CN107435063B

CN107435063B - Method for rapidly preparing sulfhydryl modified DNA nano gold complex (DNA-AuNP)

Info

Publication number: CN107435063B
Application number: CN201610364787.9A
Authority: CN
Inventors: 娄新徽; 徐擎
Original assignee: Capital Normal University
Current assignee: Capital Normal University
Priority date: 2016-05-27
Filing date: 2016-05-27
Publication date: 2020-12-22
Anticipated expiration: 2036-05-27
Also published as: CN107435063A

Abstract

The invention relates to a method for rapidly preparing a sulfhydryl modified DNA nano gold compound (DNA-AuNP). The rapid preparation of DNA-AuNP was performed using thiol-modified DNA with oligo ethylene glycol spacer chains (OEG) of different lengths as spacer chains. The loading rate of DNA on the surface of the nano-gold can be obviously improved, and the loading rate is increased along with the increase of the salt concentration. The method is a general method for preparing DNA-AuNP, and is applicable to DNA with different sulfydryl modification positions (5 'or 3' ends), lengths and sequences. The method is not only suitable for uploading linear single-stranded DNA on the nanogold, but also suitable for uploading DNA probes with secondary structures on the surface of the nanogold, and the DNA probes with the secondary structures, such as molecular beacons, can be directly uploaded on the surface of the nanogold in one step. The method is suitable for the nano-gold with different sizes, particularly the large-size nano-gold with relatively poor stability.

Description

Method for rapidly preparing sulfhydryl modified DNA nano gold complex (DNA-AuNP)

Technical Field

The invention relates to a method for rapidly preparing a sulfhydryl-modified DNA nanogold complex (DNA-AuNP) by utilizing oligo-ethylene glycol spacer chain (OEG) modified sulfhydryl DNA, belonging to the technical field of biology.

Background

The sulfhydryl modified DNA nano-gold complex (DNA-AuNP) has very important application in the fields of nano self-assembly structure construction, biosensing technology, drug transportation and the like. Efficient molecular recognition of DNA at the nanogold interface is the basis for these applications. Extensive research shows that the number, density, conformation, composition of a protective layer, shape and size of a composite probe and other factors of DNA on the DNA-AuNP obviously influence the efficiency of molecular recognition. The technical problem of poor application repeatability based on the DNA-AuNP is solved, and the key is the preparation of the DNA-AuNP, and the quantity and conformation of the DNA on an AuNP interface must be accurately controlled.

However, existing methods for DNA-AuNP preparation do not allow accurate control of the amount and conformation of DNA at the AuNP interface even under idealized laboratory conditions. And the existing DNA-AuNP preparation technology has various problems and defects. The existing preparation method of DNA-AuNP mainly utilizes a surfactant or a small molecule with affinity to nanogold to improve the salt tolerance of the DNA-AuNP, and then carries out the uploading of sulfhydryl DNA on the nanogold under the condition of high salt. DNA-AuNP was first prepared by the Mirkin project group of the university of northwest in 1996, and the DNA-AuNP was prepared by an aging-salting preparation method, wherein the electrostatic repulsion between DNA and citrate-coated negatively-charged nanogold is weakened by continuously increasing the NaCl concentration of a buffer solution, so that high-density assembly of DNA on nanogold is realized (Nature 1996,382(6592), 607-609). Because the stability of the citrate coated nano gold is poor, aggregation often occurs in the process of adding salt, and the salt adding speed needs to be carefully controlled. In addition, the stability of the DNA-AuNP is improved by a little compared with the citrate coated nano gold, but the DNA-AuNP cannot bear higher salt concentration, so that the application of the DNA-AuNP in some occasions is limited. The Alivisatos project group at the university of California, 1999 proposed a method of replacing citrate coated on the surface of nanogold with bis (p-sulfonylphenyl) phenylphosphine (BSPP) and then assembling thiol-modified DNA at high salt concentration, which greatly improved 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-Amplifier 1812). However, the DNA probe loading of the DNA-AuNP prepared by this method is much lower than that of the "aging-salting-in" method. Similarly, in 2009 and 2012, it was reported that a method of replacing citrate coated on the surface of nanogold with a nonionic fluorosurfactant (anal. chem.2009,81(20), 8523-. In addition, the classical "aging-salting" method requires 1-2 days. In 2009, it was reported that the preparation time was shortened to several hours by replacing citrate coated on the surface of nanogold with a nonionic fluorosurfactant or dATP, respectively. The simple method based on pH regulation reported by Juewen Liu project group at the university of luugu, canada, 2012 effectively weakens the electrostatic repulsion between DNA and nanogold by lowering the pH of the buffer solution to 3, and only 3 minutes of incubation is needed to achieve rapid quantitative coating of thiol-modified DNA on nanogold (j.am.chem.soc.2012,134(17), 7266-.

However, the methods have the problems that downstream application (such as cytotoxicity) can be influenced, the loading error of the DNA probe is large, and the steric hindrance of macromolecular PEG can influence the molecular recognition of the DNA probe. Moreover, the most widely used "aging-salting" method and the recently reported method for rapidly preparing DNA-AuNPs under low pH conditions are not suitable for the loading of DNA capable of forming stable secondary structures both intramolecularly and intermolecularly. Such as molecular beacons that are favored in the context of biosensing technology. The molecular beacon has the structural characteristic of molecular internal partial complementation, and is often subjected to the phenomenon of aggregation of nanogold when the DNA-AuNP is prepared according to an aging-salting method. And the method for rapidly preparing the DNA-AuNP under the condition of low pH value is only suitable for uploading linear DNA on the nanogold because hydrogen bonds can not be formed under the condition of low pH value. In 2009, the chunhai project group achieved successful preparation of molecular beacon-AuNP complexes by a method of mixed coating with a short-chain helper probe, but the loading of DNA probes was relatively low (angelw. chem. int.ed.2009,48(46), 8670-. Therefore, it is highly desirable to find a preparation method that can be used for rapidly, stably, efficiently, quantitatively and without interfering with downstream applications to solve the problem of uploading DNA with secondary structure on the surface of nanogold.

Disclosure of Invention

The invention aims to provide a new method for rapidly preparing DNA-AuNP in one step, and the method can be simultaneously applied to linear DNA and DNA with a secondary structure, such as a molecular beacon. According to the method, a sulfydryl is modified at the end of 5 'or 3', the position connected with the sulfydryl is modified with oligoethylene glycol spacer chains (OEG) with different lengths, then, a long-time aging process is not needed, the DNA is directly added into the nano-gold coated with citrate under the required salt concentration for incubation, and the loading capacity of the DNA on the AuNP is regulated and controlled by adjusting the metering ratio of the DNA to the nano-gold.

The invention provides a method for rapidly preparing a sulfhydryl modified DNA nanogold complex (DNA-AuNP), which can be simultaneously suitable for linear DNA and DNA with a secondary structure, and comprises the following steps: the method comprises the following steps: modifying the DNA to be uploaded with sulfydryl at the 5 'or 3' end, and modifying oligo ethylene glycol spacer chains (OEG) with different lengths at the positions connected with the sulfydryl; step two: the DNA is directly incubated with the nano-gold coated with the citrate under the required salt concentration without an aging process; step three: the loading amount of the DNA on the AuNP is regulated and controlled by adjusting the stoichiometric ratio of the DNA to the nanogold.

The method is characterized in that the method is suitable for one-step direct assembly of DNA with secondary structure.

The method is characterized in that the DNA having a secondary structure is a molecular beacon.

The method is characterized in that the first step is as follows: the DNA to be loaded is modified at the 5 'or 3' end with a thiol group, to which a modified oligo-ethylene glycol spacer (OEG) of different length is attached.

The above method is characterized in that the oligoethylene glycol spacer (OEG) may contain 6,12 or 18 ethylene glycol units.

The method is characterized in that the second step is as follows: the purified DNA was directly mixed with nanogold (AuNP) in a quantitative ratio in a phosphate buffer (10mM PB, pH7.4) containing the desired salt concentration without aging, and incubated at room temperature (25 ℃) for a period of time (2 minutes to 2 hours as required).

The method is characterized in that the salt concentration is required to be 0 to 300mM NaCl.

The above method is characterized in that the method is applicable to DNA and large-sized nanogold (50 and 100nm) having different thiol modification positions (5 'or 3' end), lengths and various labels.

The method is characterized in that the method can realize quantitative DNA uploading under the condition of a certain salt concentration.

The method is characterized in that the method can realize rapid DNA loading in a buffer solution with a neutral pH value.

The method is characterized in that the method does not need a surfactant or other reagents to coat the nanogold in advance.

The method is characterized in that the method can obtain different DNA loading capacity by adjusting the length of OEG, the incubation time, the salt concentration and the molar ratio of DNA to nano-gold.

The method has the following advantages: 1) OEG is an electric neutral group, and can effectively shield the repulsion between the nanogold carrying negative charges and the DNA carrying negative charges in the process of loading the DNA on the surface of the nanogold, thereby greatly accelerating the loading rate; the assembly time of 1-2 days of the traditional aging-salting method is reduced to be within 2 minutes; 2) the rapid uploading process of DNA with OEG spacer strand can be performed in neutral buffer solution with higher salt concentration, which is required for secondary structure formation, which makes the method suitable for one-step direct assembly of DNA with secondary structure on the surface of nanogold; 3) under the same assembly condition, the loading amount of the DNA with the OEG spacer chain on the surface of the nanogold is far higher than that of the DNA modified by only sulfydryl; 4) when the input proportion is less than 150/1, the DNA with OEG spacer chain can be quantitatively loaded on the nanogold, the residual amount of free DNA in the solution at the end of loading is less than 10%, the DNA consumption is greatly reduced, and the quantitative determination of the DNA loading amount on the surface is not needed. 5) The method is suitable for the nano-gold with different sizes, particularly the large-size nano-gold with relatively poor stability. The invention provides a novel method which is obviously superior to the prior art for the controllable preparation of the nano gold-sulfhydryl modified DNA compound.

The specific experimental steps of the invention are as follows:

(1) modifying the DNA to be uploaded with sulfydryl at the 5 'or 3' end, and modifying oligo ethylene glycol spacer chains (OEG) with different lengths at the positions connected with the sulfydryl; OEGs may contain 6,12, or 18 ethylene glycol units;

(2) reducing and purifying the DNA modified by the sulfhydryl group with the OEG spacer chain; the DNA was reduced in a solution containing Dithiothreitol (DTT) and 2% (V/V) Triethylamine (TEA), and then purified twice using NAP-5 column;

(3) without an aging process, the purified DNA and the nanogold (AuNP) are directly mixed in a phosphate buffer (10mM PB, pH7.4) containing a required salt concentration according to a certain substance quantity ratio, and incubated for a period of time (2 minutes to 2 hours as required) at room temperature (25 ℃);

(4) determination of the concentration of DNA-AuNP: centrifuging the mixture obtained in the step (3), re-dispersing the red substance at the bottom of the centrifuge tube in the required buffer solution, and determining the concentration of the nanogold by using ultraviolet visible absorption measurement;

(5) determination of DNA loading on nanogold N (bars/AuNP): and centrifuging the DNA-AuNP subjected to the coating process, taking the supernatant, measuring the concentration of the supernatant by using ultraviolet, and calculating the coating amount according to the concentration and the volume of the DNA and the nanogold which are put in during coating and the centrifuged supernatant. The corresponding formula is as follows:

drawings

FIG. 1 is a graph comparing the method, effect and principle of the present invention with those of the prior art, wherein FIG. 1A is the method, effect and deficiency of the "aging-salting" method of the prior art; FIG. 1B illustrates the method, effect and deficiency of the prior art "use of acidic buffer solution method"; FIG. 1C illustrates the method of the present invention and its effect; FIG. 1D is a comparison of the principles of the three methods described above.

FIG. 2 is a graph showing the particle size change of DNA-AuNP in the preparation of DNA-AuNP by the "aging-salting-out" method using dynamic light scattering for real-time measurement of thiol-modified DNA having spacer chains of the same length and different compositions in the present invention. The aging-salting process comprises the following three steps: aging, adding salt, and incubating. The aging process is the first 18 hours of the initial coating, and no additional salt is added in the process; after the salt adding process, namely the aging process is finished, gradually dropwise adding a phosphate buffer solution (pH7.4) with the concentration of 1M sodium chloride (NaCl) into the mixed solution of the 13nm nanogold and the DNA to slowly increase the salt concentration to 0.3M; after the incubation process, namely the salt adding process is completed, the incubation is continued for a period of time, so that the DNA is more completely coated on the surface of the nanogold. And selecting a plurality of time points to measure the DNA-AuNP particle size in the three steps. 15-A₁₀-SH，15-T₁₀-SH，15-EG₁₈The sequence of-SH is shown in Table 1. The molar ratio of each DNA to the nanogold is 500: 1.

FIG. 3 shows the present inventionIn the figure, the particle size variation graph of DNA-AuNP when DNA-AuNP was prepared by aging-salting-in method using different lengths of OEG as thiol-modified DNA of spacer chain and 13nm nanogold (A in FIG. 3); directly performing 2-hour incubation on thiol-modified DNA with OEG of different lengths as spacer chains and 13nm nanogold under different salt concentrations to obtain a DNA-AuNP particle size variation graph (B, C and D in FIG. 3); loading of thiol-modified DNA with different lengths of OEG as spacer strand with 13nm nanogold after 2 hours incubation in 10mM phosphate buffer (pH7.4) with and without 0.3M NaCl, respectively (E in FIG. 3); photograph (F in FIG. 3) of thiol-modified DNA having different spacer chains incubated with 13nm nanogold in 10mM phosphate buffer solution (pH7.4) containing 0.3M NaCl after 2 hours; 15-EG₁₈UV-visible spectrum of-SH (FIG. 3G) before and after 2 hours incubation with 13nm nanogold in 10mM phosphate buffer (pH7.4) containing 0.3M NaCl. The molar ratio of DNA to nanogold is 500: 1. 15-EG₆-SH，15-EG₁₂-SH,15-EG₁₈-SH，15-A₁₀-SH,15-T₁₀The sequence of-SH is shown in Table 1.

FIG. 4 shows 15-EG in the present invention at a ratio of amounts of different substances₁₈The change curve of the particle size of the DNA-AuNP with time during aging incubation of the-SH with 13nm nanogold for 2 hours (A in FIG. 4) and the loading amount after 2 hours of incubation (B in FIG. 4). 15-EG₁₈The molar ratio of-SH to nanogold is 100:1, 200:1, 300:1, 400:1 and 500: 1. The incubation solution was 10mM phosphate buffered saline (pH 7.4). The sequence of 15-EG18-SH is shown in Table 1.

FIG. 5 shows 15-EG in the present invention₁₈Agarose gel electrophoresis and DNA loading after 2 hours incubation of-SH directly with 13nm nanogold in buffer solutions containing 10mM phosphate (pH7.4) at different NaCl concentrations.

FIG. 6 shows thiol-modified and fluorescently labeled DNA (FAM-15-A) having spacer chains of the same length and different compositions in the present invention₁₀-SH，FAM-15-T₁₀-SH，FAM-15-EG₁₈-SH, table 1) curve of fluorescence intensity over time during 2 hours of aging of the nanogold surface (a in fig. 6, molar ratio of DNA to nanogold 75: 1) and different molar ratios (DNA: AuNP) condition, uploadedFAM-15-EG of₁₈The proportion of-SH to the total input amount (B in FIG. 6). FAM-15-EG₁₈The molar ratio of-SH to AuNP was 25: the NaCl concentrations at 1,50:1, 75:1, 100:1,125:1, 150:1 and 200:1 were 10,20,30,40,60,80 and 100mM, respectively.

FIG. 7 is a photograph showing that the method of the present invention is applied to DNAs having different thiol modification positions (5 'or 3' ends), lengths and large-sized nanogold (50nm) and the change in nanogold particle size before and after loading of the DNAs, wherein in FIG. 7, A is the 5 'thiol-modified DNA, B is the 30-base-length thiol-modified DNA, C is the 3' thiol-modified DNA, and D is the 50nm nanogold to which the method of the present invention is applied.

FIG. 8 shows the confirmation of the fluorescence-labeled molecular beacon (MB 1-EG) by the particle size change in each step in the present invention₁₈-SH, table 1) successful assembly on 13nm nanogold. (1 XPB) MB1-EG in 10mM phosphate buffer solution₁₈the-SH groups assemble in a linear structure onto the nanogold (. smallcircle.), so there is no change in particle size after incubation in 15mM NaOH. MB1-EG in 10mM phosphate buffer solution (0.15M NaCl, PBS) containing 0.15M NaCl₁₈-SH assembles onto nanogold in a molecular beacon structure in one step (■), thus particle size increases significantly after incubation in 15mM NaOH. The nonspecific adsorption of the linear structure DNA on the surface of the nanogold due to the base adsorption is larger than that of the DNA with the molecular beacon structure, so that the particle size reduction of the linear structure DNA after washing is obviously larger than that of the linear structure DNA.

FIG. 9 shows the confirmation of fluorescently labeled molecular beacon (FAM-MB 2-EG) in the present invention₁₈-SH, table 1) has a very good ability to hybridize to complementary DNA after successful assembly on 13nm nanogold: FIG. 9A is a schematic diagram of hybridization of a molecular beacon; FIG. 9B shows the change in particle size before and after hybridization of the molecular beacon with complementary DNA; c in FIG. 9 is a fluorescence spectrum in which the fluorescence intensity increases with the increase in the concentration of complementary DNA, and D in FIG. 9 shows the operating curve.

Detailed Description

FIG. 1 is a graph comparing the method, effect and principle of the present invention with those of the prior art, wherein FIG. 1A is the method, effect and deficiency of the "aging-salting" method of the prior art; FIG. 1B illustrates the method, effect and deficiency of the prior art "use of acidic buffer solution method"; the above prior art is already described in the background art, and is not described in detail. FIG. 1C illustrates the method of the present invention and its effect; FIG. 1D is a comparison of the principles of the three methods described above. As shown in fig. 1D, the prior art is to reduce the net charge on DNA and AuNP by introducing cations, and the electrostatic repulsive force is thus reduced, thereby achieving the loading of thiol-modified DNA on AuNP. The principle of the method of the invention is as follows: the OEG can effectively shield the electrostatic repulsive force between the DNA and the AuNP due to the electric neutrality and poor conductivity of the OEG, so that the rapid quantitative uploading of the sulfhydryl modified DNA on the AuNP is realized.

Table 1: sequence information of the DNA used in the present invention.

Example 1. monitoring in real time the changes in the particle size of DNA-AuNP when DNA-AuNP preparation was performed according to the "age-salting" method using dynamic light scattering for thiol-modified DNA having spacer chains of the same length and different composition.

Linear DNA (15-A) having 3' -end thiol-modified spacer chains of the same length and different composition₁₀-SH，15-T₁₀-SH，15-EG₁₈-SH, Table 1) was mixed with 40mM Dithiothreitol (DTT) and 2% (V/V) Triethylamine (TEA) solutions, respectively, incubated at room temperature for 30 minutes, the mixture was purified twice using NAP-5 column, and DNA was eluted in 10mM phosphate buffer solution (pH 7.4). Adding the purified DNA into nanogold with the diameter of 13nm prepared by a citric acid reduction method, wherein the mass ratio of the added substances is 500:1 (DNA: AuNP). The preparation of DNA-AuNP was carried out by three steps of aging according to the "aging-salting" method, adding salt, and incubating. The aging process is 18 hours after the DNA is added with the nanogold, and no salt is additionally added in the process; after the salt adding process, namely the aging process is finished, gradually dropwise adding a phosphate buffer solution with the concentration of 1M sodium chloride (NaCl) into the mixed solution of the 13nm nano-gold and the DNA to slowly increase the final concentration of the NaCl to 0.3M; after the incubation process, namely the salt adding process is completed, the incubation is continued to ensure that the DNA is more completely coated on the surface of the nano-gold. To be provided withThe particle size of DNA-AuNP was determined by dynamic light scattering at several time points selected in each of the above three steps (FIG. 2).

The DNA-AuNP particle size results measured by the dynamic light scattering instrument in figure 2 show that the coating process of the sulfhydryl modified DNA with the same length and different composition spacing chains on the surface of the nanogold is similar, and the improvement of the salt concentration of the system is beneficial to the improvement of the loading capacity of the DNA on the surface of the nanogold. In the aging stage (0-18h), 15-EG₁₈The self-assembly rate of the-SH on the surface of the 13nm nano-gold is far greater than 15-A₁₀-SH and 15-T₁₀-SH. 15-EG after only 2 hours of aging incubation₁₈The particle size of the (SH) -AuNP reaches 15-A₁₀Equivalent levels of-SH-AuNP (26nm) after 28 hours of salt incubation.

Example 2. measurement of coating amount and particle size of thiol-modified DNA with different lengths of OEG spacer chains on nanogold at different salt concentrations during 2 hours of aging.

The DNA was reduced and purified according to the method in example 1. In this example, 15-EG modified with a 3' thiol group was used₆-SH、15-EG₁₂-SH、15-EG₁₈Preparation of DNA-AuNP was carried out using-SH as a spacer strand DNA. The 2 hour incubation of DNA with AuNP was performed in phosphate buffered solutions containing different salt concentrations. The change in the particle size of the DNA-AuNP was determined during incubation using dynamic light scattering. After the incubation, photographing is carried out, ultraviolet-visible spectrum testing is carried out, then centrifugation is carried out at 15000rpm, 100 mu L of supernatant is taken, the concentration of the probe in the supernatant is quantitatively determined according to the ultraviolet-visible spectrum, and the coating amount is calculated.

The result shows that OEG as a spacer chain can effectively improve the assembly efficiency of DNA on the nanogold, and the traditional aging process, the salting process and the incubation process can be completed by nearly 80% only in 2 hours (FIG. 2A). 15-EG incubation in phosphate buffered saline without NaCl₁₈The coating amount of the-SH on each nano-gold particle after 2 hours of coating process reaches 129 bars of 15-EG₁₈-SH, which is sufficient to meet the loading requirement of DNA-AuNP as a nanosensor. Moreover, even short OEG (6 ethylene glycol units) can greatly improve the loading speed of the sulfhydryl modified DNA on the nanogold，15-EG₆-SH and 15-EG₁₂The coating amount of-SH on each nano-gold particle after 2 hours of coating process reaches 99 and 107 DNA respectively. With increasing salt concentration, both the DNA loading rate and the loading amount increased dramatically (fig. 3 BCDE). And AuNP still keeps red after being incubated for 2 hours under high salt condition, which indicates that the AuNP has good stability. And 15-A as an alignment₁₀-SH and 15-T₁₀Under the same conditions, AuNP aggregated (F in FIG. 3). Further 15-EG₁₈The high stability of AuNP was also confirmed by UV-visible spectra (G in FIG. 3) of-SH and 13nm nanogold before and after 2 hours incubation in 10mM phosphate buffer solution (pH7.4) containing 0.3M NaCl. The AuNP did not decrease in the intensity of the absorption peak at 520nm, but the position of the maximum absorption peak was red-shifted, sufficiently confirming the assembly of a large amount of DNA on AuNP.

Example 3 determination of 15-EG in the proportions of the amounts of the various substances₁₈The change curve of the particle size of the DNA-AuNP along with time in the aging incubation process of 2 hours of-SH and 13nm nanogold and the loading amount after the 2-hour incubation process.

15-EG was reduced and purified according to the method of example 1₁₈-SH. 15-EG added with different substance quantity ratios detected by DLS₁₈The grain diameter of the-SH and the nano-gold changes in the aging process of 2 hours, and the mass ratio of the-SH to the nano-gold is respectively 100: 1. 200: 1. 300, and (2) 300: 1. 400: 1. 500:1, selecting points with the time of 0,0.5,1,1.5 and 2 hours for particle size measurement. After aging for 2 hours, the sample was centrifuged at 15000rpm, 100. mu.L of the supernatant was collected, and the probe concentration in the supernatant was quantitatively determined by UV-visible spectroscopy and the amount of coating was calculated.

The results show that the loading amount after aging for 2h is increased as the particle size of the compound is changed in the coating process along with the increase of the amount ratio of the input substances (fig. 3). Therefore, the final coating amount can be conveniently controlled by regulating and controlling the input proportion.

Example 4 according to the results obtained in example 3, the input ratio 400 was chosen: 1 (15-EG)₁₈AuNP-SH) was subjected to 15-EG at various salt concentrations₁₈SH assembly, the success of the coating process is verified by gel electrophoresis and the coating amount is quantitatively determined by UV-visible spectroscopy.

15-EG was reduced and purified according to the method of example 1₁₈-SH, conversion of 15-EG₁₈-SH are respectively prepared in phosphate buffer solutions with different NaCl concentrations (0mM,30mM,60mM,90mM,120mM,150mM,300mM), and the solutions are directly added into the nanogold. After the 2-hour aging process is finished, centrifuging at 13000rpm, discarding the supernatant, quantitatively measuring the concentration of the probe of the supernatant by using an ultraviolet-visible spectrum, and calculating the calculated loading capacity. Another 6. mu.L of the precipitate was subjected to 2% agarose gel electrophoresis with 4. mu.L of glycerol.

The results show that as the salt concentration increases, 15-EG₁₈The loading amount of-SH on the surface of the nanogold is further increased (figure 5). As can be seen from the agarose gel, 15-EG₁₈the-SH-AuNP still keeps stable under higher salt concentration, and the nanogold keeps red. No. 1 is nano gold, and the nano gold is aggregated under the electrophoresis condition, and the color is blue black. This example illustrates that the method of the present invention can directly perform DNA assembly in nanogold under high salt conditions without causing nanogold aggregation, without a low salt aging process.

Example 5 demonstration of FAM-15-EG₁₈the-SH can be rapidly and quantitatively adsorbed on the surface of the nanogold.

The DNA was reduced and purified according to the method in example 1. In this example, DNA was loaded at a ratio of 75:1 AuNP in a 30mM 1 XPBS buffer system, and the loading process was monitored by fluorescence kinetics (wherein the excitation wavelength of a fluorescence spectrometer was 467nm and the emission wavelength was 520nm and the slit width was 10nm, and the scanning interval was 5 minutes).

According to the Fluorescence Resonance Energy Transfer (FRET) phenomenon, nanogold is a strong fluorescence quenching group due to the existence of extremely strong plasma resonance absorption (SPR), and after fluorescence labeling DNA is assembled on the surface of nanogold, the fluorescence group of the nanogold is quenched by the nanogold, so that the fluorescence signal of a system is reduced. After the end of the loading, a standard curve of DNA concentration and fluorescence amount is designed and measured according to the residual fluorescence amount of the system. The proportion of free DNA not involved in the system was calculated from the standard curve.

The results show that FAM-15-EG added with the same amount of DNA₁₈The residual amount of-SH fluorescence is significantly lower than that of FAM-15-A₁₀-SH and FAM-15-T₁₀SH, and the time for fluorescence to fall and reach equilibrium is minimal, reaching equilibrium almost immediately (fig. 6A). According to a standard curve, FAM-15-EG₁₈Residual FAM-15-EG in solution after the end of the loading process of the-SH₁₈The amount of-SH is only 5% of the input amount, and the equivalent load amount is up to 72 at the input ratio of 75/1. At the same time, because of FAM-15-EG₁₈Comparison of-SH with FAM-15-T₁₀-SH and FAM-15-A₁₀Less non-specific adsorption of-SH on nanogold, FAM-15-EG₁₈the-SH is better in the upright state, the FRET is relatively weak, and the stronger background signal is also caused, so that only 5% of the signal remains indicate FAM-15-EG₁₈the-SH is loaded on the nanogold almost completely and quantitatively. While the residual FAM-15-A in the solution was present under the same assembly conditions₁₀-SH and FAM-15-T₁₀and-SH is 24 percent and 12 percent of the input amount. The quantitative uploading can not only avoid the complicated quantitative process, but also greatly avoid the waste of DNA. This example further demonstrates that OEG spacer strands can increase both assembly rate and loading during DNA-AuNP self-assembly.

In addition, we tested the loaded FAM-15-EG under different molar ratios (DNA: AuNP)₁₈The proportion of-SH to the total input amount (B in FIG. 6). FAM-15-EG₁₈The molar ratio of-SH to AuNP was 25: the NaCl concentrations at 1,50:1, 75:1, 100:1,125:1, 150:1 and 200:1 were 10,20,30,40,60,80 and 100mM, respectively. Under the above conditions, when FAM-15-EG₁₈When the molar ratio of-SH to AuNP is lower than 150:1, more than 90% of DNA is adsorbed on AuNP, and quantitative uploading is realized.

Example 6. the method of the present invention is applicable to DNA and large-sized nanogold (50nm) having different thiol modification positions (5 'or 3' end), lengths.

The DNA was reduced and purified according to the method in example 1. Probe 15-EG was incubated for 2 hours of aging in example 2₁₈-SH、HS-EG₁₈-15、30-EG₁₈-SH (Table 1) was loaded on gold nanoparticles of 13nm and 50nm, respectively, and the particle size change of the composite before and after loading was measured by a dynamic light scattering apparatus. Coating unmodified nano-gold and sulfhydryl modified DNA with nano-goldPhotographs were taken immediately after addition of 0.2M NaCl (final concentration).

The result shows that the method for rapidly preparing the DNA-AuNP by using the OEG as the sulfhydryl-modified DNA of the spacer chain has good universality, and the method is suitable for DNAs with different sulfhydryl modification positions (5 'or 3' ends) and lengths and large-size nanogold (50nm) (figure 7), wherein in the figure 7, A is the DNA modified by the sulfhydryl at the 5 'end, B is the DNA modified by the sulfhydryl with the length of 30 bases, C is the DNA modified by the sulfhydryl at the 3' end, and D is the DNA modified by the sulfhydryl applied to the nanogold with the length of 50 nm. The loading process has the advantages of high speed, high stability, uniform particle size distribution of the compound and strong salt resistance. The unmodified nano-gold in 0.2M NaCl immediately aggregates, the color changes to grey, and the nano-gold coated with the sulfhydryl modified DNA keeps red, thus confirming that the DNA is successfully coated on the nano-gold.

Example 7 confirmation of fluorescently labeled molecular Beacon (MB 1-EG)₁₈-SH, table 1) successful assembly on 13nm nanogold.

MB1-EG was incubated in phosphate buffer (10mmol PB pH7.4) and phosphate buffer containing NaCl at a concentration of 150mmol/L (10mmol PB pH7.4) for 2h₁₈Preparation of-SH-AuNP with a feed ratio of 500:1(MB 1-EG)₁₈AuNP is-SH). The composite particle size at the beginning and end of aging was monitored using a dynamic light scattering instrument. After the aging process was completed, the pellets were centrifuged at 13000rpm, washed three times with the corresponding buffer solution to remove DNA in a free state, and the change in particle size was monitored again. After washing, the precipitate was centrifuged at 13000rpm, the supernatant was discarded, and an equal volume of 15mM NaOH solution was added to the precipitate. The change in the particle size of the DNA-AuNP was monitored by a dynamic light scattering apparatus.

The result shows that the particle size of the compound has no significant difference compared with other examples under higher salt concentration, and the molecular beacon is successfully and rapidly linked on the surface of the nanogold and is not aggregated. MB1-EG in 10mM phosphate buffer solution₁₈the-SH groups assemble in a linear structure onto the nanogold (. smallcircle.), so there is no change in particle size after incubation in 15mM NaOH. MB1-EG in 10mM phosphate buffer solution (0.15M NaCl, PBS) containing 0.15M NaCl₁₈One-step assembly of SH onto nanogold in a molecular beacon structure (S) ((S))■) and thus the particle size increased significantly after incubation in 15mM NaOH. The nonspecific adsorption of the linear structure DNA on the surface of the nanogold due to the base adsorption is larger than that of the DNA with the molecular beacon structure, so that the particle size reduction of the linear structure DNA after washing is obviously larger than that of the linear structure DNA. This result fully demonstrated MB1-EG₁₈the-SH can be directly linked to the surface of the nanogold through a molecular beacon structure under the condition of high salinity, and the MB1-T with the same base sequence₁₀the-SH groups aggregate during the conventional "aging-salting" process. This example fully demonstrates the superiority of OEG as a spacer chain.

Example 8 confirmation of fluorescently labeled molecular Beacon (FAM-MB 2-EG)₁₈-SH, table 1) has a good ability to hybridize to complementary DNA after successful assembly on 13nm nanogold.

The self-assembly and washing of the thiol DNA on the surface of the nanogold were performed according to the experimental procedure in example 7. After washing, a certain amount of the complexes were mixed with a series of concentration gradient DNA targets (FAM-MB2Target, table 1), hybridization incubation was performed, and after incubation, the fluorescence intensity of each group of complexes was quantitatively measured. Wherein, the total volume of each group is 110 muL, the volume of the compound is 50 muL, DNA targets with different concentrations are added to lead the final concentration of the system target to be 1000nM, 500nM, 250nM, 100nM, 50nM, 10nM, 5nM and 0nM, and the volume is increased to 110 muL by utilizing washing buffer solution. The fluorescence measurement conditions were the same as in example 5.

The results show that FAM-MB2-EG coated by the method of the invention₁₈the-SH-AuNP has a good biomolecule recognition function (FIG. 9). The DNA-AuNP with the molecular beacon structure is prepared by the method, the assembly process is simple, rapid, efficient and stable, and the molecular recognition performance of the compound is excellent.

Claims

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.