Analysis method for sensitive detection of virus DNA based on thulium nanoprobe

Abstract

The invention aims to provide an analysis method for ultrasensitive detection of virus DNA based on a thulium nanoprobe. The method comprises synthesis of the thulium nanoprobe and stable isotope detection. The principle of the invention is as follows: thulium nanoparticles with uniform morphology are synthesized by using a high-temperature thermal precipitation method; and then acid pickling is carried out, so that the thulium nanoparticles have water solubility and electropositivity and can generate an electrostatic adsorption effect with DNA, and the nanoprobe is formed. DNA detection is characterized in that a target object is extracted from a matrix by using magnetic beads connected with captured DNA, and after a nanoprobe is added, nanoparticles are successfully marked on the target object due to specific recognition of complementary pairing of bases between the DNA; and finally, after the thulium element is digested by aqua regia, detecting an intensity signal of the thulium element by an inductively coupled plasma mass spectrometer (ICP-MS) and carrying out quantitative analysis. By utilizing the advantages of low detection limit, excellent stability, low lanthanide element biological background, easy ionization and the like of ICP-MS, high-sensitivity, stable and accurate detection on HBV-DNA can be realized, and the method can be used for detecting a target object in actual human serum.

Description

Technical Field

[0001] The present invention belongs to the field of detection in analytical chemistry and relates to the field of mass tags sensing of viral DNA, and particularly designs an analytical method for detecting viral DNA (HBV-DNA) by inductively coupled plasma mass spectrometry using stable isotopes generated by thulium nanoparticles (NaTmF4 NPs) synthesized by a high-temperature coprecipitation method. Background Art

[0002] So far, mass spectrometry (MS) has become a widely used tool in the biological sciences. Molecular mass spectrometry includes matrix-assisted laser desorption / ionization mass spectrometry and electrospray ionization mass spectrometry, which stand out in proteomics because they can accurately identify a large number of different mass-to-charge ratio fragments. However, since soft ionization sources are often used in molecular mass spectrometry, certain requirements are imposed on the sample pretreatment procedure in molecular quantification to eliminate signal interference caused by the sample matrix. At this time, elemental mass spectrometry can make up for the deficiencies of molecular mass spectrometry in the quantitative detection of biomolecules, and ICP-MS is the best one among elemental mass spectrometers. ICP-MS has significant advantages, including 1. easy ionization of various elements; 2. high selectivity and outstanding sensitivity; 3. having the ability of isotope analysis and providing isotope distribution; 4. large linear dynamic range; 5. accurate absolute quantification can be carried out even in complex substrates; 6. multi-element analysis can be performed. Therefore, ICP-MS has broad application prospects in the analysis and detection of biomolecules as long as appropriate element labeling is combined.

[0003] In the element labeling of ICP-MS, metal stable isotope labeling has excellent compatibility with ICP-MS and plays a huge role in the precise quantitative analysis of various biomolecules, including small molecules, proteins, enzymes, nucleic acids and single cells. However, high-sensitivity analysis results cannot be obtained only through single-element labeling. Therefore, researchers have carried out various attempts, including using polymer markers, which can provide a lanthanide element loading 30 times higher than that of typical DOTA complexes and can also increase the detection sensitivity by 10 times. Another development direction is to obtain excellent analytical sensitivity through signal amplification methods, including catalytic hairpin assembly (CHA), DNA hybridization chain reaction (HCR) and DNA Walkers, etc. Although these amplification methods can improve the sensitivity and detection limit of bioanalysis detection, they have very high requirements for synthesis conditions, DNA design and detection reaction environment.

[0004] The advantages of combining metal nanoparticles with ICP-MS are the expected high sensitivity and low detection limit, which is due to the large number of metal stable isotopes in metal nanoparticles, making the analytical method of combining metal nanolabels with ICP-MS have a self-amplification effect. It is worth noting that lanthanide element labeling is still the most promising metal isotope labeling to date due to its low biological background, small polyatomic interference, high ionization efficiency, etc. Therefore, synthesizing lanthanide nanoparticles with good morphology and perfect biological functions can greatly improve the biological analysis performance of ICP-MS.

[0005] In this invention, by optimizing the synthesis and modification methods, lanthanide nanoparticles (NaTmF4) with good morphology and uniform particle size were obtained, and a high-sensitivity analytical detection method combined with ICP-MS was achieved using HBV-DNA as an analytical detection model. Summary of the Invention

[0006] This invention provides a method for analyzing viral DNA based on the detection of thulium nanoparticles, which realizes a high-sensitivity analytical detection method after a high-performance combination with ICP-MS.

[0007] The principle of this invention is: using streptavidin magnetic beads as an analytical detection platform. First, the magnetic beads are mixed with the sample and incubated at room temperature for a period of time, so that the capture DNA modified on the magnetic beads can successfully extract the target from the sample. On this basis, a certain amount of thulium nanoparticle probe is added for DNA base complementary pairing. The nanoprobe is successfully located on the target. Therefore, a sandwich structure is formed by the magnetic beads, the target, and the thulium nanoparticles. According to the magnetism of the magnetic beads, the remaining impurities in the sample are magnetically separated, and aqua regia is added to dissolve the sandwich structure. Finally, ICP-MS is used to detect the digestion solution, so as to obtain the content information of thulium, in order to quantitatively analyze HBV-DNA and achieve the purpose of high-sensitivity detection and analysis of HBV.

[0008] To achieve the above object, the present invention provides the following technical solutions: The principle of preparing the thulium nanoprobe in this invention is: using the high-temperature co-precipitation synthesis method to synthesize thulium nanoparticles with uniform morphology. First, thulium elements form a precipitate with sodium salts and fluoride salts at room temperature, and then in the presence of oleic acid (OA), the precipitate forms uniform nanoparticles at high temperature. The oleic acid molecules on the surface of the nanoparticles are removed by pickling, making it positively charged, which can directly generate electrostatic adsorption with the phosphate groups at the end of DNA, and synthesize the required nanoprobe for detection.

[0009] The principle of detecting HBV-DNA is as follows: Magnetic beads modified with capture DNA are mixed with the sample. Through the base complementary pairing principle of DNA and the magnetic separation ability of the magnetic beads, the target is accurately extracted from the sample matrix. Then, by adding nanoparticles modified with detection DNA to bind to the target DNA, HBV-DNA is quantitatively labeled.

[0010] The method for digesting and performing stable isotope detection on the labeled thulium nanoprobe is as follows: After magnetic separation, aqua regia with a certain concentration is used to digest the thulium nanoprobe, and the thulium element in the nanoparticles is digested into the solution. Then, high-sensitivity detection of the thulium element is carried out by ICP-MS, and 169 Tm is selected as the detection object for high-sensitivity quantitative analysis. Linear regression analysis is performed through the change in the intensity value of the thulium element caused by different concentrations of HBV-DNA, and thus the HBV-DNA to be detected can be quantitatively analyzed.

[0011] Among them, when the magnetic beads react with the target DNA, the solution used is TBS buffer (25 mM, pH = 7.4), the time is 30 min, and the temperature is 25 °C; when the thulium nanoprobe reacts with the target DNA, the solution used is TBS buffer (25 mM, pH = 7.4), the time is 15 min, and the temperature is 25 °C.

[0012] The present invention has the following beneficial effects: The present invention provides an analytical method that can sensitively detect HBV-DNA. Through the high-temperature co-precipitation synthesis method, thulium nanoparticles with uniform morphology can be synthesized as probes. The thulium nanoparticles with good uniformity provide as many attachment sites as possible for the detection DNA, improving the ability to recognize the target. When combined with ICP-MS, the sensitivity of the detection method is greatly improved. At the same time, by using the base complementary pairing principle of DNA and the magnetic selection performance of the magnetic beads, interference in the detection process can be reduced, ensuring the accuracy of the analysis results. It is worth noting that the detection limit of the detection method designed in this study is even better than some studies using amplification methods to enhance the detection signal, proving that by optimizing the synthesis of nanoparticles, the detection sensitivity of the probe can be improved, thereby simplifying the detection process and achieving the purpose of being easy to promote. Description of the Drawings

[0013] Figure 1 It is a schematic diagram of the analytical method for detecting HBV-DNA based on thulium nanoparticles; Figure 2 It is an exploration of the feasibility of the analytical method for detecting HBV-DNA based on thulium nanoparticles; Figure 3 It is all the DNA sequences used in the analytical method for detecting HBV-DNA based on thulium nanoparticles; Figure 4 It is an exploration of the selectivity of the analytical method for detecting HBV-DNA based on thulium nanoparticles; Figure 5For the detection performance of the analytical method for detecting HBV-DNA based on thulium nanoparticles; Figure 6 For the spike recovery in serum of the analytical method for detecting HBV-DNA based on thulium nanoparticles. Specific implementation manner

[0014] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The water used in the following examples is all ultrapure water, which is processed by a Milli-Q ultrapure water purification system. All samples in the following examples were not purified before use.

[0015] The present invention is carried out according to the following specific steps:

[0016] a. Synthesis of oleic acid-coated thulium nanoparticles a1 Weigh 1 mmol of thulium chloride salt, 6 mL of oleic acid, and 12 mL of 1-octadecene. Under an oxygen-free and water-free environment, mix the three and heat them up to 150 °C to ensure that the thulium chloride salt is completely dissolved in the organic solvent; a2 Wait for the solution to cool naturally to room temperature, add 10 ml of methanol containing 100 mg of NaOH and 148 mg of NH4F, and stir at room temperature for 50 min; a3 Heat up to 100 °C to remove the excess methanol and water, and continue to heat up to 280 °C under nitrogen protection for 1 h, and then wait for the solution to cool naturally to room temperature; a4 Mix the reaction solution and acetone in a ratio of 1:1, and centrifuge at 10000 rpm for 30 min. Then redisperse the precipitate in a mixed solution of ethanol and cyclohexane, and centrifuge twice under the same centrifugation conditions. Finally, disperse the precipitate in cyclohexane.

[0017] b. Synthesis of thulium nanoprobes b1 Take 1 ml of the above nanoparticle solution and mix it with 1 ml of hydrochloric acid solution (1 M), and sonicate for 30 min; b2 Centrifuge at 15000 rpm for 20 min, redissolve in 1 ml of hydrochloric acid solution (0.1 M), and sonicate for 30 min; b3 Centrifuge at 15000 rpm for 20 min, redissolve in ethanol solution, wash twice under the same centrifugation conditions, and finally disperse the nanoparticles in deionized water; b4 Mix 100 μL of DNA (6 μM) with 125 μL of nanoparticles and react overnight in an incubator at 37 °C; b5 Centrifuge at 14,000 rpm for 5 min, redissolve in TBS solution, wash once with TBS buffer under the same centrifugation conditions, then disperse in TBS buffer containing 5% (w / v) BSA. The blocking procedure is completed by incubating at room temperature for 1 h, then wash once with TBS buffer and disperse in TBS buffer, and store at 4 °C.

[0018] c. Magnetic bead labeling to capture DNAc1 Take 9.5 μL of magnetic bead suspension and wash it 3 times with B&W buffer, and remove the washing solution by magnetic separation; c2 Incubate the magnetic beads with 9.5 μL of capture DNA solution (5 μM) at room temperature for 1 h, and label the capture DNA with biotin-streptavidin reaction; c3 Remove the excess DNA by magnetic separation, disperse the magnetic beads in B&W buffer containing 10% (w / v) BSA for blocking. The blocking procedure is completed by incubating at room temperature for 1 h. After washing 4 times with B&W, redissolve the magnetic beads labeled with capture DNA in TBS buffer and store at 4 °C until use.

[0019] d. Analysis and detection of HBV-DNA d1 Take 5 μL of the previously prepared magnetic bead mixture as the capture unit and incubate it with different concentrations of HBV-DNA at 25 °C for 30 min; d2 React the magnetic bead-capture DNA-target with the nanoprobe mixture at 25 °C for 15 min, and then rinse 3 times with TBS buffer; d3 After magnetic separation, remove the supernatant, and digest the magnetic beads and lanthanide nanoparticles with 50 μL of aqua regia at room temperature.

[0020] e. ICP-MS detection e1 Take 50 μL of the digested solution, dilute it to 8 mL, and transfer it to an EP tube; e2 Detect 169 Tm in ICP-MS in SRD mode; e3 Using 169 Tm as the standard, substitute the measured intensity into the linear equation to calculate the concentration.

[0021] The following is a further description in conjunction with the accompanying drawings of the specification, but the analysis method of the present invention is not limited to the following embodiments.

[0022] Example 1. Comparative experiment on whether a DNA sandwich sandwich structure is formed To accurately detect the concentration of biomarkers in a sample, the formation of a sandwich structure is crucial. For HBV-DNA, the necessity of the sandwich structure and the feasibility of the analytical method of the present invention were compared under different conditions.

[0023] In the experiment, the concentration of the target HBV-DNA was 100 pM. The sample solution added to the group lacking the target (blank group) was TBS buffer solution, and the other control groups were HBV-DNA solutions (100 pM); the probe solution added to the group lacking the nanoprobe was TBS buffer solution, and the other control groups were thulium nanoparticle solutions; the solution added to the group lacking the capture DNA was a solution of bare magnetic beads, and the other control groups were magnetic bead solutions modified with capture DNA. Keeping all other conditions exactly the same, the target was detected by mixing 5 μL of the magnetic bead mixture and reacting at 25 °C for 30 min, then adding 10 μL of the nanoprobe solution and reacting at 25 °C for 15 min. After washing 3 times with TBS buffer solution and magnetic separation, 50 μL of aqua regia was added for digestion, and ICP-MS was used to detect the lanthanide element 169 Tm signal (as Figure 1 shown).

[0024] Figure 2 It can be seen that no effective intensity signal of thulium element can be detected whether under the conditions of lacking target DNA, nanoprobe or the capture DNA not being connected to the magnetic bead. Only when the target DNA, nanoprobe and the magnetic bead connected with capture DNA are all present can an obvious thulium element signal be detected, which can be used for the detection of HBV-DNA. Therefore, this example also proves the conditions required for the formation of the sandwich structure and the feasibility of the analytical method of the present invention.

[0025] Example 2. Explore the specific detection of HBV-DNA by the analytical method of the present invention. Taking HBV-DNA as an example, this example used an analytical method based on the detection of thulium nanoparticles to detect several mismatch types and random sequences of HBV-DNA to explore the specificity of this method.

[0026] Using the above steps, thulium nanoparticles were used as probes to detect HBV-DNA (1 nM), mis-1 (single-base mismatch, 1 nM), mis-2 (double-base mismatch, 1 nM), mis-3 (triple-base mismatch, 1 nM), rs (random sequence, 1 nM) respectively (the sequences are shown in detail in Figure 3 ), keeping all other conditions exactly the same. The target DNA was mixed with 5 μL of the magnetic bead mixture and reacted at 25 °C for 30 min, then 10 μL of the nanoprobe solution was added and reacted at 25 °C for 15 min. After washing 3 times with TBS buffer solution and magnetic separation, 50 μL of aqua regia was added for digestion, and ICP-MS was used to detect the lanthanide element 169 Tm signal.

[0027] The results are as Figure 4 shown. Except that HBV-DNA can significantly enhance the signal of lanthanide elements, other several mismatch types and random sequences cannot cause obvious signal changes, proving that the specific recognition of base complementary pairing between DNAs in the present invention can perform highly selective detection of HBV-DNA.

[0028] Example 3. Explore the sensitivity detection of HBV-DNA by the analysis method of the present invention This example explored the sensitivity and linearity of the analysis method based on the detection of thulium nanoparticles for the detection of HBV-DNA.

[0029] In the experiment, 5 μL of magnetic bead solution was added to 25 μL of HBV-DNA solutions with different concentrations, reacted at 25 °C for 30 min, then 10 μL of nanoprobe solution was added and reacted at 25 °C for 15 min, washed 3 times with TBS buffer solution, after magnetic separation, 50 μL of aqua regia was added for digestion, and ICP-MS was used to detect the 169 Tm signal.

[0030] As Figure 5 can be seen, according to the relationship that the concentration is proportional to the response signal, the linear relationship between the HBV-DNA concentration and the 169 Tm response signal was obtained. When the HBV-DNA concentration was within 10 - 10 6 fM, 169 the Tm response signal showed a linear relationship, the linear correlation coefficient was 0.999, and the calculated detection limit was 0.473 pM (n = 12, 3σ). It was proved that this analysis method had good sensitivity.

[0031] Example 4. Explore the detection performance of the analysis method of the present invention in serum

[0032] Sample collection: Human serum samples were collected from the Seventh People's Hospital of Chengdu.

[0033] Sample pretreatment: In view of the complex composition and viscosity of serum samples, 1% bovine serum albumin (BSA) TBS buffer solution was used to dilute the serum samples.

[0034] The analysis method based on the detection of thulium nanoparticles was used to detect human serum samples: 5 μL of magnetic bead mixture was added to 25 μL of human serum sample, reacted at 25 °C for 30 min, then 10 μL of nanoprobe solution was added and reacted at 25 °C for 15 min, washed 3 times with TBS buffer solution, after magnetic separation, 50 μL of aqua regia was added for digestion, and ICP-MS was used to detect the 169 Tm signal.

[0035] Detection results The results are shown in Figure 6 , and the recoveries of HBV-DNA in serum samples are all concentrated in the range of 95.91 - 111.8%, showing good recoveries. It indicates that this analytical method can adapt to the complex environment of actual samples and ensure accuracy.

Claims

1. An analytical method for sensitive detection of viral DNA based on thulium nanoprobes, characterized in that: (1) Synthesis of thulium nanoprobes; (2) Modification of magnetic beads as capture units; (3) Detection and analysis process of the analyte; (4) Stable isotope detection; (1) Synthesis of thulium nanoprobes: Weigh 1 mmol of thulium chloride salt, 6 mL of oleic acid, and 12 mL of 1-octadecene. Under an oxygen-free and water-free environment, mix the three and heat them up to 150 °C to ensure the complete dissolution of thulium chloride salt. After the solution cools to room temperature, add 10 ml of methanol containing 100 mg of NaOH and 148 mg of NH4F, stir at room temperature for 50 min, then heat up to 100 °C to remove the excess methanol and water. Under nitrogen protection, continue to heat up to 280 °C and react for 1 h. After the solution cools to room temperature, thulium nanoparticles wrapped with OA are obtained. Wash the nanoparticles with ethanol and cyclohexane. Finally, these nanoparticles can be stored in cyclohexane for several months. Take 1 ml of the above nanoparticle solution and mix it with 1 ml of hydrochloric acid solution (1 M), sonicate for 30 min, centrifuge at 15000 rpm for 20 min, redissolve in 1 ml of hydrochloric acid solution (0.1 M), sonicate for 30 min, centrifuge at 15000 rpm for 20 min, redissolve in ethanol solution, wash twice under the same centrifugation conditions, and finally disperse the nanoparticles in deionized water to obtain naked thulium nanoparticles. Mix 100 μL of DNA (6 μM) with 125 μL of naked nanoparticles, react overnight in an incubator at 37 °C, centrifuge at 14000 rpm for 5 min, redissolve in TBS solution, wash once with TBS buffer under the same centrifugation conditions, then disperse in TBS buffer containing 5% (w / v) BSA, incubate at room temperature for 1 h, wash once with TBS buffer, and disperse in TBS buffer for storage at 4 °C. (2) Modification of magnetic beads as the capture unit: Take 9.5 μL of magnetic bead suspension and wash it 3 times with B&W buffer, and remove the washing solution by magnetic separation. Incubate the magnetic beads with 9.5 μL of capture DNA solution (5 μM) at room temperature for 1 h, and label the capture DNA with biotin-streptavidin reaction. Remove the excess DNA by magnetic separation, disperse the magnetic beads in B&W buffer containing 10% (w / v) BSA for blocking, incubate at room temperature for 1 h, wash 4 times with B&W buffer, add the magnetic beads labeled with capture DNA to TBS buffer, and store at 4 °C until use. (3) Analytical process for detecting the target: Use 5 μL of the previously prepared magnetic beads as the capture unit, incubate with different concentrations of HBV-DNA target at 25 °C for 30 min, mix the magnetic bead-capture DNA-target DNA conjugate with the nanoprobe, react at 25 °C for 15 min, then rinse 3 times with TBS buffer. After magnetic separation, digest the magnetic beads and thulium nanoparticles with 50 μL of aqua regia at room temperature for subsequent ICP-MS analysis. (4) Stable isotope detection: Take 50 μL of the digested solution, dilute it to 8 mL, and transfer it to an EP tube. Detect 169 Tm in ICP-MS in SRD mode; using 169 Tm as the standard, substitute the measured intensity into the linear equation to calculate the concentration.

2. The analysis method according to claim 1, characterized in that: The analysis method uses ICP-MS stable isotope analysis. The thulium nanoprobes are combined with magnetic beads and used for the sensitive detection of viral DNA. By performing linear regression analysis on the ICP-MS intensity signal of thulium element, highly sensitive and stable detection of the analyte can be achieved; The concentration range of the HBV-DNA detection by the said analysis method is 10-10 6 fM, and the detection limit is 0.473 pM.

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