阅读说明：本技术 一种基于sers光谱技术的白血病融合基因检测方法 (leukemia fusion gene detection method based on SERS spectroscopy technology ) 是由 冯尚源 王运燚 贾香港 林学亮 许云超 于 2019-10-31 设计创作，主要内容包括：本发明构建一个特异性检测BCR-ABL融合基因(b3a2)的生物传感器,能够实现超高灵敏度白血病融合基因定量检测。主要的技术思路是利用表面增强拉曼光谱(SERS)极高的检测灵敏度,通过组装二维SERS基底后利用融合基因DNA的互补配对将AgNPs与SERS基底上的银纳米球耦连起来形成纳米间隙,两个纳米球互相耦合导致纳米间隙中电磁场非常强,这样就可以构筑具有极强SERS效应的热点结构,进而使标记分子拉曼信号获得巨大增强,最终可以得到超高灵敏度的SERS光谱检测结果。该检测方法能够很好的解决目前临床上检测方法灵敏度低、特异性弱等问题,为准确诊断慢性髓系白血病提供一种简单、快速、有效的检测手段。(The invention constructs biosensors for specifically detecting BCR-ABL fusion genes (b3a2), and can realize the quantitative detection of the leukemia fusion genes with ultrahigh sensitivity, the main technical idea is to utilize the extremely high detection sensitivity of Surface Enhanced Raman Spectroscopy (SERS), couple AgNPs and silver nanospheres on an SERS substrate by utilizing the complementary pairing of fusion gene DNA after assembling a two-dimensional SERS substrate to form a nanogap, and the mutual coupling of the two nanospheres leads to the extremely strong electromagnetic field in the nanogap, so a hot spot structure with extremely strong SERS effect can be constructed, further the marked molecular Raman signal is greatly enhanced, and finally the SERS spectrum detection result with ultrahigh sensitivity can be obtained.)

1, leukemia fusion gene detection method based on SERS spectroscopy, characterized by comprising the following steps:

preparing an SERS substrate;

1-1) synthesizing AgNPs with negatively charged surfaces: reducing silver nitrate by using a reducing agent sodium citrate to synthesize colloidal silver: mixing AgNO₃Heating the solution under vigorous stirring, boiling, and adding sodium citrate; keeping the liquid boiling, continuously heating and stirring for 60 minutes, and changing the color into yellow green;

1-2) preparation of surface hydroxylated silica flakes: cutting the silicon dioxide sheet into the size of 4mm multiplied by 4mm, and then respectively carrying out ultrasonic treatment in acetone, ethanol and ultrapure water for 10 minutes; taking out and drying, soaking the silicon dioxide slices in a freshly prepared piranha solution, heating, cooling, cleaning with ultrapure water, standing and drying in the air to finish surface hydroxylation of the silicon dioxide slices;

1-3) protonation of the amino group of the silane coupling agent is completed: dispersing a silane coupling agent in ethanol, then soaking a silica sheet with a hydroxylated surface in the solution, taking out, washing with a large amount of water, taking out the unmodified silane coupling agent, airing the silica sheet, and then soaking in ultrapure water to complete protonation of an amino group of the silane coupling agent;

1-4) assembly of substrates: taking the silver nanoparticles newly synthesized in the step 1-1) and having the surfaces with negative electricity, soaking the protonated silicon dioxide sheet in the silver nanoparticles, taking out the silver nanoparticles and cleaning the silver nanoparticles with ultrapure water to obtain an SERS substrate;

2) synthetic AgNPS with positively charged surface

Taking AgNO₃Putting 90ml of the solution into a conical flask and stirring; the hydroxylamine hydrochloride and the sodium hydroxide are respectively taken and mixed and then are rapidly added into the AgNO₃In the solution, the solution in the conical flask changes from colorless to gray;

3) assembly of SERS detection sensor

3-1) specifically combining the signal sequence and the capture sequence with AgNPs and an SERS substrate respectively, wherein the capture sequence and the signal sequence are respectively put into a mixed solution of TCEP (5 mM) and Tris-HCl buffer solution to activate sulfydryl;

3-2) completing the assembly of the SERS detection sensor: adding the signal sequence modified AgNPs solution with positive charges into a silicon dioxide SERS substrate, adding target sequences with different concentrations, cleaning the silicon dioxide SERS substrate after reaction, and preparing for the next SERS measurement after drying at normal temperature.

2. The leukemia fusion gene detection method based on SERS spectroscopy according to claim 1, wherein:

preparation of SERS substrate

1-1) synthesizing AgNPs with negatively charged surfaces: reducing silver nitrate by using a reducing agent sodium citrate to synthesize colloidal silver: AgNO at a concentration of 1mM in a volume of 100mL₃Heating the solution under vigorous stirring, boiling, and adding 3mL of 1% sodium citrate; keeping the liquid boiling, continuously heating and stirring for 60 minutes, and changing the color into yellow green;

1-2) preparation of surface hydroxylated silica flakes: cutting the silicon dioxide sheet into the size of 4mm multiplied by 4mm, and then respectively carrying out ultrasonic treatment in acetone, ethanol and ultrapure water for 10 minutes; taking out and drying, soaking the silicon dioxide slices in a freshly prepared piranha solution, and heating to 150 ℃ for 30 minutes; after cooling, washing with ultrapure water, standing and airing to finish the surface hydroxylation of the silicon dioxide sheet;

the piranha solution is a mixed solution of concentrated sulfuric acid and 30% hydrogen peroxide in a ratio of 7: 3;

1-3) protonation of the amino group of the silane coupling agent is completed: dispersing 150 mu L of silane coupling agent in 150mL of ethanol, then soaking the silica plate with the hydroxylated surface in the solution for 12h, taking out, washing with a large amount of water, and taking out the unmodified silane coupling agent; drying the silicon dioxide sheet, and soaking the silicon dioxide sheet in ultrapure water for 8 hours to complete protonation of amino groups of the silane coupling agent;

1-4) preparing a substrate: taking 370 mu L of newly synthesized silver nanoparticles obtained in the step 1), soaking the protonated silicon dioxide sheet in the silver nanoparticles for 4h, taking out the silicon dioxide sheet, and cleaning the silicon dioxide sheet with ultrapure water to obtain an SERS substrate;

2) synthetic AgNPS with positively charged surface

Taking the concentration of 1.1 × 10^-3mol/L AgNO₃Putting 90ml of the solution into a conical flask and stirring; respectively taking 6 × 10^-25ml of hydroxylamine hydrochloride in mol/L and 4.5ml of sodium hydroxide in 0.1mol/L, and mixing the twoQuickly added to 90ml AgNO after combination₃In the solution, the solution in the conical flask changes from colorless to gray;

3) assembly of SERS detection sensor

3-1) taking 20. mu.L of 1. mu.M capture sequence and 20. mu.L of 1. mu.M signal sequence, respectively, and putting into a mixed solution of 20. mu.L of 5mM TCEP and 50. mu.L of 1M Tris-HCl buffer, pH =7.4, to activate thiol groups; after reacting for 1 hour, soaking the silicon dioxide SERS substrate by using a capture sequence solution, mixing a signal sequence solution with AgNPs with positive charges, standing overnight at room temperature, respectively adding 20 mu L of 1 mu M6-sulfydryl-1-hexanol for reacting for 3 hours, and respectively washing by using a large amount of water to remove sequences which are not specifically combined, thereby finishing the modification of sulfydryl;

3-2) adding the positively charged AgNPs solution modified with the signal sequence into a silicon dioxide SERS substrate, adding target sequences with different concentrations, reacting for 4h, cleaning the silicon dioxide SERS substrate, and drying at normal temperature to prepare for the next SERS measurement.

3. The leukemia fusion gene detection method based on SERS spectroscopy according to claim 2, wherein the base sequences of the capture sequence, the signal sequence and the target sequence are as follows:

(1) capture sequence: 5 '-SH-TTTTTCCCAACCCAACCCTCCTTGGAGTTCCAACGAGCGGCTTCACTCAGACCCTGAGGCTCAAAGTCAGATGCTACTGGCCGCTGAAGGGCTT-3';

(2) signal sequence:

5’- Cy5-TTGAACTCTGCTTAAATC-SH-3’;

(3) the target sequence is: GATTTAAGCAGAGTTCAA and merges into point AAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAGGGTTGGGTTGGGAAAAA.

Technical Field

The invention belongs to the field of biomedical leukemia gene detection, and particularly relates to methods for realizing the ultra-sensitive detection of leukemia fusion genes (b3a2) based on an SER (serial enhanced Raman scattering) technology.

Background

In recent years, the incidence of global leukemia has been increasing year by year, wherein the incidence of leukemia is ranked eighth in terms of incidence of all cancers in the United states, in China, leukemia is ranked eighth in terms of male incidence, and in terms of incidence and mortality in adolescent populations . at present, the medicine is classified into acute and chronic leukemia according to the differentiation degree of leukemia Cells.Chronic Myelogenous Leukemia (CML) is of chronic leukemia, the mechanism of pathological formation is that Philadelphi chromosomes located between chromosome 9 and chromosome 22 long arms are translocated.A specific molecular mechanism is that ABL genes located on chromosome 9 long arms of normal human and BCR genes located on chromosome 22 of normal human are recombined to generate BCR-ABL fusion genes after chromosome translocation.A gene ABL gene, also called ABL1 gene, is protooncogenes, which are fused with various genes in tumor cells, wherein the fusion with BCR genes is the most common, the fusion of the gene genes, the gene is also called ABL1 gene, the gene, which is also has strong sensitivity to the gene encoding tyrosine-encoding the gene, the gene is fused with the gene, the gene has the gene-induced by the gene-induced fusion of the gene, the gene has the high sensitivity of the gene, the gene has the high gene-induced fusion of the high-induced fusion of the gene-induced cell growth and the high-induced fusion of the gene, the gene has the high-induced fusion of the gene, the gene has the high-induced fusion of the gene, the gene has the high-induced cell growth of the gene, the gene has the high-induced cell growth of the gene has the high-induced cell growth of the gene of.

Since the advent of Surface Enhanced Raman Scattering (SERS), there has been interest from many scientists in many fields, mainly because SERS spectra have high detection sensitivity, strong specificity, narrow half-peak width of the spectral peak and can provide abundant molecular fingerprint information, low damage, short time consumption, easy operation and convenient carrying, at present, SERS has been widely applied to the fields of disease diagnosis, drug detection, food safety, material characterization, etc. we have recently made many studies on the diagnosis of SERS spectral diseases.

Disclosure of Invention

Compared with the method for clinically detecting the BCR-ABL fusion gene, the method has the advantages that SERS can just solve problems faced by the BCR-ABL fusion gene, silver nanoparticles (AgNPs) with negative electricity are firstly synthesized, meanwhile, a silane coupling agent is used for modifying a hydroxylated silica plate, then, the silicon wafer is put into water for protonation, the AgNPs with negative electricity are assembled on the silica plate, then, AgNPs with positive electricity are synthesized, two sections of complementary sequences with the BCR-ABL fusion gene are respectively assembled on the SERS substrate of the silica plate and the AgNPs with positive electricity, a DNA fragment assembled on the AgNPs with positive electricity is modified with Cy5 Raman marker molecules, when a target sequence BCR-ABL fusion gene appears, Raman marker molecules Cy5 are just positioned at a 'hot spot' position formed by two silver nanospheres, BCR-ABL fusion gene (b3a2) samples with different concentrations are arranged, and high-sensitivity and quantitative detection of the BCR-ABL fusion gene (b3a2) can be realized according to the change of the SERS spectrum intensity of the Raman marker molecules Cy 5.

In order to achieve the purpose, the invention adopts the following technical scheme:

method for detecting leukemia fusion gene (b3a2) based on SERS technology, comprising the following steps:

1) preparation of SERS substrate

1-1) synthesizing AgNPs with negatively charged surfaces: reducing silver nitrate by using a reducing agent sodium citrate to synthesize colloidal silver: AgNO at a concentration of 1mM in a volume of 100mL₃The solution was boiled under vigorous stirring and 3mL of 1% sodium citrate was added. Keeping the liquid boiling, continuously heating and stirring for 60 minutes, and changing the color into yellow green

1-2) preparation of surface hydroxylated silica flakes: the silica sheet was cut into a size of 4mm × 4mm, and then subjected to ultrasonic treatment in acetone, ethanol, and ultrapure water for 10 minutes each. After being taken out and dried, the silicon dioxide slices are soaked in a freshly prepared piranha solution (a mixed solution of concentrated sulfuric acid and 30% hydrogen peroxide in a ratio of 7: 3) and heated to 150 ℃ for 30 minutes. After cooling, the silicon dioxide sheet is cleaned by ultrapure water, placed and dried, and the surface hydroxylation of the silicon dioxide sheet is completed.

1-3) protonation of the amino group of the silane coupling agent is completed: 150 mu L of silane coupling agent is dispersed in 150mL of ethanol, then the silica piece with the surface hydroxylated is soaked in the solution for 12h, and after being taken out, the unmodified silane coupling agent is taken out by washing with a large amount of water. And (3) airing the silicon dioxide sheet, and then soaking the silicon dioxide sheet in ultrapure water for 8 hours to complete protonation of amino groups of the silane coupling agent.

1-4) preparing a substrate: taking 370 mu L of newly synthesized silver nanoparticles obtained in the step 1), soaking the protonated silicon dioxide sheet in the silver nanoparticles for 4h, taking out the silicon dioxide sheet, and cleaning the silicon dioxide sheet with ultrapure water to obtain the SERS substrate.

2) Synthetic AgNPS with positively charged surface

Taking the concentration of 1.1 × 10^-3mol/L AgNO₃90ml of the solution was put into an Erlenmeyer flask and stirred. Respectively taking 6 × 10^-25ml of hydroxylamine hydrochloride in mol/L and 4.5ml of sodium hydroxide in 0.1mol/L are mixed and then quickly added into 90ml of AgNO₃In the solution, the solution in the Erlenmeyer flask changed from colorless to gray.

3) Assembly of SERS detection sensor

3-1) taking 20 μ L of 1 μ M capture sequence and 20 μ L of 1 μ M signal sequence respectively, putting into a mixed solution of 20 μ L of TCEP (5 mM) and 50 μ L of Tris-HCl buffer (1M, PH =7.4) to activate sulfydryl, after 1 hour of reaction, soaking a silicon dioxide SERS substrate with the capture sequence solution, mixing the signal sequence solution with quantitative positively charged AgNPs, standing at room temperature overnight, adding 20 μ L of 1 μ M6-sulfydryl-1-hexanol respectively, reacting for 3 hours, and then washing with a large amount of water to remove the sequences which are not specifically bound, namely finishing the modification of sulfydryl.

3-2) adding the positively charged AgNPs solution modified with the signal sequence into a silicon dioxide SERS substrate, adding target sequences with different concentrations, reacting for 4h, cleaning the silicon dioxide SERS substrate, and drying at normal temperature to prepare for the next SERS measurement.

The base sequences of the capture sequence, the signal sequence and the target sequence are as follows:

(1) capture sequence:

5’-SH-TTTTTCCCAACCCAACCCTCCTTGGAGTTCCAACGAGCGGCTTCACTCAGACCCTGAGGCTCAAAGTCAGATGCTACTGGCCGCTGAAGGGCTT-3’；

(2) signal sequence:

5’- Cy5-TTGAACTCTGCTTAAATC-SH-3’;

(3) the target sequence is:

GATTTAAGCAGAGTTCAA (fusion point) AAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAGGGTTGGGTTGGGAAAAA.

The method has the advantages that silicon dioxide is used as a substrate, silver nanoparticles (AgNPs) with negative electricity are used as a substrate for surface enhanced Raman scattering detection, biosensors for specifically detecting BCR-ABL fusion genes (b3a2) are formed through subsequent treatment, a silicon dioxide sheet is modified by using a silane coupling agent and is protonated to adsorb the synthesized silver nanoparticles with negative electricity, and finally, chemical bonds formed by silver and amino groups are firmly assembled on the silicon dioxide sheet to construct a two-dimensional SERS substrate, a signal chain and a capturing chain are respectively modified on AgNPs and SERS substrates with positive electricity, after a signal chain with Cy5 Raman marker molecules is added, three links are combined together at to form detection sensors, quantitative detection on the BCR-ABL fusion genes (b3a2) is realized by detecting Raman spectrum signals of Cy5, meanwhile, complementary matching of DNA can couple AgNPs with the AgNPs on the SERS substrate with positive electricity and AgNPs marked with the Cy molecules to form nanogaps with nano gaps with similar to 5 marker molecules, a 'strong resonance structure with a' strong resonance effect is constructed, and a hot spot enhanced Raman spectrum detection result is finally obtained, and the excimer detection is in a huge high sensitivity detection effect.

Drawings

FIG. 1 is an experimental schematic diagram of detecting BCR-ABL fusion gene by SERS spectroscopy.

FIG. 2 is a UV-VIS absorption spectrum and scanning electron microscope image of AgNPs with negative and positive charges; wherein, (A) is the ultraviolet-visible absorption spectrum of the AgNPs with negative electricity and positive electricity, (B) is the transmission image of the AgNPs with negative electricity, and (C) is the TEM image of the AgNPs with positive electricity.

FIG. 3 is a scanning electron micrograph of a silica plate assembled with negatively charged AgNPs.

FIG. 4 shows the result of SERS spectrum detection of R6G solution; wherein (A) is SERS spectrum of R6G measured by dropping method and soaking method, and (B) is spectrum 611cm of R6GSERS measured by dropping method^-1Peak intensity variation, and (C) detecting R6G SERS spectrum 611cm by soaking method^-1The peak intensity changes.

FIG. 5 shows the result of SERS spectroscopy quantitative detection of BCR-ABL fusion gene (b3a 2); wherein (A) is BCR-ABL fusion gene (B3a2) SERS spectrogram with different concentrations, and (B) is Lg (C)_Target) And linear relationship between spectral peak intensities.

Detailed Description

The present invention will now be described with reference to specific embodiments and with reference to the accompanying drawings, at .

1. Reagent

Silver nitrate (AgNO)₃) Hydroxylamine hydrochloride (HO-NH)₂HCl), sulfuric acid (H)₂SO₄) Anhydrous ethanol and hydrochloric acid (HCl) were purchased from national pharmaceutical group chemical reagents, Inc., silane coupling agent (. gamma. -aminopropyltriethoxysilane) was purchased from Shandong Youso chemical technology, Inc., trisodium citrate dihydrate (Na)₃C₆H₅O₇.2H₂O), sodium hydroxide (NaOH), 30% hydrogen peroxide (H)₂0₂) The DNA sequence used in this experiment was obtained from Shanghai glass boat plastic, Inc. and was analyzed for all reagents and was not purified , which were purchased from West Gansu science, Inc., 4-aminothiophenol (4-ATP), Tris (2-carboxyethyl) phosphine (TCEP), and Tris (Tris) aminomethane (Tris) from Shanghai Aladdin Biotechnology, Inc., 6-mercapto-1-hexanol (MCH) from Bailingwei science, Inc., rhodamine 6G (R6G) from Sigma, and silica plate from Jiangsu glass boat plastic, Inc.

2. Synthesis of AgNPs with negative surface

Lee and Meisel (Lee P C, Meisel D. Adsorption and surface-enhanced ramanian of dies on silver and gold gases [ J ] were used]The Journal of Physical Chemistry, 1982,86(17): 3391-3395), etc., and The colloidal silver is synthesized by reducing silver nitrate with a reducing agent sodium citrate. The invention has the volume of 100mL and the concentration of 1mM AgNO₃The solution was boiled under vigorous stirring and 3mL of 1% sodium citrate was added. The liquid was kept boiling and stirring was continued for 60 minutes, and the color changed to yellow-green.

Preparation of SERS substrate

1) The silica sheet was cut into a size of 4mm × 4mm, and then subjected to ultrasonic treatment in acetone, ethanol, and ultrapure water for 10 minutes each. After being taken out and dried, the silicon dioxide slices are soaked in a freshly prepared piranha solution (a mixed solution of concentrated sulfuric acid and 30% hydrogen peroxide in a ratio of 7: 3) and heated to 150 ℃ for 30 minutes. After cooling, the silicon dioxide sheet is cleaned by ultrapure water, placed and dried, and the surface hydroxylation of the silicon dioxide sheet is completed.

2) 150 mu L of silane coupling agent (gamma-aminopropyltriethoxysilane) is dispersed in 150mL of absolute ethanol, then the silica piece with the surface hydroxylated is soaked in the solution for 12h, and after taking out, the unmodified silane coupling agent is taken out by washing with a large amount of water.

3) And (3) airing the silicon dioxide sheet, and then soaking the silicon dioxide sheet in ultrapure water for 8 hours to complete protonation of amino groups of the silane coupling agent. And (3) taking 370 mu L of silver nano particles newly synthesized in the step (2) with negative surfaces, soaking the protonated silicon dioxide sheet in the silver nano particles for 4h, taking out the silicon dioxide sheet and cleaning the silicon dioxide sheet by using ultrapure water.

4. AgNPS synthesis with positively charged surface

Taking the concentration of 1.1 × 10^-3mol/L AgNO₃90ml of the solution was put into an Erlenmeyer flask and stirred. Respectively taking 6 × 10^-25ml of hydroxylamine hydrochloride in mol/L and 4.5ml of sodium hydroxide in 0.1mol/L are mixed and then quickly added into 90ml of AgNO₃In the solution, the solution in the Erlenmeyer flask changed from colorless to gray.

Assembly of SERS detection sensor

1) mu.L of 1. mu.M capture sequence and 20. mu.L of 1. mu.M signal sequence were taken and put into a mixed solution of 20. mu.L of TCEP (5 mM) and 50. mu.L of Tris-HCl buffer, respectively, to activate thiol groups. After 1 hour of reaction, the silica SERS substrate was soaked with the capture sequence solution and the signal sequence solution was mixed with 500 μ L of positively charged AgNPs while standing overnight at room temperature.

2) mu.L of 1. mu.M 6-mercapto-1-hexanol was added to react for 3 hours, and then the sequences not specifically bound were washed away with a large amount of water, respectively, to complete the modification of the mercapto group. Adding a signal sequence modified positively charged AgNPs solution into a silicon dioxide SERS substrate, and adding target sequences (10) with different concentrations^-6To 10^-12Seven BCR-ABL fusion gene (b3a2) standard samples with concentration gradient) react for 4 hours, then the silicon dioxide SERS substrate is cleaned, and the Raman spectrum detection is carried out after the silicon dioxide SERS substrate is dried at normal temperature. The Raman spectrometer used was a Renishaw inVia Raman microchip (Renishaw plc, UK), the laser being a semiconductor laser with a wavelength of 785 nm.

The base sequences of the capture sequence, the signal sequence and the target sequence are as follows:

(1) capture sequence:

5’-SH-TTTTTCCCAACCCAACCCTCCTTGGAGTTCCAACGAGCGGCTTCACTCAGACCCTGAGGCTCAAAGTCAGATGCTACTGGCCGCTGAAGGGCTT-3’；

(2) signal sequence:

5’- Cy5-TTGAACTCTGCTTAAATC-SH-3’;

(3) the target sequence is:

GATTTAAGCAGAGTTCAA (fusion point) AAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAGGGTTGGGTTGGGAAAAA.

The invention characterizes the synthesized AgNPs with negative charge and positive charge by ultraviolet-visible absorption spectrum and a scanning electron microscope. Fig. 2 (a) is an ultraviolet-visible absorption spectrum of negatively charged AgNPs, whose plasmon resonance peak is at 412nm, and positively charged AgNPs whose plasmon resonance peak is at 423 nm. As can be seen from the TEM image, the average particle size of the negatively charged AgNPs is 77 + -5 nm (FIG. 2B), and the average particle size of the positively charged AgNPs is 27 + -10 nm (FIG. 2C).

As the silane coupling agent is modified on the silicon dioxide sheet, amino groups are exposed outside, the protonation treatment enables the surface of the silicon dioxide sheet to be positively charged, the silicon dioxide sheet is mutually attracted with the AgNPs with negative charges and finally forms a bond with the amino groups, and the assembly efficiency is improved, the AgNPs with negative charges are uniformly assembled on the silicon dioxide sheet, and the AgNPs with negative charges are mutually repelled and dispersedly arranged among particles, so that fixed intervals are maintained, the aggregation of the particles is less, and the particle assembly density on the whole silicon dioxide sheet is high.

In order to make the experimental data obtained in the present invention more reliable and reproducible, we tested using two methodsComparison was made to rhodamine (R6G) SERS spectra. The prepared silicon dioxide SERS substrate is used for detection by adopting two methods of soaking and dropping 10 respectively^-6The results of SERS spectroscopy detection of the solution of R6G and R6G in mol/L are shown in FIG. 4. In fig. 4 (a), the black curve is a SERS spectrum obtained by the spot method, and the red curve is a SERS spectrum obtained by the immersion method of R6G solution, each spectrum being an average value taken from ten random points. FIG. 4 (B) is 611cm corresponding to SERS spectra of R6G solution obtained by randomly taking ten points in the spot-drop method^-1The peak intensity changes, and FIG. 4 (C) is 611cm corresponding to SERS spectra of R6G solution randomly taken ten points in the soaking method^-1Peak intensity variation of 611cm^-1The peak may be attributed to an in-plane bending vibration model of the C-C-C ring. Combining the three data plots in fig. 4, although the intensity of the spectral peaks is stronger for the droplet method, the droplet method is more affected by the "coffee ring" effect, and the infusion method is less affected by the "coffee ring" effect, as analyzed from the Relative Standard Deviation (RSD). Therefore, the subsequent data of the study all adopt a soaking method. Meanwhile, RSD data also prove that the SERS substrate in the research has better uniformity and reproducibility.

This study took 10^-6To 10^-12SERS spectrum quantitative detection of the BCR-ABL fusion gene (b3a2) is carried out on seven standard samples of the BCR-ABL fusion gene (b3a2) with concentration gradients, and the obtained result is shown in FIG. 5. Selecting 691cm^-1Performing linear fitting on the Cy5SERS spectral characteristic peak at the wave number position and the target chain concentration to obtain the logarithm Lg (C) of the Raman signal intensity I and the target chain concentration_-Target) The linear relationship between:

I=5.9832×10^-4Lg(C_-Target) +0.00822, the limit of detection (LOD) of this detection method was calculated to be 42.28fM, which is reported to be 10 for real-time fluorescent quantitative PCR detection^-5~10^-6M, compared with the detection limit of the method, the detection limit is improved by 8 orders of magnitude. Therefore, the method can realize the ultrasensitive SERS spectrum detection of the BCR-ABL fusion gene (b3a 2).

SEQUENCE LISTING

<110> university of Fujian profession

<120> leukemia fusion gene detection methods based on SERS spectroscopy technology

<130>3

<160>3

<170>PatentIn version 3.3

<210>1

<211>94

<212>DNA

<213> Artificial sequence

<400>1

tttttcccaa cccaaccctc cttggagttc caacgagcgg cttcactcag accctgaggc 60

tcaaagtcag atgctactgg ccgctgaagg gctt 94

<210>2

<211>18

<212>DNA

<213> Artificial sequence

<400>2

ttgaactctg cttaaatc 18

<210>3

<211>112

<212>DNA

<213> Artificial sequence

<400>3

gatttaagca gagttcaaaa gcccttcagc ggccagtagc atctgacttt gagcctcagg 60

gtctgagtga agccgctcgt tggaactcca aggagggttg ggttgggaaa aa 112