CN1219075C - Preparation and Application of Core/Shell Magnetic Nanoparticle Labeled Gene Probe - Google Patents

Abstract

The invention relates to a gene detection technology, in particular to an ultra-sensitive detection method for nucleic acid by using magnetic and non-magnetic nano-particles to mark gene probes. Magnetic Fe₃O₄The (core)/Au (shell) nanoparticle marker gene is used as a capture probe, and a hybrid containing the target is separated through magnetic field control; the connecting chain is designed to replace a target chain, and the connecting chain and the nano probe are repeatedly added to a hybrid to form a continuously stacked hybrid aggregate, so that nano amplification of a detection signal is realized.

Description

Preparation and Application of Core/Shell Magnetic Nanoparticle-labeled Gene Probes--Methods for Establishing a Detection System of HBV Deoxyribose Nucleic Acid

technical field

The invention relates to the technical field of virus gene detection. Specifically, a method for detecting viral DNA and its variation is established by using Fe ₃ O ₄ (core)/gold (shell) nanoparticle virus deoxyribonucleic acid (DNA) probes through secondary amplification technology.

Background technique

Nanoparticles are particles with a diameter of 1-100nm. In this scale range, nanomaterials have many new properties, so it has become a hot field of scientific research. Utilizing the unique characteristics of force, electricity, light, and magnetism of nanomaterials, a highly interdisciplinary nanobiomedical technology has been formed, which has a wide range of application values in reality. In 1996, Chad A Mirkin et al covalently bound alkanethiol-modified oligonucleotides to the surface of gold nanoparticles through gold-sulfur (Au-S) bonds to form DNA probes labeled with gold nanoparticles, and Using this probe, target DNA is detected. They found that: when the gold nanoparticle aggregates are formed, the detection limit of the target single chain can reach 10fMol; when the gold nanometer monolayer is formed and Ag ⁺ -hydroquinone solution is added for amplification, the detection limit of the target single chain can reach 50fMol . The research results were published in Science.

Viral hepatitis (especially hepatitis B) is an important disease that seriously endangers human health. It is estimated that there are 400-500 million hepatitis B virus (hepatitis B, HBV) carriers in the world. my country is an area with a high incidence of hepatitis B infection, and the HBV carrier rate of the population is 10-20%. The direct economic loss caused by viral hepatitis to the country is about 50-100 billion yuan each year, and the resulting social burden and other negative effects are even greater. for huge. For hepatitis B patients (including before and after liver transplantation) after antiviral treatment, the titer of wild-type hepatitis B virus decreased or mutated (especially in patients treated with lamivudine), traditional biochemical methods (ELISA, enzyme-linked Immunoadsordent assay) cannot be detected, and PCR (polymorase chain reaction) technology has its application limitations due to the existence of certain false positives. HBV mutation can produce immune escape, anti-vaccine and drug resistance, etc., and the condition is easy to deteriorate and seriously threaten the life of patients. At present, there is no document disclosing the method of using Fe ₃ O ₄ (core)/gold (shell) nanoparticle virus DNA probe to detect virus DNA and its variation through secondary amplification technology.

Contents of the invention

The purpose of the present invention is to integrate the specificity of DNA binding with the unique characteristics of nanomaterials, establish a novel DNA detection system, and realize ultrasensitive detection of viral DNA and its variation.

The present invention is achieved like this:

a. Design an oligonucleotide probe with viral DNA as the target sequence. The probe contains 21-60 nucleotides and consists of two parts, the main part is 15-50 nucleotides long, and the other small part is 6 -10 nucleotides, modify the 5' end or 3' end of the oligonucleotide with alkanethiol; chemically synthesize the oligonucleotide, and purify it by polyacrylamide gel electrophoresis. The main part of the oligonucleotide is complementary to the mutant or conserved region of the viral DNA, and is 15-50 nucleotides long; the other small part is 6-10 nucleotides T to ensure that the main part of the oligonucleotide Some fully bind to their complementary regions.

b. Alkanethiol-modified probes are covalently bound to Au nanoparticles or Fe ₃ O ₄ (core)/Au (shell) nanoparticles through Au-S bonds;

c. Using a magnetic field to control the capture probe;

d. In the detection system, a connecting chain is added to replace the target chain, and a signal amplification substance is added.

The main advantages of this invention are threefold:

1. The secondary amplification effect of the signal realizes the ultra-sensitive detection of viral DNA. The gold nanoparticle DNA probe designed for viral DNA hybridizes with the target single strand in solution, and on this basis, the connecting strand is designed to replace the target single strand to form a gold nanoparticle aggregate, which greatly reduces the amount of the target single strand (signal amplified once); then silver (Ag ⁺ )-hydroquinone solution was added, the Ag ⁺ ion captured an electron from the surface of the gold nanoparticle or the solution and was reduced to an Ag atom, and attached to the surface of the gold nanoparticle (signal two second magnification, magnification about 10 ⁵ times). Two times of amplification can make the detection limit of DNA less than 0.1fMol (currently, the detectable sensitivity in the world is 10fMol). Through the analysis of the gray scale of the polymer, the virus DNA fragments of known sequence can be detected qualitatively or semi-quantitatively, thereby Achieve ultrasensitive detection of viral DNA. At the same time, the plasmon resonance signal of Au nanoparticles is extremely sensitive in the DNA denaturation temperature range: the light absorption curve of the oligonucleotide-modified Au nanoparticle system changes with temperature very sharply near the DNA denaturation temperature. The Full Width at Half Maximum (FWHM) of the primary differential is ~2°C, while the FWHM of DNA is usually ~15°C, thus improving the specific selectivity of DNA hybridization.

2. We invented and applied Fe ₃ O ₄ (core)/Au (shell) nanoparticles, which greatly reduced the steric hindrance effect of hybridization to ensure the hybridization rate. Traditional Au nanoparticles have no core. The viral DNA capture probe we constructed contains Fe ₃ O ₄ core and is controlled by a magnetic field. It can not only pre-separate the target single strand from the sample to be tested, but also use the magnetic field to A monolayer of _Fe3O4 ( _core )/Au(shell)-Au nanoparticles with controllable coverage is formed on the substrate, which greatly reduces the steric effect of hybridization and ensures the detection of the target single strand in viral DNA samples The lower limit reaches 0.1fMol.

3. Another significant advantage of this technology is the low detection cost. Its main consumable is alkanethiol-oligonucleotide-modified gold nanoparticles. Assuming that each polymer has nearly a thousand gold nanoparticles, each gold nanoparticle (about 15nm in diameter) is connected with about 220 oligonucleotides (the actual number can be lower by 30-50%), if the detection system contains nearly a thousand polymers body, the total number of oligonucleotides required is about 2.2×10 ⁸ . The price of purchasing alkanethiol-modified oligonucleotides (2 OD value, HPLC purification) is about 1100-1500 yuan, which contains about 1×10 ¹⁶ oligonucleotides. Moreover, the preparation cost of gold nanoparticles is also very low. Consumables (alkanethiol-modified oligonucleotides, gold nanoparticles) cost less than 1 yuan per detection. If this technology can realize integrated and automated instrument detection, it will have great profit margins and price advantages.

Description of drawings

Accompanying drawing is a schematic diagram of the detection process of Fe ₃ O ₄ (core)/gold (shell) nanoparticles HBV DNA probe

Detailed ways

Example 1. Ultrasensitive detection of HBV DNA by Fe ₃ O ₄ (core)/Au (shell) nanoparticles HBV DNA probe

Oligonucleotide design and synthesis:

The oligonucleotide probe consists of 26 nucleotides and consists of two parts, the main part is complementary to the mutation region or conserved region of HBV DNA, and is 20 nucleotides long; the other small part is 6 nucleosides acid T to ensure that the main part of the oligonucleotide is fully combined with its complementary region.

1. Screening, cloning and purification of variant strains and wild strains of various types of hepatitis B HBV DNA (acute, chronic/severe, mild and virus carriers). Find out the mutation region related to the occurrence and development of hepatitis B and conduct systematic research, design probes for common mutation regions; at the same time design probes for the conserved sequence region of HBV DNA. Design probes with HBV DNA (accession: X75658) 1873-1912 as the target sequence:

①The designed probe c (probe c) is complementary to the 1893-1912 position of the target single strand, and the probe c sequence is:

5' -ttt ttt gtc aat gtc cat gcc cca aa-3'.

②The design of the connecting chain: the length of the connecting chain is 40bp, and the sequence is:

5’ -caa gct gtg cct tgg gtg gcg ggg ggg ggg ggg ggg ggg g-3’

The first 20bp is the same as the part of the target single strand that is not hybridized with probe c (ie 1873-1892 positions), and the last 20bp is not complementary or similar to probe c. Using connecting strands instead of target single strands to form gold nanoparticle aggregates can greatly reduce the required amount of target single strands and achieve ultra-sensitive detection of DNA.

③Design probe a (probe a) and probe b (probe b) respectively for the connecting chain,

The probe a sequence is: 5’-gcc acc caa ggc aca gct tgt ttt tt-3’

The probe b sequence is: 5’-ttt ttt ccc ccc ccc ccc ccc ccc cc-3’

They are strictly complementary to the connecting strand.

The result of the probe design is: probe a and probe c can complementarily bind to different segments on the target single strand, and probe a and probe b can complementarily bind to different segments on the connecting strand. Modify the 5' or 3' end of the oligonucleotide with an alkanethiol. Oligonucleotides were chemically synthesized and purified by polyacrylamide gel electrophoresis.

2. Covalently bind the alkanethiol-modified probe to Au nanoparticles or Fe ₃ O ₄ (core)/Au (shell) nanoparticles through the Au-S bond to form a probe chain:

The probe c modified by alkanethiol is coupled with superparamagnetic Fe ₃ O ₄ (core)/Au (shell) nanoparticles to form a target single-strand capture probe. In the sample to be tested, the capture probe captures the target single strand through hybridization (heating, annealing), and through magnetic field manipulation and cleaning, the preliminary separation of the target single strand is achieved, and the steric hindrance caused by the chip design can be avoided. .

Au nanoparticles were coupled with alkanethiol-modified probe a or probe b to form Au nanoparticle aggregates.

The Fe ₃ O ₄ (core)/Au (shell) nanoparticles were linked to oligonucleotides modified with alkanethiol, and the linking method was as follows:

(1) 5ml of citric acid-treated 10nM Fe ₃ O ₄ (core)/Au (shell) nanoparticles solution was mixed with 2.5 OD alkanethiol-modified oligonucleotides (final concentration was 3.61 μM), and left at room temperature for 24 hours ;

(2) Add 0.1M sodium chloride, 10mM phosphate buffer (pH=7), leave at room temperature for 40 hours, 14,000 rpm, 25 minutes, remove the supernatant;

(3) Add 5ml of 0.1M sodium chloride, 10mM phosphate buffer (pH=7), wash the red oily precipitate, 14,000 rpm, 25 minutes, remove the supernatant;

(4) The precipitate was resuspended in 5 ml of fresh 0.3M sodium chloride, 10 mM phosphate buffer solution pH=7, 0.01% sodium azide solution.

The method for linking Au nanoparticles to alkanethiol-modified oligonucleotides is similar.

3. Detection process:

① Probe c[Fe ₃ O ₄ (core)/Au (shell)] is mixed with the sample to be tested, hybridized with the target single strand, separated by a magnetic field, and washed.

② After adding probe a and hybridizing with the other segment of the target single strand, the probe a particle is connected to probe c [Fe ₃ O ₄ (core)/Au (shell)], and the magnetic field is strengthened, and the superparamagnetic Fe ₃ O ₄ (core )/Au (shell) nanoparticles will pull the Au nanoparticles of probe a to sink and be absorbed at the bottom, arranged and not easy to be washed away.

③ Add linking chain, probe b, probe a in sequence, and repeat several times, so that the Au nanoparticles of probea in the previous step are fixed points, forming a parachute-like layered Au nanoparticle aggregate containing probe a, probe b ( The signal is amplified once).

④ Add Ag ⁺ -hydroquinone solution (silver-hydroquinone), Ag ⁺ will get an electron from the surface of gold nanoparticles and reduce to Ag, and attach to the surface of gold nanoparticles, the film surface will appear gray-black color immediately , the more targets, the darker the color (the signal is amplified twice, and the magnification is about 10 ⁵ times).

⑤ Qualitatively or semi-quantitatively detect HBV DNA fragments with known sequences by analyzing the gray scale of aggregates.

Claims (7)

1, a kind of magnetic nanoparticle mark gene probe detects the method for gene, this method adopts the secondary singal amplifying technique, it is characterized in that this probe is to be made of 100-500 oligonucleotide of magnetic nanoparticle surface covalent attachment, nano particle is a core/shell structure, and its nuclear is Fe ₃O ₄, shell is Au, is Fe ₃O ₄/ Au nano particle.

2, the described detection method of claim 1 is characterized in that preparing by the following method the magnetic nanoparticle gene probe:

(1) Au, Fe ₃O ₄, Fe ₃O ₄The preparation of/Au nano particle;

(2) design of oligonucleotide probe, synthetic, modification;

(3) Au, Fe ₃O ₄The assembling of/Au nano gene label probe.

3, the described detection method of claim 1 is characterized in that this probe is applied to gene recombination and detects.

4, the described detection method of claim 2, the preparation that it is characterized in that the magnetic nanoparticle gene probe are following carrying out:

The preparation particle diameter is the Fe of 10-100nm ₃O ₄/ Au nano particle, be complementary to target to be checked and modify through alkane hydrogen mercaptan, concentration is that the oligonucleotide of 1-100 μ M mixes.Placed 8-16 hour at 4-37 ℃, add sodium-chlor, phosphate buffered saline buffer pH=7 to final concentration and be respectively 0.025-0.1M, 10mM, placed 24-48 hour for 4-37 ℃, 10,000-15, the centrifugal 20-40min of 000rpm, remove supernatant, add 1ml 0.025-0.1M sodium-chlor, 10mM phosphate buffered saline buffer pH=7, wash red oily precipitation, 10,000-15, the centrifugal 20-40min of 000rpm removes supernatant, repeat to wash once, precipitation is resuspended in and contains in 0.3M sodium-chlor, 10mM phosphate buffered saline buffer pH=7, the 0.01% sodium azide solution.

5, the described detection method of claim 3 is characterized in that:

Magnetic Fe ₃O ₄/ Au nanoparticle label gene is caught probe as the prisoner, controls by magnetic field to be fixed, by forming crossbred with complementary target hybridization.

6, the described detection method of claim 3 is characterized in that:

The design connection chain replaces the target chain, adds connection chain and Au nanoparticle mark gene probe on crossbred repeatedly, forms the continuous stacked hybridization aggregate on the crossbred basis, realizes that the nanometer of detection signal is amplified, i.e. " signal amplifies for the first time ".

7, the described detection method of claim 3 is characterized in that:

In the hybridization system, add AgNO ₃With Resorcinol liquid, Ag ⁺Be reduced to Ag by the Au in the hybridization aggregate, be deposited on around the hybridization aggregate, demonstrate macroscopic beige, be i.e. " signal amplifies for the second time ".