 x PB) MB1-EG₁₈ -SH assembled on gold nanoparticles in a linear structure (○), so there was no change in particle size after incubation in 15 mM NaOH. MB1-EG₁₈ -SH was assembled to gold nanoparticles in one step with molecular beacon structure in 10 mM phosphate buffered saline (0.15 M NaCl, PBS) containing 0.15 M NaCl (■), thus resulting in significant particle size after incubation in 15 mM NaOH Increase. The non-specific adsorption of linear structure DNA on the surface of gold nanoparticles due to base adsorption is greater than that of DNA with molecular beacon structure, so the particle size reduction of the former is significantly greater than that of the latter after washing.

图9,是本发明中，证实荧光标记分子信标(FAM-MB2-EG₁₈-SH，表1)在13nm纳米金上的成功组装后具有很好的与互补DNA进行杂交的能力：图9中A是分子信标的杂交原理图；图9中B示出分子信标与互补DNA进行杂交前后粒径的变化；图9中C是荧光强度随着互补DNA浓度的增加而增加的荧光光谱图，和图9中D示出工作曲线。Figure 9, in the present invention, confirms that the fluorescently labeled molecular beacon (FAM-MB2-EG₁₈ -SH, Table 1) has a good ability to hybridize with complementary DNA after the successful assembly on 13nm gold nanoparticles: Figure 9 A is the hybridization schematic diagram of the molecular beacon; B in Fig. 9 shows the change of particle size before and after the hybridization of the molecular beacon and complementary DNA; C in Fig. 9 is the fluorescence spectrum graph showing that the fluorescence intensity increases with the increase of the complementary DNA concentration , and D in Figure 9 shows the working curve.

具体实施方式Detailed ways

图1是本发明与现有技术的方法、效果及原理比较图，其中图1A是现有技术中“老化-加盐”法的方法、效果和不足；图1B是现有技术中“使用酸性缓冲溶液法”的方法、效果和不足；上述现有技术已在背景技术中进行了描述，不再赘述。图1C是本发明的方法及其效果；图1D是上述三种方法的原理比较。如图1D所示，现有技术是通过引入阳离子来减小DNA和AuNP上的净电荷，静电排斥力因此减小，从而实现巯基修饰DNA在AuNP上的上载。而本发明的方法原理是：OEG由于其电中性和差的导电能力，可以有效屏蔽DNA与AuNP间的静电排斥力，从而实现巯基修饰DNA在AuNP上的快速定量上载。Fig. 1 is the method, effect and principle comparison diagram of the present invention and the prior art, wherein Fig. 1A is the method, effect and deficiency of the "aging-salting" method in the prior art; Fig. 1B is the prior art "use acid The methods, effects and deficiencies of the "buffer solution method"; the above-mentioned prior art has been described in the background art, and will not be repeated. FIG. 1C is the method of the present invention and its effect; FIG. 1D is the principle comparison of the above three methods. As shown in Figure 1D, in the prior art, the net charge on DNA and AuNPs is reduced by introducing cations, and thus the electrostatic repulsion is reduced, thereby realizing the uploading of thiol-modified DNA on AuNPs. The principle of the method of the present invention is that OEG can effectively shield the electrostatic repulsion between DNA and AuNP due to its electrical neutrality and poor conductivity, thereby realizing rapid quantitative uploading of thiol-modified DNA on AuNP.

表1：本发明中所用的DNA的序列信息。Table 1: Sequence information of DNA used in the present invention.

实施例1.利用动态光散射实时监测具有相同长度和不同组成的间隔链的巯基修饰DNA按照“老化-加盐”法进行DNA-AuNP制备时的DNA-AuNP的粒径变化。Example 1. Real-time monitoring of thiol-modified DNA with spacer chains of the same length and different compositions by dynamic light scattering. The particle size changes of DNA-AuNPs when DNA-AuNPs were prepared according to the "aging-salting" method.

将具有3’端巯基修饰的,具有相同长度和不同组成的间隔链的线性DNA(15-A₁₀-SH，15-T₁₀-SH，15-EG₁₈-SH，表1)分别与40mM二硫苏糖醇(DTT)和2％(V/V)的三乙胺(TEA)溶液混合，在室温下孵育30分钟，混合物利用NAP-5柱进行两次纯化，DNA洗脱在10mM的磷酸盐缓冲溶液(pH 7.4)中。将纯化后的DNA加入柠檬酸还原法制备的直径为13nm的纳米金，投入物质的量比例为500:1(DNA：AuNP)。按照“老化-加盐”法的老化，加盐，温育这三步进行DNA-AuNP的制备。老化过程为DNA加入纳米金后的18个小时，该过程不另外添加盐；加盐过程即老化过程完成后，逐渐向13nm纳米金与DNA的混合溶液中滴加浓度为1M氯化钠(NaCl)的磷酸盐缓冲溶液使得NaCl的终浓度缓慢增至0.3M；温育过程即加盐过程完成之后继续孵育使DNA更加完全地包被于纳米金表面。以上三步均选取数个时间点利用动态光散射测定DNA-AuNP的粒径(图2)。The linear DNAs (15-A₁₀ -SH, 15-T₁₀ -SH, 15-EG₁₈ -SH, Table 1) with the same length and different composition of spacer strands with 3'-terminal thiol groups were mixed with 40 mM bismuth, respectively. Thithreitol (DTT) and 2% (v/v) triethylamine (TEA) solution were mixed and incubated at room temperature for 30 minutes. The mixture was purified twice using a NAP-5 column, and the DNA was eluted in 10 mM phosphoric acid. in salt buffer solution (pH 7.4). The purified DNA was added to gold nanoparticles with a diameter of 13 nm prepared by the citric acid reduction method, and the ratio of the amount of the input substances was 500:1 (DNA:AuNP). The preparation of DNA-AuNP was carried out according to the three steps of aging, adding salt and incubation according to the "aging-salting" method. The aging process is 18 hours after DNA is added to the nano-gold, and no additional salt is added in this process; the salt-adding process, that is, after the aging process is completed, is gradually added to the mixed solution of 13nm nano-gold and DNA with a concentration of 1M sodium chloride (NaCl). ) of phosphate buffer solution to slowly increase the final concentration of NaCl to 0.3M; the incubation process, that is, after the salt addition process is completed, continue to incubate so that the DNA is more completely coated on the surface of the gold nanoparticles. In the above three steps, several time points were selected to determine the particle size of DNA-AuNPs by dynamic light scattering (Fig. 2).

由图2中动态光散射仪测定的DNA-AuNP粒径结果说明，具有相同长度和不同组成的间隔链的巯基修饰DNA在纳米金表面的包被过程是相似的，提高体系盐浓度有利于DNA在纳米金表面上上载量的提高。在老化阶段(0-18h)，15-EG₁₈-SH在13nm纳米金表面上的自组装速率远远大于15-A₁₀-SH和15-T₁₀-SH。仅2个小时的老化孵育之后，15-EG₁₈-SH—AuNP的粒径就达到了与15-A₁₀-SH—AuNP经28小时加盐孵育后相当的水平(26nm)。The particle size of DNA-AuNPs measured by dynamic light scattering in Figure 2 shows that the coating process of sulfhydryl-modified DNA with spacer chains of the same length and different compositions on the surface of gold nanoparticles is similar, and increasing the system salt concentration is beneficial to DNA Increased loading on nanogold surfaces. During the aging stage (0-18h), the self-assembly rate of 15-EG₁₈ -SH on the 13 nm nano-gold surface was much higher than that of 15-A₁₀ -SH and 15-T₁₀ -SH. After only 2 hours of aging incubation, the particle size of 15-EG₁₈ -SH-AuNPs reached a level (26 nm) comparable to that of 15-A₁₀ -SH-AuNPs after 28 hours of salt incubation.

实施例2.测定2小时老化过程中具有不同长度OEG间隔链的巯基修饰DNA在不同盐浓度下在纳米金上包被量及粒径。Example 2. Determination of the coating amount and particle size of sulfhydryl-modified DNA with different lengths of OEG spacer chains on gold nanoparticles under different salt concentrations during 2-hour aging.

按照实施例1中的方法还原和纯化DNA。本实施例中使用具有3’端巯基修饰的分别以15-EG₆-SH、15-EG₁₂-SH、15-EG₁₈-SH作为间隔链的DNA进行DNA-AuNP的制备。在含有不同盐浓度的磷酸盐缓冲溶液中进行DNA与AuNP的2小时孵育。在孵育过程中利用动态光散射测定DNA-AuNP粒径的变化。孵育结束后拍照，进行紫外可见光谱测试，然后进行15000rpm离心，取上清液100μL，根据紫外可见光谱定量测定其上清液中探针浓度并计算包被量。The DNA was reduced and purified as in Example 1. In this example, DNA-AuNPs were prepared by using DNAs modified with 3'-terminal thiols and using 15-EG₆ -SH, 15-EG₁₂ -SH, and 15-EG₁₈ -SH as spacer strands, respectively. A 2-hour incubation of DNA with AuNPs was performed in phosphate buffered solutions containing different salt concentrations. The change in particle size of DNA-AuNPs was measured by dynamic light scattering during incubation. After the incubation, take pictures, conduct UV-Vis spectroscopy test, then centrifuge at 15,000 rpm, take 100 μL of the supernatant, quantitatively determine the probe concentration in the supernatant according to UV-Vis spectroscopy, and calculate the coating amount.

结果说明，OEG作为间隔链能有效提高DNA在纳米金上的组装效率，仅用2小时的老化过程就完成了传统老化、加盐、孵育过程近80％的包被进程(图2A)。在不含NaCl的磷酸盐缓冲溶液中孵育，15-EG₁₈-SH经过2小时的包被过程每个纳米金颗粒上的包被量达129条15-EG₁₈-SH，这足以满足DNA-AuNP作为纳米传感器的上载量要求。而且即使是很短的OEG(6个乙二醇单元)也可以大幅提高巯基修饰DNA在纳米金上的上载速度，15-EG₆-SH和15-EG₁₂-SH经过2小时的包被过程每个纳米金颗粒上的包被量分别达99条和107条DNA。随着盐浓度的提高，DNA的上载速度和上载量均大幅提高(图3BCDE)。而且AuNP在高盐条件下孵育2小时后仍然保持红色，说明具有良好的稳定性。而作为比对的15-A₁₀-SH和15-T₁₀-SH在相同条件下AuNP发生聚集(图3中F)。另外15-EG₁₈-SH与13nm纳米金在含有0.3M NaCl的10mM磷酸盐缓冲溶液(pH7.4)中孵育2小时前后的紫外可见光谱(图3中G)也证实了AuNP的高稳定性。AuNP在520nm处的吸收峰的强度没有降低，只是最大吸收峰的位置发生红移，充分证实了大量的DNA组装在AuNP上。The results showed that OEG as a spacer chain can effectively improve the assembly efficiency of DNA on gold nanoparticles, and only 2 hours of aging process completed nearly 80% of the coating process of traditional aging, salt addition, and incubation (Figure 2A). Incubated in NaCl-free phosphate buffer solution, 15-EG₁₈ -SH coated 129 strips of 15-EG₁₈ -SH on each gold nanoparticle after a 2-hour coating process, which was sufficient for DNA- Loading requirements for AuNPs as nanosensors. And even a very short OEG (6 ethylene glycol units) can greatly improve the uploading speed of thiol-modified DNA on gold nanoparticles, 15-EG₆ -SH and 15-EG₁₂ -SH after 2 hours of coating process The amount of coating on each gold nanoparticle reached 99 and 107 DNAs, respectively. With the increase of salt concentration, the upload speed and the amount of DNA were greatly improved (Fig. 3BCDE). Moreover, AuNPs remained red after 2 hours of incubation under high salt conditions, indicating good stability. In contrast, 15-A₁₀ -SH and 15-T₁₀ -SH, which were compared, aggregated AuNPs under the same conditions (F in FIG. 3 ). In addition, the UV-Vis spectra (G in Figure 3) before and after 15-EG₁₈ -SH incubation with 13 nm gold nanoparticles in 10 mM phosphate buffer solution (pH 7.4) containing 0.3 M NaCl for 2 h also confirmed the high stability of AuNPs . The intensity of the absorption peak of AuNPs at 520 nm did not decrease, but the position of the maximum absorption peak shifted red, which fully confirmed that a large amount of DNA was assembled on AuNPs.

实施例3.测定在不同物质的量的比例下，15-EG₁₈-SH与13nm纳米金老化孵育2小时过程中DNA-AuNP粒径随时间变化曲线及孵育2小时后的上载量。Example 3. Determination of the change curve of DNA-AuNP particle size with time during 15-EG₁₈ -SH and 13 nm nano-gold aging and incubation for 2 hours under the ratio of the amount of different substances and the loading amount after incubation for 2 hours.

按照实施例1中的方法还原和纯化15-EG₁₈-SH。利用DLS检测投入不同物质的量比例的15-EG₁₈-SH与纳米金在2小时老化过程中粒径变化，其物质的量比例分别为100：1、200：1、300：1、400：1、500：1，选取时间为0,0.5,1,1.5,2h时的点进行粒径测量。在老化2h后对样品进行15000rpm离心，取上清液100μL，根据紫外可见光谱定量测定其上清液中探针浓度并计算包被量。15-EG₁₈ -SH was reduced and purified as in Example 1. The particle size changes of 15-EG₁₈ -SH and gold nanoparticles in different proportions of materials were detected by DLS during the 2-hour aging process, and the proportions of materials were 100:1, 200:1, 300:1, and 400:1, respectively. 1. 500:1, select the point when the time is 0, 0.5, 1, 1.5, 2h for particle size measurement. After aging for 2 hours, the samples were centrifuged at 15,000 rpm, and 100 μL of the supernatant was taken. The probe concentration in the supernatant was quantitatively determined according to UV-Vis spectroscopy, and the coating amount was calculated.

结果表明，随着投入物质的量比例不断提高，包被过程中复合物粒径变化越大，老化2h后的上载量不断增加(图3)。因此，可以方便地通过调控投入比例来控制最终的包被量。The results showed that with the increasing proportion of the amount of the input material, the larger the change in the particle size of the composite during the coating process, the larger the loading amount after 2 h of aging (Figure 3). Therefore, it is convenient to control the final coating amount by adjusting the input ratio.

实施例4.根据实施例3中得到的结果，选取投入比例400：1(15-EG₁₈-SH:AuNP)，进行不同盐浓度下的15-EG₁₈-SH组装，利用凝胶电泳验证包被过程的成功和紫外可见光谱定量测定包被量。Embodiment 4. According to the result obtained in embodiment 3, choose input ratio 400:1 (15-EG₁₈ -SH:AuNP), carry out 15-EG₁₈ -SH assembly under different salt concentrations, utilize gel electrophoresis to verify package The amount of coating was quantified by the success of the process and by UV-Vis spectroscopy.

按照实施例1中的方法还原和纯化15-EG₁₈-SH，将15-EG₁₈-SH分别配置在具有不同NaCl浓度(0mM,30mM,60mM,90mM,120mM,150mM,300mM)的磷酸盐缓冲溶液中，直接将上述溶液加入纳米金中。2小时老化过程结束后，13000rpm离心，弃去上清液，紫外可见光谱定量测量上清液探针浓度，计算上载量。另取6μL沉淀物加入4μL甘油进行2％的琼脂糖凝胶电泳。15-EG₁₈ -SH was reduced and purified according to the method in Example 1, and 15-EG₁₈ -SH was prepared in phosphate buffer with different NaCl concentrations (0mM, 30mM, 60mM, 90mM, 120mM, 150mM, 300mM), respectively In the solution, the above solution is directly added to the gold nanoparticles. After the 2-hour aging process, centrifuge at 13,000 rpm, discard the supernatant, quantitatively measure the probe concentration of the supernatant by UV-Vis spectroscopy, and calculate the uploading amount. Another 6 μL of the precipitate was added to 4 μL of glycerol for 2% agarose gel electrophoresis.

结果表明随着盐浓度的升高，15-EG₁₈-SH在纳米金表面的上载量进一步提高(图5)。由琼脂糖凝胶可以看出，15-EG₁₈-SH—AuNP在较高盐浓度下仍然保持稳定，纳米金保持红色。1号为纳米金，在电泳条件下发生聚集，颜色为蓝黑色。该实施例说明本发明的方法无需低盐老化过程，可以直接在高盐条件下进行DNA在纳米金的组装而不引起纳米金的聚集。The results showed that with the increase of salt concentration, the loading of 15-EG₁₈ -SH on the surface of gold nanoparticles was further increased (Fig. 5). It can be seen from the agarose gel that 15-EG₁₈ -SH-AuNP remains stable at higher salt concentration, and the gold nanoparticles remain red. No. 1 is gold nanoparticles, which aggregated under electrophoresis conditions, and the color was blue-black. This example shows that the method of the present invention does not require a low-salt aging process, and can directly perform the assembly of DNA in nano-gold under high-salt conditions without causing the aggregation of nano-gold.

实施例5.证实FAM-15-EG₁₈-SH可以快速定量吸附在纳米金表面。Example 5. It was confirmed that FAM-15-EG₁₈ -SH can be rapidly and quantitatively adsorbed on the surface of gold nanoparticles.

按照实施例1中的方法还原和纯化DNA。本实施例中，投入比例为DNA:AuNP＝75:1，在30mM 1×PBS缓冲体系下进行上载，利用荧光动力学监测上载过程(其中，荧光光谱仪激发波长为467nm狭缝宽度5nm、检测发射波长为520nm狭缝宽度10nm，扫描间隔为5分钟)。The DNA was reduced and purified as in Example 1. In this example, the input ratio is DNA:AuNP=75:1, uploading is carried out in a 30mM 1×PBS buffer system, and the uploading process is monitored by fluorescence kinetics (wherein, the excitation wavelength of the fluorescence spectrometer is 467nm, the slit width is 5nm, the detection emission is The wavelength is 520 nm, the slit width is 10 nm, and the scanning interval is 5 minutes).

根据荧光共振能量转移(FRET)现象，纳米金由于存在极强的等离子共振吸收(SPR)，为强荧光猝灭基团，荧光标记DNA组装到纳米金表面后，其荧光基团被纳米金猝灭，导致体系荧光信号下降。上载结束后，根据体系残余荧光量设计并测定DNA浓度与荧光量标准曲线。根据标准曲线计算体系中未参与上载游离DNA比例。According to the phenomenon of fluorescence resonance energy transfer (FRET), gold nanoparticles are strong fluorescence quenching groups due to the extremely strong plasmon resonance absorption (SPR). off, resulting in a decrease in the fluorescence signal of the system. After uploading, a standard curve of DNA concentration and fluorescence was designed and determined according to the residual fluorescence of the system. Calculate the proportion of cell-free DNA not involved in uploading in the system according to the standard curve.

结果表明，DNA在投入量相同条件下，FAM-15-EG₁₈-SH荧光残余量明显低于FAM-15-A₁₀-SH和FAM-15-T₁₀-SH，且荧光下降并达到平衡的时间最短，几乎立即达到平衡(图6A)。根据标准曲线，FAM-15-EG₁₈-SH在上载过程结束后，溶液中残余的FAM-15-EG₁₈-SH仅为投入量的5％，在75/1的投入比例下，折合上载量高达72。同时由于FAM-15-EG₁₈-SH相比FAM-15-T₁₀-SH和FAM-15-A₁₀-SH在纳米金上的非特异性吸附更小，FAM-15-EG₁₈-SH更好的处于直立状态，FRET相对较弱，也会造成较强的背景信号，因此仅有5％的信号残余表明FAM-15-EG₁₈-SH近乎完全定量地上载到纳米金上。而相同组装条件下，溶液中残余的FAM-15-A₁₀-SH和FAM-15-T₁₀-SH为投入量的24％与12％。定量上载不仅可以免去繁琐的定量过程，也极大避免了DNA的浪费。本实施例进一步证明OEG间隔链可以在DNA-AuNP自组装过程中既提高组装速率又提高上载量。The results showed that under the same amount of DNA input, the residual fluorescence of FAM-15-EG₁₈ -SH was significantly lower than that of FAM-15-A₁₀ -SH and FAM-15-T₁₀ -SH, and the fluorescence decreased and reached equilibrium. The time was the shortest and equilibrium was reached almost immediately (Fig. 6A). According to the standard curve, after the uploading process of FAM-15-EG₁₈ -SH, the residual FAM-15-EG₁₈ -SH in the solution is only 5% of the input amount. Under the input ratio of 75/1, it is equivalent to the upper loading amount. Up to 72. At the same time, because FAM-15-EG₁₈ -SH has less non-specific adsorption on gold nanoparticles than FAM-15-T₁₀ -SH and FAM-15-A₁₀ -SH, FAM-15-EG₁₈ -SH is better In the upright state, FRET is relatively weak and also causes a strong background signal, so only 5% of the signal residual indicates that FAM-15-EG₁₈ -SH is loaded onto gold nanoparticles almost quantitatively. Under the same assembly conditions, the residual FAM-15-A₁₀ -SH and FAM-15-T₁₀ -SH in the solution were 24% and 12% of the input amount. Quantitative uploading not only eliminates the tedious quantitative process, but also greatly avoids the waste of DNA. This example further proves that the OEG spacer chain can improve both the assembly rate and the loading capacity during DNA-AuNP self-assembly.

另外我们测试了不同摩尔比例(DNA：AuNP)条件下，所上载的FAM-15-EG₁₈-SH占总投入量的比例(图6中B)。FAM-15-EG₁₈-SH与AuNP的摩尔比为25：1,50:1，75:1，100:1,125:1，150:1和200:1时的NaCl的浓度分别是10,20,30,40,60,80和100mM。在上述条件下，当FAM-15-EG₁₈-SH与AuNP的摩尔比低于150:1时，大于90％的DNA都吸附在AuNP上，实现了定量上载。In addition, we tested the ratio of the uploaded FAM-15-EG₁₈ -SH to the total input amount under different molar ratios (DNA: AuNP) (B in Figure 6 ). The molar ratios of FAM-15-EG₁₈ -SH to AuNPs were 25:1, 50:1, 75:1, 100:1, 125:1, 150:1 and 200:1, and the NaCl concentrations were 10, 20, 30, 40, 60, 80 and 100mM. Under the above conditions, when the molar ratio of FAM-15-EG₁₈ -SH to AuNP was lower than 150:1, more than 90% of the DNA was adsorbed on AuNP, realizing quantitative uploading.

实施例6.本发明的方法适用于具有不同巯基修饰位置(5'或者3'端)、长度的DNA和大尺寸纳米金(50nm)。Example 6. The method of the present invention is applicable to DNA with different sulfhydryl modification positions (5' or 3' end) and length and large-sized gold nanoparticles (50 nm).

按照实施例1中的方法还原和纯化DNA。根据实施例2中2小时老化孵育的条件，对探针15-EG₁₈-SH、HS-EG₁₈-15、30-EG₁₈-SH(表1)分别进行在13nm及50nm纳米金上进行上载，利用动态光散射仪测定上载前后复合物粒径变化。并将未修饰的纳米金和巯基修饰DNA包被后纳米金分别加入0.2M NaCl(终浓度)后立即拍照。The DNA was reduced and purified as in Example 1. According to the conditions of 2-hour aging incubation in Example 2, the probes 15-EG₁₈ -SH, HS-EG₁₈ -15, 30-EG₁₈ -SH (Table 1) were uploaded on 13 nm and 50 nm gold nanoparticles, respectively , and the particle size changes of the complexes before and after uploading were measured by dynamic light scattering. The unmodified gold nanoparticles and the thiol-modified DNA were coated with 0.2M NaCl (final concentration), respectively, and the photos were taken immediately.

结果表明，利用OEG作为间隔链的巯基修饰DNA进行快速DNA-AuNP制备的方法具有良好的通用性，本发明的方法适用于具有不同巯基修饰位置(5'或者3'端)、长度的DNA和大尺寸纳米金(50nm)(图7)，其中图7中A为5'端巯基修饰的DNA，B为30个碱基长度的巯基修饰的DNA，C为3'端巯基修饰的DNA，D为本发明应用于50nm纳米金。上载过程速度快、稳定性高，复合物粒径分布均一，抗盐能力强。0.2M NaCl中的未修饰的纳米金立即发生聚集，颜色变为灰色，而巯基修饰DNA包被后的纳米金保持红色，也证实了DNA成功地包被到了纳米金上。The results show that the method for rapid DNA-AuNP preparation using OEG as spacer chain sulfhydryl modified DNA has good versatility, and the method of the present invention is suitable for DNA and Large-sized gold nanoparticles (50nm) (Fig. 7), wherein A in Fig. 7 is sulfhydryl-modified DNA at the 5' end, B is sulfhydryl-modified DNA with a length of 30 bases, C is the 3'-end sulfhydryl-modified DNA, D The invention is applied to 50nm nano-gold. The uploading process is fast and stable, the composite particle size distribution is uniform, and the salt resistance is strong. The unmodified gold nanoparticles in 0.2M NaCl immediately aggregated and turned gray, while the gold nanoparticles coated with thiol-modified DNA remained red, which also confirmed that the DNA was successfully coated on the gold nanoparticles.

实施例7.证实荧光标记分子信标(MB1-EG₁₈-SH，表1)在13nm纳米金上的成功组装。Example 7. Demonstration of successful assembly of fluorescently labeled molecular beacons (MB1-EG₁₈ -SH, Table 1 ) on 13 nm gold nanoparticles.

分别在磷酸盐缓冲溶液(10mmol PB pH＝7.4)和含有NaCl浓度为150mmol/L的磷酸盐缓冲溶液(10mmol PB pH＝7.4)中通过2h的孵育进行MB1-EG₁₈-SH—AuNP的制备，投入比例为500:1(MB1-EG₁₈-SH:AuNP)。利用动态光散射仪监测老化开始和结束时的复合物粒径。老化过程结束后，13000rpm离心，沉淀物以相应的缓冲溶液洗涤三次以除去游离态的DNA，再次监测粒径变化。洗涤干净后，13000rpm离心，弃去上清，在沉淀物中加入等体积的15mM NaOH溶液。利用动态光散射仪监测DNA-AuNP粒径变化。The preparation of MB1-EG₁₈ -SH-AuNP was carried out in phosphate buffer solution (10mmol PB pH=7.4) and phosphate buffer solution (10mmol PB pH=7.4) containing NaCl concentration of 150mmol/L by 2h incubation, respectively. The input ratio was 500:1 (MB1-EG₁₈ -SH:AuNP). The particle size of the complexes at the beginning and end of aging was monitored using dynamic light scattering. After the aging process, centrifuge at 13,000 rpm, wash the precipitate three times with the corresponding buffer solution to remove free DNA, and monitor the particle size change again. After washing, centrifuge at 13,000 rpm, discard the supernatant, and add an equal volume of 15 mM NaOH solution to the pellet. The particle size changes of DNA-AuNPs were monitored by dynamic light scattering.

结果表明，较高盐浓度下，复合物粒径与其他实施例相比无显著性差异，证明分子信标成功快速链接在纳米金表面，且未发生聚集现象。在10mM磷酸盐缓冲溶液中MB1-EG₁₈-SH以线性结构组装到纳米金上(○)，因此在15mM NaOH中孵育后粒径没有变化。在含有0.15M NaCl的10mM磷酸盐缓冲溶液中(0.15M NaCl，PBS)MB1-EG₁₈-SH以分子信标结构一步组装到纳米金上(■)，因此在15mM NaOH中孵育后粒径显著增加。线性结构DNA在纳米金表面上由于碱基吸附造成的非特异性吸附大于具有分子信标结构的DNA，因此洗涤后前者的粒径下降明显大于后者。该结果充分证明了MB1-EG₁₈-SH在高盐度条件下能快速的以分子信标结构直接链接在纳米金表面，而具有相同碱基序列的MB1-T₁₀-SH在传统的“老化-加盐”过程中发生聚集。该实施例充分证明了OEG作为间隔链的优异性。The results show that under higher salt concentration, the particle size of the composite has no significant difference compared with other examples, which proves that the molecular beacon is successfully and quickly linked to the surface of gold nanoparticles without aggregation. MB1-EG₁₈ -SH assembled on gold nanoparticles in a linear structure in 10 mM phosphate buffer (○), so there was no change in particle size after incubation in 15 mM NaOH. MB1-EG₁₈ -SH was assembled to gold nanoparticles in one step with molecular beacon structure in 10 mM phosphate buffered saline (0.15 M NaCl, PBS) containing 0.15 M NaCl (■), thus resulting in significant particle size after incubation in 15 mM NaOH Increase. The non-specific adsorption of linear structure DNA on the surface of gold nanoparticles due to base adsorption is greater than that of DNA with molecular beacon structure, so the particle size reduction of the former is significantly greater than that of the latter after washing. This result fully proves that MB1-EG₁₈ -SH can quickly link to the surface of gold nanoparticles with molecular beacon structure under high salinity conditions, while MB1-T₁₀ -SH with the same base sequence can be directly linked to the surface of gold nanoparticles under high salinity - Aggregation occurs during the addition of salt. This example fully demonstrates the superiority of OEG as a spacer chain.

实施例8.证实荧光标记分子信标(FAM-MB2-EG₁₈-SH，表1)在13nm纳米金上的成功组装后具有很好的与互补DNA进行杂交的能力。Example 8. Confirmation that the fluorescently labeled molecular beacon (FAM-MB2-EG₁₈ -SH, Table 1 ) has a good ability to hybridize with complementary DNA after successful assembly on 13 nm gold nanoparticles.

根据实施例7中实验方法进行巯基DNA在纳米金表面的自组装及洗涤。洗涤结束后，取一定量复合物与系列浓度梯度的DNA靶标(FAM-MB2Target，表1)混合，进行杂交孵育，孵育结束后，对各组复合物的荧光强度进行定量测定。其中，每一组总体积110μL，复合物体积为50μL，加入不同浓度的DNA靶标使体系靶标终浓度为1000nM、500nM、250nM、100nM、50nM、10nM、5nM和0nM，利用洗涤缓冲溶液定容至110μL。荧光测定条件同实施例5。According to the experimental method in Example 7, the self-assembly and washing of sulfhydryl DNA on the surface of gold nanoparticles were carried out. After washing, a certain amount of complexes was mixed with a series of concentration gradient DNA targets (FAM-MB2Target, Table 1) for hybridization incubation. After incubation, the fluorescence intensity of each group of complexes was quantitatively determined. Among them, the total volume of each group was 110 μL, and the volume of the complex was 50 μL. Different concentrations of DNA targets were added to make the final concentrations of the system targets 1000nM, 500nM, 250nM, 100nM, 50nM, 10nM, 5nM and 0nM, and the washing buffer solution was used to make up the volume to 110 μL. The fluorescence measurement conditions were the same as those in Example 5.

结果表明，通过本发明方法包被的FAM-MB2-EG₁₈-SH—AuNP具有良好的生物分子识别功能(图9)。通过本方法制备具有分子信标结构的DNA-AuNP，组装过程简单、快速、高效、稳定，复合物的分子识别性能优越。The results show that the FAM-MB2-EG₁₈ -SH-AuNP coated by the method of the present invention has a good biomolecular recognition function ( FIG. 9 ). The DNA-AuNP with molecular beacon structure is prepared by the method, the assembly process is simple, fast, efficient and stable, and the molecular recognition performance of the complex is excellent.

Claims (6)

1. A method for rapidly preparing a sulfhydryl modified DNA nanogold complex (DNA-AuNP), which can be simultaneously applied to linear DNA and DNA with a secondary structure, comprises the following steps: the method comprises the following steps: modifying a thiol group at the 5 'or 3' end of the DNA to be uploaded, and modifying an oligoethylene glycol spacer chain (OEG) with different lengths at the position connected with the thiol group, wherein the oligoethylene glycol spacer chain (OEG) with different lengths is 18 ethylene glycol units; step two: the DNA is directly incubated with the nano-gold coated with the citrate in a 10mM phosphate buffer solution with 0-0.3M NaCl and pH7.4 without an aging process, wherein the nano-gold is 13nm nano-gold or 50nm nano-gold; step three: and regulating the loading amount of the DNA on the AuNP by adjusting the stoichiometric ratio of the DNA to the nanogold, wherein the molar ratio of the DNA to the AuNP is lower than 150: 1.

2. The method of claim 1, wherein the method is adapted for direct assembly of DNA having secondary structure in one step.

3. The method according to claim 2, wherein the DNA having a secondary structure is a molecular beacon.

4. The method of claim 1, wherein step two is as follows: the DNA was incubated directly with citrate coated nanogold in 0-0.3M NaCl pH7.4 in 10mM phosphate buffer for 2 minutes to 2 hours at 25 ℃ without the need for an aging process.

5. The method according to claim 1 or 2, wherein the method does not require a surfactant to pre-coat the nanogold.

6. The method of claim 1 or 2, wherein the method can be used to obtain different DNA loading by adjusting the length of the oligo-ethylene glycol spacer, the incubation time, the salt concentration and the molar ratio of DNA to nanogold.

Images