CN107064485B

CN107064485B - Application of decahedral nano silver probe in screening aptamer by using surface plasmon resonance imaging technology

Info

Publication number: CN107064485B
Application number: CN201710023541.XA
Authority: CN
Inventors: 许丹科; 贾文超; 李慧
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2017-01-13
Filing date: 2017-01-13
Publication date: 2020-05-19
Anticipated expiration: 2037-01-13
Also published as: CN107064485A

Abstract

The invention discloses an application of a decahedral nano silver probe in screening aptamer by using surface plasmon resonance imaging technology, wherein the decahedral nano silver probe comprises the following components in parts by weight: decahedral nano silver with the particle size of 50-60 nm is used as an inner core, two nucleotide chains are arranged on the outer surface of the nano silver, a chain A is a complementary chain of a library reverse primer, and the complementary chain of the library reverse primer is bonded on the outer surface of the nano silver; the B chain is a nucleic acid library sequence which is combined with the complementary pairing of the reverse primer of the library; wherein the complementary strand of the reverse primer of the library has the following structure: the 3 'end is a complementary sequence of a reverse primer of the library, the middle is 10A basic groups which are used as a spacer arm, and the 5' end is modified by sulfydryl. The decahedral nano silver provided by the invention can load a nucleic acid library, can improve the signal sensitivity in the aptamer screening process, and further improves the screening efficiency of aptamers.

Description

Application of decahedral nano silver probe in screening aptamer by using surface plasmon resonance imaging technology

Technical Field

The invention belongs to the technical field of biological monitoring, and particularly relates to an aptamer-modified nano silver pair SPRI signal amplification technology and application thereof in lactoferrin real-time screening.

Background

Compared with nano gold, nano silver has excellent photochemical properties, and can generate stronger and narrower plasma resonance peak. Therefore, the local plasma on the surface of the nano silver can be better coupled with the plasma wave on the surface of a Surface Plasma Resonance (SPR) chip, and stronger signal output is generated.

Surface plasmon resonance imaging (surface plasmon resonance imaging) is an optical detection instrument with no need of marking samples, real time and high flux. The method can be used for carrying out real-time quantitative determination on a plurality of target samples on the surface of the chip. Therefore, the screening target can be loaded on the surface of the chip, the nucleic acid library is introduced, the positive screening/negative screening target can be collected in real time, and the binding condition of the positive screening/negative screening channel library in the aptamer screening process is observed, so that the aptamer with high affinity and high specificity is screened out.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a decahedral nano silver probe for signal amplification of SPRI, which is beneficial to observing the combination condition of a positive sieve/negative sieve channel library in an aptamer screening process, so that a high-affinity and high-specificity aptamer can be screened out.

The invention also aims to solve the technical problem of providing a preparation method of the decahedral nano silver probe.

The invention also aims to solve the technical problem of providing a decahedral nano silver probe for screening the lactoferrin nucleic acid aptamer.

The technical problem to be solved by the invention is to provide the preparation method of the decahedral nano silver probe for screening the lactoferrin nucleic acid aptamer.

The invention finally solves the technical problem of providing the application of the decahedral nano silver probe in screening of the lactoferrin nucleic acid aptamer.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the decahedral nano silver probe is applied to screening of aptamer by using surface plasmon resonance imaging technology.

The local plasmas on the surface of the decahedron nano silver can be electromagnetically coupled with the plasmons on the surface of the sensing chip, so that the coupling effect of the SPRI light source and the surface plasmas can be enhanced; therefore, stronger difference between the front and the back of the reflected light is generated, and the sensitivity of sensing can be further improved.

In the screening process of the aptamer, the decahedral nano silver particles are modified on the nucleic acid library, so that the molecular weight of a detection target can be increased, the electromagnetic coupling between the decahedral nano silver particles and the surface is enhanced, the detection sensitivity of the nucleic acid library is increased, and the number of screening rounds of the aptamer is further shortened.

Wherein, the decahedral nano silver probe comprises: decahedral nano silver with the particle size of 50-60 nm is used as an inner core, and two nucleotide chains are arranged on the outer surface of the nano silver;

the A chain is a complementary chain of a library reverse primer, and the complementary chain of the library reverse primer is bonded on the outer surface of the nano silver;

the B chain is a nucleic acid library sequence which is combined with the complementary pairing of the reverse primer of the library;

wherein the complementary strand of the reverse primer of the library has the following structure: the 3 'end is a complementary sequence of a reverse primer of the library, 10-18A basic groups are arranged in the middle and serve as spacer arms, and the 5' end is modified by sulfydryl. The 3 'end is a complementary sequence of a reverse primer of the library and is used for being complementarily combined with a sequence of a nucleic acid library, a certain steric hindrance effect exists due to the fact that a complementary chain and the library are hybridized on the surface of decahedral nano-silver, 10A basic groups are added between a hybridized chain segment and the decahedral nano-silver, the steric hindrance effect can be reduced, the hybridization efficiency is improved, the 5' end is a mercapto group and is used for being bonded with the outer surface of the nano-silver, and the length of the complementary sequence is preferably 10A basic groups.

The preparation method of the decahedral nano silver probe comprises the following steps:

(1) synthesis of decahedral nano silver: adding sodium citrate, polyvinylpyrrolidone, L-arginine and silver nitrate into 100-300 mL of deionized water according to a molar ratio of (0.1-70): (0.1-10): (0.2-30), and then adding 0.1-1M of sodium borohydride aqueous solution to ensure that the molar ratio of sodium borohydride to silver nitrate is (1-100): 1, reacting for 16-72 hours under the irradiation of a blue light lamp to obtain a decahedral nano silver solution;

(2) modification of decahedral nano silver: taking the decahedral nano silver solution obtained in the step (1), adding a library reverse primer complementary strand and sodium chloride,

wherein, the molar ratio of the nano-silver, the complementary chain of the reverse primer of the library and the sodium chloride is (0.1-10 pmol): (0.1-10 nmol): 10-200 mu mol), the Tween-20 is dripped, and the volume ratio of the Tween-20 to the nano-silver solution is (0.001-0.01): 1, uniformly mixing, and shaking for 1-5 h at 37 ℃;

(3) standing the mixed system obtained in the step (2) for 10-24 hours, preferably 24 hours;

(4) centrifuging the solution obtained in the step (3), adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2-4 times, wherein the volume of the 1 × PBS buffer solution added each time is 0.1-5 times of the volume of the precipitate; finally, resuspending the precipitate obtained by centrifugation by using a 1 XPBSM buffer solution;

the centrifugation condition is 10000-20000 rpm for 10-30 minutes, preferably 15000rpm for 15 minutes;

(5) adding a nucleic acid library solution into the mixed system obtained in the step (4), so that the molar ratio of the nucleic acid library to the decahedral nano silver modified with the reverse primer complementary strand is (100-2000): and 1, carrying out oscillation reaction for 10-60 min to obtain the decahedral nano silver probe.

In step (1), preferably 15mL of 50mM sodium citrate, 15mL (6%) of polyvinylpyrrolidone (PVP), 2.5mL of 10mM L-arginine, 10mL of 10mM silver nitrate, are added to 175mL of deionized water, followed by 0.8mL of 0.5M aqueous sodium borohydride solution.

In the step (2), 1mL of the decahedral nano silver (Ag) is preferably synthesized₁₀) To this solution, 70. mu.L of library reverse primer hybrid strand and oligonucleotide strand at a concentration of 10. mu.M were added, 64. mu.L of 2M aqueous sodium chloride solution at a concentration of 2M was added, and 1 drop of Tween-20 was added.

The decahedral nano silver probe for screening the lactoferrin nucleic acid aptamer takes 50-60 nm decahedral nano silver as an inner core, a library reverse primer complementary strand is bonded on the outer surface of the nano silver, the library reverse primer complementary strand is complementarily paired with a nucleic acid library sequence,

wherein the complementary strand of the library reverse primer is:

5’-SH-(CH₂)₆-AAAAAAAAAAGAAGAGGAGGGAGGT-3’

the nucleic acid library sequence is:

5 '-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3', wherein N40 represents 40 random bases.

The preparation method of the decahedral nano silver probe for screening the lactoferrin nucleic acid aptamer comprises the following steps:

(1a) synthesis of decahedral nano silver: adding sodium citrate, polyvinylpyrrolidone, L-arginine and silver nitrate into 100-300 mL of deionized water according to a molar ratio of (0.1-70): (0.1-10): (0.2-30), and then adding 0.1-1M of sodium borohydride aqueous solution to ensure that the molar ratio of sodium borohydride to silver nitrate is (1-100): 1, reacting for 16-72 hours under the irradiation of a blue light lamp to obtain a decahedral nano silver solution;

(2a) modification of decahedral nano silver: taking the decahedral nano silver solution obtained in the step (1), adding a library reverse primer complementary strand and sodium chloride,

the complementary strand of the library reverse primer is:

5’-SH-(CH₂)₆-AAAAAAAAAAGAAGAGGAGGGAGGT-3’

(3a) standing the mixed system obtained in the step (2a) for 10-24 hours;

(4a) centrifuging the solution obtained in the step (3a), adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the centrifuging and resuspending steps for 2-4 times, wherein the volume of the 1 × PBS buffer solution added each time is 0.1-5 times of the volume of the precipitate; finally, resuspending the precipitate obtained by centrifugation by using a 1 XPBSM buffer solution;

(5a) adding a nucleic acid library sequence solution into the mixed system obtained in the step (4a), so that the molar ratio of the nucleic acid library sequence to the decahedral nano silver modified with the reverse primer complementary strand is 100-2000: 1, carrying out oscillation reaction for 10-60 min to obtain a decahedral nano silver probe;

the nucleic acid library sequence is:

In the step (1a), the preferred molar ratio of the sodium citrate, the polyvinylpyrrolidone, the L-arginine and the silver nitrate is (0.1-70): (0.1-10): (0.2-30): 0.2-30).

In the step (2a), the molar ratio of the nano-silver, the complementary strand of the library reverse primer and the sodium chloride is preferably (0.1 to 10pmol): (0.1 to 10nmol): 10 to 200 μmol).

The application of the decahedral nano silver probe for screening the lactoferrin nucleic acid aptamer in screening the lactoferrin nucleic acid aptamer.

Wherein, the steps of screening the aptamer by using the decahedral nano silver probe are as follows:

(1b) the SPRI sensor chip surface sulfhydryl alkyl acid self-assembly:

soaking a gold sheet with a gold film thickness of 45-52 nm in an ethanol solution of 11-mercaptoalkyl acid with the concentration of 0.5-10 mM for reaction for 12-36 h; rinsing the chip surface with copious amounts of ethanol and ultra-pure water, finally N₂Drying for later use;

(2b) preparing a solution:

positive sieve protein solution: dissolving lactoferrin in 1 XPBSM buffer solution, wherein the concentration of lactoferrin is 250 mug/mL;

the negative sieve protein solution is prepared by dissolving BSA, α -lactalbumin, β -lactoglobulin and casein in 10mM sodium acetate solution, wherein the pH value is 4.5, and the concentration of the BSA, the α -lactalbumin, the β -lactoglobulin and the casein is 160 mu g/mL;

screening buffer solution: MgCl was added to 1 XPBS₂·6H₂O solid, 1 x PBSM formed as screening buffer;

chip activation reagent: 0.8M EDC aqueous solution, 0.2M NHS aqueous solution;

blocking reagent: 1M ethanolamine solution;

(3b) transferring 200 μ L of 0.8M EDC, mixing with 200 μ L of 0.2M NHS to form 400 μ L of mixed solution of 0.4M EDC and 0.1M NHS, and performing activation reaction on the surface of the chip for 40min at a flow rate of 10 μ L/min; then respectively reacting positive sieve protein and negative sieve protein in the two channels at the flow rate of 10 mu L/min for 40 min; finally, sealing 200 mu L of 1M ethanolamine at the flow rate of 20 mu L/min for 10 min; then using 1 XPBS buffer solution to balance the chip for 30 min;

(4b) after the baseline is stable, the positive sieve channel and the negative sieve channel are connected in series, the six-way valve is connected to the inlet of the negative sieve channel, a constant-flow injection pump is used for transmitting a screening buffer solution at the flow rate of 20 mu L/min, the six-way valve is used for injecting a 200 mu L decahedral nano-silver load library, and meanwhile, the signals are collected and stored in the positive sieve channel and the negative sieve channel, and the reaction time is 40 min;

(5b) separating the positive sieve channel from the negative sieve channel, washing the positive sieve channel for 15min by using a screening buffer solution, then washing the positive sieve channel by using 90 mu L of 50mM NaOH solution at the flow rate of 10-50 mu L/min, collecting an eluted sample, and then adding 37.5 mu L of 120mM hydrochloric acid solution for neutralization;

(6b) PCR amplification and ssDNA purification:

and (3) PCR amplification: carrying out 7-9 rounds of pre-amplification on the collected and neutralized product, dividing the solution into 40 parts for carrying out 9 rounds of amplification, and combining the 40 parts of solution after obtaining the product;

magnetic bead cleaning: washing streptavidin-labeled magnetic beads (SA-MB) with the particle size of 3 μm twice by using 0.1mg/mL BSA solution, and removing the supernatant;

and (4) purifying and sequencing the ssDNA to obtain the lactoferrin nucleic acid aptamer.

In step (1b), the optimal thickness of gold film is 48nm, the concentration of 11-mercaptoalkyl acid is 1mM, and the gold flake is obtained

The reaction time was chosen to be 24 h.

Has the advantages that: the decahedral nano silver provided by the invention loads the nucleic acid library, can greatly increase the sensing signal of SPRI, and can be used for screening the aptamer of lactoferrin by combining a high-throughput real-time imaging aptamer screening technology, so that the affinity state between the nucleic acid library and a target can be monitored in real time, and a brand-new high-efficiency screening platform is provided for the aptamer screening technology.

The invention utilizes the decahedral nano silver to amplify and enhance the SPRI signal by about 128 times, and is a simple and rapid amplification technology.

Drawings

FIG. 1 is a transmission electron microscope topography of decahedral nanosilver.

Fig. 2 is a graph of the effect of using decahedral nanosilver on SPRI signal amplification. The large graph shows an SPR sensorgram of reaction of modifying a biotin-containing nucleic acid chain to a decahedral nano-silver surface and Streptavidin (Streptavidin) fixed on the surface of the SPRI chip, and the inset shows an SPR sensorgram of reaction of an unmodified decahedral nano-silver biotin nucleic acid chain and Streptavidin fixed on the surface of the SPRI chip. Through response value comparison, the signal of the modified decahedral nano silver is 128 times that of the unmodified signal.

Fig. 3 uses SPRI screening curves and imaging plots. In FIG. 3, R1-R5 represent 1-5 rounds; FIG. 3a is a positive sieve curve of 1-5 rounds, and FIG. b is a negative sieve curve of 1-5 rounds. The imaging graph represents the SPRI imaging graph of 1-5 screening processes, the left channel of the graph represents a negative screen channel, and the right channel of the graph represents a positive screen channel.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1: and preparing a decahedral nano silver probe.

(1) Synthesis of decahedral nano silver: adding 15mL of 50mM sodium citrate, 15mL (6%) of polyvinylpyrrolidone (PVP), 2.5mL of 10mM L-arginine, 10mL of 10mM silver nitrate into 175mL of deionized water, then adding 0.8mL of 0.5M aqueous solution of sodium borohydride, wherein the color of the solution immediately becomes dark yellow, and irradiating the reaction flask from 4 sides for 48 hours by using 4 blue lamps with 24W;

(2) adding oligonucleotide chains or library reverse primer hybrid chains screened by lactoferrin into the decahedral nano-silver solution obtained in the step (1), and standing for 18 hours. The ratio of the total molar amount of oligonucleotide strands or lactoferrin screening library reverse primer hybrid strands to the molar amount of decahedral nanosilver was 288: 1;

the oligonucleotide chain is as follows:

5’-SH-(CH₂)₆-TTTGGGTTTGGGTTTGGGTTT-biotin, which is modified at its 5 'end with a thiol group and at its 3' end with biotin;

the reverse primer hybrid chain is as follows:

5’-SH-(CH₂)₆AAAAAAAAAAGAAGAGGAGGGAGGT-3 ', the 5' end of which is modified by sulfhydryl and the end has 10A bases as spacer.

(3) Adding 1 XPBS (137mM NaCl,2.7mM KCl,10mM, Na) to the mixed system obtained in the step (2)₂HPO₄·12H₂O,2mM KH₂PO₄) The solution was allowed to stand for 6 hours.

(4) And (4) adding 2mol/L sodium chloride solution into the mixed system obtained in the step (3) to enable the final concentration of the sodium chloride to be 0.2mol/L, and then adding 5 mu L of Tween-20.

(5) And (4) standing the mixed system obtained in the step (4) for 48 hours.

(6) Centrifuging the solution obtained in the step (5) to remove supernatant, adding 1 XPBS solution into the precipitate for resuspension, repeating the steps of centrifuging and resuspending for 3 times, wherein the adding volume of PBS buffer solution is 1 time of the volume of the mixed system to be added each time; finally, the precipitate obtained by centrifugation is re-suspended by 1 xPBSM to obtain the decahedral nano silver probe modified by the aptamer.

Modification of decahedral nano silver: taking 1mL of the decahedral nano silver (Ag) synthesized by the step (1)₁₀) Adding 70 mu L of lactoferrin screening library reverse primer hybrid chain or oligonucleotide chain with the concentration of 10 mu M into the solution, adding 64 mu L of 2M sodium chloride aqueous solution with the concentration, adding 1 drop of Tween-20, uniformly mixing, vibrating in a constant-temperature oscillator at 37 ℃ for 2h, and standing for 18 h.

Example 2: preparing a decahedral nano silver probe of a load lactoferrin nucleic acid library.

(1) Synthesis of decahedral nano silver: 15mL of 50mM sodium citrate, 15mL (6%) of polyvinylpyrrolidone (PVP), 2.5mL of 10mM L-arginine, 10mL of 10mM silver nitrate were added to 175mL of deionized water, followed by 0.8mL of 0.5M aqueous sodium borohydride, which immediately turned dark yellow in color, and the reaction flask was irradiated from 4 sides with 4 24W blue lamps for 48 hours.

(2) Adding the reverse primer complementary strand into the decahedral nano silver solution prepared in the step (1), and standing for 18 hours, wherein the ratio of the total molar amount of the reverse primer complementary strand to the molar amount of the decahedral nano silver is 288: 1.

The reverse primer complementary strand is as follows:

5’-SH-(CH₂)₆AAAAAAAAAAGAAGAGGAGGGAGGT-3 'the 5' end is modified by sulfhydryl and the end has 10A bases as spacer arm;

(3) adding 1 XPBS (137mM NaCl,2.7mM KCl,10mM, Na) to the mixed system obtained in the step (2)₂HPO₄·12H₂O,2mM KH₂PO₄) Buffer, and standing for 6 hours.

(4) And (4) adding 2mol/L sodium chloride solution into the mixed system obtained in the step (3) to enable the final concentration of sodium chloride to be 0.2mol/L, and then adding 5 mu L of Tween-20.

(5) And (4) standing the mixed system obtained in the step (4) for 48 hours.

(6) Centrifuging the solution obtained in the step (5) to remove supernatant, adding the precipitate into 1 xPBSM buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 3 times, wherein the adding volume of the 1 xPBSM buffer solution is 1 time of the volume of the mixed system to be added; the pellet from the final centrifugation was resuspended in 1 XPBSM buffer.

(7) Adding the lactoferrin nucleic acid library dissolved in the 1 xPBSM buffer solution into the solution obtained in the step (6), and carrying out shake reaction in a constant-temperature shake table at 37 ℃ for 0.5h to obtain a standby library load probe;

the lactoferrin nucleic acid library is:

5 '-GACAGGCAGGACACCGTAAC-N40-CTGCTACCTCCCTCCTCTTC-3', wherein N40 is 40 random bases.

Example 3: SPRI signal amplification using decahedral nanosilver

(1) The SPRI sensor chip surface sulfhydryl alkyl acid self-assembly:

soaking a gold sheet with the thickness of 48nm in an ethanol solution of 11-mercapto alkyl acid with the concentration of 1mM for reaction for 24 h; rinsing the chip surface with copious amounts of ethanol and ultra-pure water, finally N₂Drying for later use;

(2) preparing a solution:

streptavidin protein solution: transferring 2.5. mu.L of SA solution with concentration of 10mg/mL, adding 247.5. mu.L of 10mM sodium acetate buffer solution (pH4.5) to form 250. mu.L of SA protein solution with concentration of 100. mu.g/mL;

bovine serum albumin solution: mu.L of 10mg/mL BSA was removed and 240. mu.L of 10mM sodium acetate solution (pH4.5) was added to form 400. mu.L BSA protein solution;

running buffer: diluting 20 XPBS with secondary water to 1 XPBS, adding MgCl to 1 XPPBS₂·6H₂O solid, 1 XPBSM as screening buffer

Chip activation reagent: 1.52g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed and dissolved in 10mL of ultra-pure water to form a 10mL, 0.8M aqueous solution of EDC; weighing 0.23g N-hydroxysuccinimide, and dissolving in 10mL of ultrapure water to form 10mL of 0.2M NHS aqueous solution;

blocking reagent: 0.98g of ethanolamine hydrochloride powder was weighed and dissolved in 10mL of ultrapure water to form 10mL of a 1M ethanolamine solution.

(3) Protein fixation:

transferring 200 μ L of 0.8M EDC, mixing with 200 μ L of 0.2M NHS to form 400 μ L of 0.4M EDC/0.1MNHS mixed solution, and performing activation reaction on the surface of the chip for 40min at a flow rate of 10 μ L/min; then SA protein and BSA protein are respectively reacted in the two channels at the flow rate of 10 mu L/min for 25 min; finally sealing the surface of the chip for 10min by 200 mu L of 1M ethanolamine at the flow rate of 20 mu L/min; then, using 1 XPBSM buffer solution to balance the chip for 30 min;

(4) monitoring a chip baseline by using self-developed Labview and Livegraph software, and connecting a positive sieve channel and a negative sieve channel in series after the baseline is stable;

(5) the six-way valve is connected to the inlet of the negative sieve channel, a constant flow injection pump is used for transmitting the running buffer solution at the flow rate of 50 mu L/min, 200 mu L of the decahedral nano silver modified by the oligonucleotide chain is injected by the six-way valve, and meanwhile, the signals of the positive channel and the negative channel are collected and stored, and the reaction time is 4 min.

Finally, the obtained data are processed to obtain the binding curve of the oligonucleotide and the SA protein modified on the decahedral nano silver surface, and the result is shown in figure 2.

Example 4: application of decahedral nano silver probe for screening library by using negative-screening lactoferrin in SPRI real-time aptamer screening technology

The test method comprises the following steps:

(1) the SPRI sensor chip surface sulfhydryl alkyl acid self-assembly:

soaking a gold sheet with the thickness of 48nm in an ethanol solution of 11-mercaptoalkyl acid with the concentration of 1mM for overnight reaction; rinsing the chip surface with copious amounts of ethanol and ultra-pure water, finally N₂Drying for later use;

(2) preparing a solution:

positive sieve protein solution: adding 200 mu L of ultrapure water into the lactoferrin powder with the bottle specification of 1mg to form 200 mu L of lactoferrin aqueous solution with the concentration of 5 mg/mL; transferring 12.5 μ L of the solution, adding 237.5 μ L of 1 XPBSM buffer solution to form 250 μ L of lactoferrin solution with a concentration of 250 μ g/mL;

transferring 2 mu L of BSA and α -lactalbumin with the concentration of 20mg/mL, transferring 8 mu L of β -lactoglobulin and casein with the concentration of 5mg/mL, finally transferring 230 mu L of 10mM sodium acetate solution (pH4.5) to the inside, and finally forming 250 mu L of BSA and α -lactalbumin, wherein 4 negative sieve protein solutions with the concentrations of 5mg/mL of β -lactoglobulin and casein being 160 mu g/mL are formed;

screening buffer solution: diluting 20 XPBS with secondary water to 1 XPBS, adding MgCl to 1 XPPBS₂·6H₂O solid, 1 x PBSM formed as screening buffer;

chip activation reagent: 1.52g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was weighed and dissolved in 10mL of ultrapure water to form 10mL of a 0.8M EDC aqueous solution; weighing 0.23g N-hydroxysuccinimide, and dissolving in 10mL of ultrapure water to form 10mL of 0.2M NHS aqueous solution;

(3) Protein fixation:

transferring 200 μ L of 0.8M EDC, mixing with 200 μ L of 0.2M NHS to form 400 μ L of 0.4M EDC/0.1MNHS mixed solution, and performing activation reaction on the surface of the chip for 40min at a flow rate of 10 μ L/min; then respectively reacting the negative sieve protein and the lactoferrin in the two channels at the flow rate of 10 mu L/min for 25 min; finally sealing the surface of the chip for 10min by 200 mu L of 1M ethanolamine at the flow rate of 20 mu L/min; then, using 1 XPBSM buffer solution to balance the chip for 30 min;

(5) the six-way valve is connected to the inlet of the negative sieve channel, a constant flow injection pump is used for transmitting the screening buffer solution at the flow rate of 20 mu L/min, the six-way valve is used for injecting the 200 mu L decahedral nano silver loading library, and meanwhile, the signals of the positive sieve channel and the negative sieve channel are collected and stored, and the reaction time is 40 min.

(6) Separating the positive sieve channel from the negative sieve channel, washing the positive sieve channel for 15min by using a screening buffer solution, then washing the positive sieve channel by using 90 mu L of 50mM NaOH solution at a flow rate of 10-50 mu L/min, collecting an eluted sample, and then adding 37.5 mu L of 120mM hydrochloric acid solution for neutralization;

(7) PCR amplification and ssDNA purification:

and (3) PCR amplification: the collected and neutralized product is pre-amplified for 7-9 times, and the PCR amplification conditions are (95 ℃,5 min; 60 ℃, 30 s; 72 ℃, 30 s; 4 ℃,10 min). Dividing the solution into 40 parts, performing amplification for 9 rounds to obtain a product, and combining the 40 parts of solution;

and (3) ssDNA purification: and adding the 40 parts of combined product into the washed magnetic beads, oscillating the mixed product in a constant-temperature oscillating table at 37 ℃ for 30min, removing supernatant by magnetic separation, washing the magnetic beads for 2-3 times by using 1 XPBS, and removing the supernatant. Then, 5. mu.L of a 50mM NaOH solution was added thereto, and after separating the supernatant, 50. mu.L of a 50mM hydrochloric acid solution was added thereto for neutralization.

Then repeating the steps (1) - (7) to perform the next several rounds of screening, and then sending the samples of the 3 rd, 4 th and 5 th rounds to perform clone sequencing to obtain a plurality of nucleic acid sequences with high repeatability. And using Surface Plasmon Resonance (SPR) to determine its affinity constant (K) for lactoferrin_d) Characterization was performed. Finally, 5 high affinity lactoferrin aptamer chains were obtained. The SPRI screening curve is shown in FIG. 3 with an imaging plot.

The decahedral nano silver is applied to the amplification of the SPRI on the sensing signal, can enhance the SPRI signal by 128 times, and has a good signal enhancement function. Under the same condition, the screening library of the decahedral nano silver loaded lactoferrin uses a real-time label-free method to screen the lactoferrin, and under the condition of 5 screening rounds, 6 aptamer chains with high affinity are obtained.

SEQUENCE LISTING

<110> Nanjing university

Application of <120> decahedral nano silver probe in screening aptamer by using surface plasmon resonance imaging technology

<130>SG20170112001

<160>4

<170>PatentIn version 3.5

<210>1

<211>25

<212>DNA

<213>Artificial Sequence

<220>

<223> library reverse primer complementary strand

<400>1

aaaaaaaaaa gaagaggagg gaggt 25

<210>2

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> nucleic acid library sequences 5' terminal sequence

<400>2

gacaggcagg acaccgtaac 20

<210>3

<211>20

<212>DNA

<213>Artificial Sequence

<220>

<223> 3' terminal sequence of nucleic acid library sequences

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ctgctacctc cctcctcttc 20

<210>4

<211>21

<212>DNA

<213>Artificial Sequence

<220>

<223> oligonucleotide chain

<400>4

tttgggtttg ggtttgggtt t 21

Claims

1. The application of the decahedral nano silver probe in screening the aptamer by using a surface plasma resonance imaging technology;

the decahedral nano silver probe comprises the following components: decahedral nano silver with the particle size of 50-60 nm is taken as an inner core, two nucleotide chains are arranged on the outer surface of the nano silver,

wherein the structure of the complementary strand of the reverse primer of the library is as follows: the 3 'end is a complementary sequence of a reverse primer of the library, 10-18A basic groups are arranged in the middle and serve as spacer arms, and the 5' end is modified by sulfydryl;

(1) synthesis of decahedral nano silver: sodium citrate, polyvinylpyrrolidone, L-arginine and silver nitrate are mixed according to a molar ratio of (0.1-70): (0.1-10): (0.2-30): (0.2-30) adding the mixture into 100-300 ml of deionized water, and then adding 0.1-1M of sodium borohydride aqueous solution to enable the molar ratio of sodium borohydride to silver nitrate to be (1-100): 1, reacting for 16-72 hours under the irradiation of a blue light lamp to obtain a decahedral nano silver solution;

wherein the molar ratio of the nano-silver to the complementary strand of the library reverse primer to the sodium chloride is (0.1-10 pmol): (0.1 to 10nmol): (10-200 mu mol), dripping Tween-20, wherein the volume ratio of the Tween-20 to the nano-silver solution is (0.001-0.01): 1, uniformly mixing, and shaking for 1-5 h at 37 ℃;

(3) standing the mixed system obtained in the step (2) for 10-24 hours;

(4) centrifuging the solution obtained in the step (3), adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the steps of centrifuging and resuspending for 2-4 times, wherein the volume of the 1 × PBS buffer solution added each time is 0.1-5 times of the volume of the precipitate; finally, the sediment obtained by centrifugation is resuspended by using 1 XPBS buffer solution;

2. The decahedral nano-silver probe for screening the lactoferrin nucleic acid aptamer is characterized in that 50-60 nm decahedral nano-silver is used as an inner core, two nucleotide chains are arranged on the outer surface of the nano-silver,

wherein the complementary strand of the library reverse primer is:

5’- SH -(CH2)₆-AAAAAAAAAAGAAGAGGAGGGAGGT-3’；

the nucleic acid library sequence is:

3. The method for preparing a decahedral nanosilver probe of claim 2, characterized by comprising the steps of:

(1a) synthesis of decahedral nano silver: sodium citrate, polyvinylpyrrolidone, L-arginine and silver nitrate are mixed according to a molar ratio of (0.1-70): (0.1-10): (0.2-30): (0.2-30) adding the mixture into 100-300 ml of deionized water, and then adding 0.1-1M of sodium borohydride aqueous solution to enable the molar ratio of sodium borohydride to silver nitrate to be (1-100): 1, reacting for 16-72 hours under the irradiation of a blue light lamp to obtain a decahedral nano silver solution;

(2a) modification of decahedral nano silver: taking the decahedral nano silver solution obtained in the step (1a), adding a library reverse primer complementary strand and sodium chloride,

the complementary strand of the library reverse primer is:

5’- SH -(CH2)₆-AAAAAAAAAAGAAGAGGAGGGAGGT-3’；

(3a) standing the mixed system obtained in the step (2a) for 10-24 hours;

(4a) centrifuging the solution obtained in the step (3a), adding the precipitate into 1 × PBS buffer solution for resuspension, repeating the centrifuging and resuspending steps for 2-4 times, wherein the volume of the 1 × PBS buffer solution added each time is 0.1-5 times of the volume of the precipitate; finally, the sediment obtained by centrifugation is resuspended by using 1 XPBS buffer solution;

(5a) adding a nucleic acid library solution into the mixed system obtained in the step (4a), so that the molar ratio of the nucleic acid library to the decahedral nano silver modified with the reverse primer complementary strand is (100-2000): 1, carrying out oscillation reaction for 10-60 min to obtain a decahedral nano silver probe;

the nucleic acid library sequence is:

4. Use of the decahedral nanosilver probe of claim 2 for screening lactoferrin nucleic acid aptamers.

5. The use according to claim 4, wherein the step of screening for aptamers using decahedral nanosilver probes is as follows:

(1b) the SPRI sensor chip surface sulfhydryl alkyl acid self-assembly:

(2b) preparing a solution:

positive sieve protein solution: dissolving lactoferrin in 1 XPBS buffer solution, wherein the concentration of lactoferrin is 250 mug/mL;

the negative sieve protein solution is prepared by respectively dissolving BSA, α -lactalbumin, β -lactoglobulin and casein in 10mM sodium acetate solution, wherein the pH value is 4.5, and the concentrations of the BSA, the α -lactalbumin, the β -lactoglobulin and the casein are respectively 160 mu g/mL;

screening buffer solution: adding MgCl to 1 XPBS₂•6H₂O solid, 1 x PBSM formed as screening buffer;

chip activation reagent: 0.8M EDC aqueous solution, 0.2M NHS aqueous solution;

blocking reagent: 1M ethanolamine solution;

(3b) protein fixation:

transferring 200 μ L of 0.8M EDC, mixing with 200 μ L of 0.2M NHS to form 400 μ L of mixed solution of 0.4M EDC and 0.1M NHS, and performing activation reaction on the surface of the chip for 40min at a flow rate of 10 μ L/min; then respectively reacting positive sieve protein and negative sieve protein in the two channels at the flow rate of 10 mu L/min for 40 min; finally, sealing 200 mu L of 1M ethanolamine at the flow rate of 20 mu L/min for 10 min; then using 1 XPBS buffer solution to balance the chip for 30 min;

(4b) after the base line is stable, connecting the positive sieve channel and the negative sieve channel in series; the six-way valve is connected to the inlet of the negative sieve channel, a constant flow injection pump is used for transmitting the screening buffer solution at the flow rate of 20 mu L/min, the six-way valve is used for injecting the 200 mu L decahedral nano silver loading library, and meanwhile, the signals of the positive sieve channel and the negative sieve channel are collected and stored, and the reaction time is 40 min;

(5b) separating the positive sieve channel from the negative sieve channel, washing the positive sieve channel for 15min by using a screening buffer solution, then washing the positive sieve channel by using 90 mu L of 50mM NaOH solution at a flow rate of 10-50 mu L/min, collecting an eluted sample, and then adding 37.5 mu L of 120mM hydrochloric acid solution for neutralization;

(6b) PCR amplification and ssDNA purification: