High-risk HPV subtype multiple sensitive detection analysis method based on combination of lanthanide nanoprobe and ICP-MS (Inductively Coupled Plasma Mass Spectrometry)

Abstract

The invention relates to the field of detection of analytical chemistry, in particular to a high-risk HPV (human papillomavirus) subtype multiple sensitive detection and analysis method based on combination of a lanthanide nanoprobe and ICP-MS. The multiple sensitive detection and analysis method comprises the following steps: synthesizing lanthanide nanoparticles wrapped by oleic acid; synthesizing the lanthanide nano biological probe by using the prepared lanthanide nano particles wrapped by the oleic acid; modifying the magnetic bead as a capture unit to obtain a modified magnetic bead; mixing target DNA with the prepared lanthanide series nano biological probe and the prepared modified magnetic beads, incubating and digesting to obtain a digesting solution; and carrying out ICP-MS detection on the digested solution. By utilizing the advantages of high sensitivity, excellent stability, low lanthanide element biological background, easy ionization and the like of the ICP-MS, multiple, sensitive, stable and accurate detection on the high-risk HPV subtypes can be realized, and the method can be used for simultaneously detecting multiple high-risk HPV subtypes in actual human serum.

Description

Technical Field

[0001] The present invention relates to the field of detection in analytical chemistry, and particularly to a multiplex sensitive detection and analysis method for high-risk HPV subtypes based on the combination of lanthanide nanoprobes and ICP-MS. Background Art

[0002] Since HPV was first discovered in 1975, as of 2024, 231 types of HPV have been identified. After decades of research, HPV has been confirmed to have carcinogenic potential. Therefore, based on the pathogenicity and carcinogenic ability of HPV, researchers have classified it into high-risk and low-risk types. It should be emphasized that cancers caused by high-risk HPV account for 5% of all human cancers. The close association between HPV and cervical cancer, anal cancer, and oropharyngeal cancer has been confirmed. In addition, there is evidence that other cancers, such as oral cancer, esophageal cancer, and lip cancer, may also be related to HPV infection. Currently, the gold standard for HPV detection is polymerase chain reaction (PCR). In the past few decades, it has shown remarkable efficacy, thus saving countless lives. However, most PCR methods use fluorescence labeling. The spectral interference problem of fluorescence labeling limits the number of targets that can be detected simultaneously. In addition, during the detection process, the nucleic acid amplification step is prone to contamination, which may lead to false positive results. At the same time, the entire detection process requires operators to have excellent professional skills and the instrument equipment to have extremely high precision. Therefore, it is crucial to develop a detection method that is simple, sensitive, and capable of multiplex analysis of HPV subtypes without any nucleic acid amplification operation.

[0003] In the process of elemental labeling by inductively coupled plasma mass spectrometry (ICP-MS), metal stable isotope labeling has played a huge role in the accurate quantitative analysis of various biomolecules. Since metal nanoparticles contain thousands of metal atoms, the nanoparticle probes themselves have an inherent amplification effect in the ICP-MS analysis method. In addition, ICP-MS has advantages such as excellent selectivity, high sensitivity, a wide linear dynamic range, and the ability for multi-element analysis. Therefore, the combination of metal nanoprobe technology and ICP-MS has great potential in enhancing multiplex analysis methods.

[0004] In order to further expand the upper limit of the number of metal nanoparticle markers, recent research has increasingly confirmed the potential of lanthanide nanoparticles. Due to the characteristics of lanthanide nanoparticles such as low biological background, high ionization efficiency, and low polyatomic interference, using them as metal markers for ICP-MS can significantly improve the detection sensitivity and upper limit, thus further highlighting the advantages of multiplex detection. Therefore, there is an urgent need to provide a multiplex sensitive detection and analysis method for high-risk HPV subtypes based on the combination of lanthanide nanoprobes and ICP-MS. Summary of the Invention

[0005] The object of the present invention is to provide a method for multiplex sensitive detection and analysis of high-risk HPV subtypes based on the combination of lanthanide nanoprobes and ICP-MS. In the present invention, lanthanide element nanoparticles (NaTbF 4 , NaHoF 4 , NaEuF 4 , NaPrF 4 , NaYF 4 and NaTmF 4 NPs) with good morphology and uniform particle size are obtained, and high-risk HPV subtypes are used as an analysis and detection model to achieve multiplex sensitivity analysis and detection in combination with ICP-MS.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] The present invention provides a method for multiplex sensitive detection and analysis based on the combination of lanthanide nanoprobes and ICP-MS. The multiplex sensitive detection and analysis method includes the following steps:

[0008] (1) Synthesize lanthanide nanoparticles encapsulated with oleic acid;

[0009] (2) Synthesize lanthanide nano-bioprobes by using the lanthanide nanoparticles encapsulated with oleic acid prepared in step (1);

[0010] (3) Modify magnetic beads as a capture unit to obtain modified magnetic beads;

[0011] (4) Mix the target DNA with the lanthanide nano-bioprobes prepared in step (2) and the modified magnetic beads prepared in step (3), incubate and digest to obtain a digested solution;

[0012] (5) Detect the digested solution by ICP-MS.

[0013] Furthermore, in step (1), the specific steps for synthesizing the lanthanide nanoparticles encapsulated with oleic acid are as follows:

[0014] (1.1) Weigh 0.5 - 2 mmol of lanthanide chloride salts, 3 - 12 mL of oleic acid and 7.5 - 30 mL of 1-octadecene, mix the three in an oxygen-free and water-free environment and heat to 140 - 180 °C to ensure that the lanthanide chloride salts are completely dissolved in the organic solvent;

[0015] (1.2) Wait for the solution to cool to room temperature naturally, slowly dropwise add 5 - 20 ml of methanol containing 50 - 200 mg of NaOH and 74 - 296 mg of NH 4 F, and stir at room temperature for 50 min;

[0016] (1.3) Heat up until the excess methanol and water in the solution are removed, and continue to heat up to 280 - 290 °C under nitrogen protection for 0.5 - 1 h, then wait for the solution to cool naturally to room temperature;

[0017] (1.4) Mix the reaction solution and acetone in a ratio of 1:1, centrifuge at 10000 rpm for 30 min, then redisperse the precipitate in a mixed solution of ethanol and cyclohexane, and wash twice under the same centrifugation conditions to obtain lanthanide nanoparticles encapsulated with oleic acid.

[0018] Furthermore, in step (2), the specific steps for synthesizing the lanthanide nanobiosensor are as follows:

[0019] (2.1) Take 1 - 2 ml of the solution of lanthanide nanoparticles encapsulated with oleic acid and mix it with 1 - 2 ml of hydrochloric acid solution, and ultrasonicate for 30 min;

[0020] (2.2) Centrifuge at 14000 - 15000 rpm for 20 - 30 min, redissolve in 1 - 2 ml of hydrochloric acid solution, and ultrasonicate for 30 min;

[0021] (2.3) Centrifuge at 14000 - 15000 rpm for 20 - 30 min, redissolve in ethanol solution, and wash twice under the same centrifugation conditions to obtain nanoparticles dispersed in deionized water;

[0022] (2.4) Mix 40 - 56 μL of DNA solution with 50 - 70 μL of nanoparticles and react in an incubator at 37 °C for 10 - 15 h;

[0023] (2.5) 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 10% BSA, complete the blocking procedure by incubating at 25 °C for 1 h, then wash once with TBS buffer and disperse in TBS buffer containing 10% BSA to obtain the lanthanide nanobiosensor.

[0024] Furthermore, in step (3), the specific steps for modification are as follows:

[0025] (3.1) Take 2 - 4 μL of magnetic bead suspension and wash it 3 times with TBS buffer, and remove the washing solution by magnetic separation;

[0026] (3.2) Incubate the magnetic beads with 2 - 4 μL of capture DNA solution at room temperature for 1 h, and label the capture DNA with biotin - streptavidin reaction;

[0027] (3.3) Remove the excess captured DNA by magnetic separation. Disperse the magnetic beads in TBS buffer containing 10% BSA for blocking. After washing 4 times with TBS, redissolve the magnetic beads labeled with captured DNA in TBS buffer to obtain the modified magnetic beads.

[0028] Further, in step (3.3), the blocking is carried out by incubating at 25 °C for 0.5 - 1.5 h.

[0029] Further, in step (4), the specific steps of incubation and digestion are as follows:

[0030] (4.1) Take 5 - 10 μL of the modified magnetic beads as the capture unit, incubate with different concentrations of HPV - DNA sample solution and 10 - 20 μL of lanthanide nanobiosensor at 25 °C for 30 min;

[0031] (4.2) Wash the magnetic beads 3 times with 200 μL of TBS buffer;

[0032] (4.3) After magnetic separation, remove the supernatant, and digest the magnetic beads and lanthanide nanoparticles with 50 - 100 μL of aqua regia at room temperature.

[0033] Further, in step (5), the specific steps of ICP - MS detection are as follows:

[0034] (5.1) Take 50 μL of the digested solution, dilute it to 5 - 10 mL, and transfer it to an EP tube;

[0035] (5.2) Detect 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm in ICP - MS, SRD mode;

[0036] (5.3) Using 89 Y, 141 Pr, 153Eu, 159 Tb, 165 Ho and 169 Tm as the standard, substitute the measured intensity into the linear equation to calculate the concentration.

[0037] Further, the multiplex sensitive detection and analysis method can be used for the detection of high - risk HPV subtypes.

[0038] The present invention also provides a lanthanide nanobiosensor, characterized in that the lanthanide nanobiosensor is synthesized by the relevant steps in any one of claims 1 - 8 of the multiplex sensitive detection and analysis method.

[0039] The present invention also provides an application of the lanthanide nanobiosensor in the preparation of a product for detecting high-risk HPV subtypes.

[0040] The principle of the present invention is to use streptavidin magnetic beads as a capture platform. The magnetic beads linked with capture DNA, the sample solution, and a variety of lanthanide nanobiosensors are mixed and incubated at 25 °C for a period of time. Through the principle of base complementary pairing, the magnetic beads successfully capture the target DNA in the sample. At the same time, the lanthanide nanosensors can also be located on the target DNA. Therefore, a sandwich structure is formed by the magnetic beads, the target DNA, and the lanthanide nanoparticles. Using the magnetism of the magnetic beads, the remaining impurities and excess lanthanide nanobiosensors in the sample are magnetically separated, and aqua regia is added for digestion. Finally, ICP-MS is used to detect the digested solution to obtain the content information of the lanthanide elements, so as to perform quantitative analysis on HPV-DNA and achieve the purpose of sensitive detection of multiple high-risk HPV subtypes.

[0041] The principle of preparing the lanthanide nanobiosensor in the present invention is as follows: Lanthanide nanoparticles with uniform morphology are synthesized by the high-temperature co-precipitation synthesis method. First, lanthanide metal ions form a precipitate with sodium ions and fluoride ions at room temperature. 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 washed away with hydrochloric acid to make them positively charged, which can directly generate electrostatic adsorption with the phosphate groups at the 5'-end of DNA, and the lanthanide nanobiosensor required for detection is synthesized.

[0042] The principle of detecting HPV-DNA in the present invention is as follows: The magnetic beads linked with capture DNA, the sample solution, and a variety of lanthanide nanobiosensors are mixed. Through the principle of base complementary pairing, the magnetic beads successfully capture the target DNA in the sample. At the same time, the lanthanide nanosensors can also be located on the target DNA. Then, using the magnetism of the magnetic beads, the remaining impurities and excess lanthanide nanobiosensors in the sample are magnetically separated to perform quantitative labeling on HPV-DNA.

[0043] Advantageous effects:

[0044] The present invention provides an analytical method capable of multiplex and sensitive detection of multiple high-risk HPV subtypes. Six lanthanide nanoparticles with good morphology can be synthesized as probes through a high-temperature coprecipitation synthesis method. The lanthanide nanoparticles with good uniformity provide as many attachment sites as possible for detecting DNA, and the distribution density of the detecting DNA is regulated by non-functional short chains, improving the ability to identify target DNA. When combined with ICP-MS, the sensitivity of the detection method is well improved. The concentration range for detecting high-risk HPV subtypes by the method in the present invention includes 0.1 - 20,000 pM, and the detection limit can be as low as 0.07 pM. Meanwhile, by using the base complementary pairing principle of DNA and the magnetic separation performance of magnetic beads, interference during 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 that of 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, thus simplifying the detection process and achieving the purpose of being easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0046] Figure 1 It is a transmission electron microscope comparison diagram of the magnetic beads in the present invention. Among them, a is the transmission electron microscope diagram of the bare magnetic beads, and b is the transmission electron microscope diagram of the magnetic beads connected with nanoparticles after the detection reaction is completed;

[0047] Figure 2 It is a schematic diagram of a multiplex analysis method for detecting high-risk HPV subtypes based on multiple lanthanide nanoparticles in the present invention;

[0048] Figure 3 It is a result diagram of the feasibility exploration of a multiplex analysis method for detecting high-risk HPV subtypes based on multiple lanthanide nanoparticles in the present invention;

[0049] Figure 4 It is a result diagram of the selectivity exploration of a multiplex analysis method for detecting high-risk HPV subtypes based on multiple lanthanide nanoparticles in the present invention;

[0050] Figure 5 It is a result diagram of the detection performance of a multiplex analysis method for detecting high-risk HPV subtypes based on multiple lanthanide nanoparticles in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0052] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0054] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0055] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0056] The chemical reagents, biochemical reagents, and materials used in the present invention can be obtained from commercial sources without special indication. The water used in the following examples is ultrapure water processed by a Milli-Q ultrapure water purification system. The relevant sequences involved in the following examples were synthesized and purified by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China).

[0057] The multiplex sensitive detection and analysis method based on the combination of lanthanide nanoprobes and ICP-MS in the present invention comprises the following steps:

[0058] (1) Synthesis of oleic acid-coated lanthanide nanoparticles

[0059] (1.1) Weigh 0.5 - 2 mmol of lanthanide chloride salts, 3 - 12 mL of oleic acid, and 7.5 - 30 mL of 1 - octadecene. Under an anaerobic and anhydrous environment, mix the three and heat them to 140 - 180 °C to ensure that the lanthanide chloride salts are completely dissolved in the organic solvent.

[0060] (1.2) Wait for the solution to cool naturally to room temperature, and slowly add dropwise 5 - 20 ml of methanol containing 50 - 200 mg of NaOH and 74 - 296 mg of NH 4 F, and stir at room temperature for 50 min.

[0061] (1.3) Heat up until the excess methanol and water in the solution are removed, and continue to heat up to 280 - 290 °C under nitrogen protection and react for 0.5 - 1 h. Then wait for the solution to cool naturally to room temperature.

[0062] (1.4) Mix the reaction solution and acetone in a ratio of 1:1, centrifuge at 10000 rpm for 30 min. Then redisperse the precipitate in a mixed solution of ethanol and cyclohexane, and wash twice under the same centrifugation conditions. Finally, disperse the precipitate in cyclohexane for storage.

[0063] (2) Synthesis of lanthanide - based nanobiosensors

[0064] (2.1) Take 1 - 2 ml of the above - mentioned nanoparticle solution and mix it with 1 - 2 ml of hydrochloric acid solution (1 M), and sonicate for 30 min.

[0065] (2.2) Under the condition of 14000 - 15000 rpm, centrifuge for 20 - 30 min, redissolve in 1 - 2 ml of hydrochloric acid solution (0.1 M), and sonicate for 30 min.

[0066] (2.3) Under the condition of 14000 - 15000 rpm, centrifuge for 20 - 30 min, redissolve in ethanol solution, wash twice under the same centrifugation conditions, and finally disperse the nanoparticles in deionized water.

[0067] (2.4) Add 40 - 56 μL of DNA solution (the detection DNA and T 10 are added in the optimal ratio, and the concentration is 4 - 8 μM each) and mix it with 50 - 70 μl of nanoparticles, and react in an incubator at 37 °C for 10 - 15 h.

[0068] (2.5) Under the condition of 14000 rpm, centrifuge for 5 min, redissolve in TBS solution, wash once with TBS buffer under the same centrifugation conditions, and then disperse in TBS buffer containing 10% (w / v) BSA. The blocking procedure is completed by incubating at 25 °C for 1 h. Then wash once with TBS buffer and disperse in TBS buffer containing 10% (w / v) BSA, and store at 4 °C.

[0069] (3) Magnetic bead labeling for DNA capture

[0070] (3.1) Take 2 - 4 μL of magnetic bead suspension and wash it 3 times with TBS buffer. Remove the washing solution by magnetic separation.

[0071] (3.2) Incubate the magnetic beads with 2 - 4 μL of capture DNA solution (5 μM) at room temperature for 1 h, and label the capture DNA with biotin - streptavidin reaction.

[0072] (3.3) Remove the excess capture DNA by magnetic separation, and disperse the magnetic beads in TBS buffer containing 10% (w / v) BSA for blocking. The blocking procedure is completed by incubating at 25 °C for 0.5 - 1.5 h. After washing 4 times with TBS, redissolve the capture DNA - labeled magnetic beads in TBS buffer and store at 4 °C until use.

[0073] (4) Analysis and detection of HPV - DNA

[0074] (4.1) Use 5 - 10 μL of the previously prepared magnetic bead mixture as the capture unit, and incubate it with different concentrations of HPV - DNA sample solution and 10 - 20 μL of lanthanide nanobiosensor at 25 °C for 30 min.

[0075] (4.2) Wash the magnetic beads 3 times with 200 μL of TBS buffer.

[0076] (4.3) After magnetic separation, remove the supernatant, and digest the magnetic beads and lanthanide nanoparticles with 50 - 100 μL of aqua regia at room temperature.

[0077] (5) ICP - MS detection

[0078] (5.1) Take 50 μL of the digested solution, dilute it to 5 - 10 mL, and transfer it to an EP tube.

[0079] (5.2) Detect 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm in ICP - MS in SRD mode;

[0080] (5.3) Using 89 Y, 141 Pr, 153Eu, 159 Tb, 165 Ho and 169 Tm as the standard, substitute the measured intensity into the linear equation to calculate the concentration.

[0081] Example 1

[0082] The multi-sensitive detection and analysis method based on the combination of lanthanide nanoprobes and ICP-MS in the present invention comprises the following steps:

[0083] (1) Synthesis of oleic acid-coated lanthanide nanoparticles

[0084] (1.1) Weigh 1 mmol of lanthanide chloride salt, 6 mL of oleic acid, and 15 mL of 1-octadecene. Under an oxygen-free and water-free environment, mix the three and heat up to 160 °C to ensure that the lanthanide chloride salt is completely dissolved in the organic solvent;

[0085] (1.2) Wait for the solution to cool naturally to room temperature, and slowly add dropwise 10 ml of methanol containing 100 mg of NaOH and 148 mg of NH 4 F, and stir at room temperature for 50 min;

[0086] (1.3) Heat up until the excess methanol and water in the solution are removed, and continue to heat up to 285 °C under nitrogen protection and react for 1 h, then wait for the solution to cool naturally to room temperature;

[0087] (1.4) 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, wash twice under the same centrifugation conditions, and finally disperse the precipitate in cyclohexane for storage.

[0088] (2) Synthesis of lanthanide nanobiosensors

[0089] (2.1) Take 1 ml of the above nanoparticle solution and mix it with 1 ml of hydrochloric acid solution (1 M), and ultrasonicate for 30 min;

[0090] (2.2) Under the condition of 15000 rpm, centrifuge for 20 min, redissolve in 1 ml of hydrochloric acid solution (0.1 M), and ultrasonicate for 30 min;

[0091] (2.3) Under the condition of 15000 rpm, centrifuge for 20 min, redissolve in ethanol solution, wash twice under the same centrifugation conditions, and finally disperse the nanoparticles in deionized water;

[0092] (2.4) Add 50 μL of DNA solution (detection DNA and T 10 are added in the optimal ratio, and the concentration is 6 μM each) and 62.5 μL of nanoparticles, and react in an incubator at 37 °C for 12 h;

[0093] Centrifuge for 5 min at 14,000 rpm under the condition of (2.5), redissolve in TBS solution, wash once with TBS buffer under the same centrifugation condition, then disperse in TBS buffer containing 10% (w / v) BSA, and complete the blocking procedure by incubating at 25 °C for 1 h. Then wash once with TBS buffer, disperse in TBS buffer containing 10% (w / v) BSA, and store at 4 °C.

[0094] (3) Magnetic bead labeling to capture DNA

[0095] (3.1) Take 3 μL of magnetic bead suspension and wash it 3 times with TBS buffer, and remove the washing solution by magnetic separation;

[0096] (3.2) Incubate the magnetic beads with 3 μL of capture DNA solution (5 μM) at room temperature for 1 h, and label the capture DNA with biotin-streptavidin reaction;

[0097] (3.3) Remove the excess capture DNA by magnetic separation, disperse the magnetic beads in TBS buffer containing 10% (w / v) BSA for blocking. The blocking procedure is completed by incubating at 25 °C for 1 h. After washing 4 times with TBS, redissolve the magnetic beads labeled with capture DNA in TBS buffer and store at 4 °C until use.

[0098] (4) Analysis and detection of HPV-DNA

[0099] (4.1) Take 5 μL of the previously prepared magnetic bead mixture as the capture unit, incubate with different concentrations of HPV-DNA sample solution and 10 μL of lanthanide nanobiosensor at 25 °C for 30 min;

[0100] (4.2) Wash the magnetic beads 3 times with 200 μL of TBS buffer;

[0101] (4.3) After magnetic separation, remove the supernatant, and digest the magnetic beads and lanthanide nanoparticles with 50 μL of aqua regia at room temperature.

[0102] (5) ICP-MS detection

[0103] (5.1) Take 50 μL of the digested solution, dilute it to 8 mL, and transfer it to an EP tube;

[0104] (5.2) Detect 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm in ICP-MS in SRD mode;

[0105] (5.3) Using 89 Y, 141 pr,153 Eu, 159 Tb, 165 Ho and 169 Tm as the standard, substitute the measured intensity into the linear equation to calculate the concentration.

[0106] The results are as Figure 1 shown. It can be seen from Figure 1 that by comparing the surface of the bare magnetic beads and the magnetic beads after reaction, it can be seen that through the method proposed in the present invention, the nanoparticles are all located on the surface of the magnetic beads for subsequent analysis and detection.

[0107] Comparative experiment on whether DNA sandwich sandwich structure is formed in Example 2

[0108] To accurately detect the concentration of biomarkers in a sample, the formation of a sandwich sandwich structure is crucial. For HPV-DNA, the necessity of the sandwich sandwich structure and the feasibility of the analysis method of the present invention were compared under different conditions.

[0109] In the experiment, the concentration of the target HPV-DNA was 1 nM. The sample solution added to the group lacking the target (blank group) was TBS buffer solution, and the other control groups were HPV-DNA solution (1 nM); the probe solution added to the group lacking the nanoprobe was TBS buffer solution, and the other control groups were six lanthanide 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 solutions of magnetic beads modified with capture DNA. Keeping other conditions exactly the same, detect the reaction of the target with a 5 μL magnetic bead mixture and a 10 μL nanoprobe mixture solution at 25 °C for 30 min, wash 3 times with TBS buffer solution, after magnetic separation, add 50 μL of aqua regia for digestion, and use ICP-MS to detect the lanthanide elements 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm signals (as Figure 2 shown).

[0110] Figure 3 It can be seen that no effective intensity signal of lanthanide elements can be detected under the conditions of lacking target DNA, nanoprobe or no capture DNA connected to the magnetic beads. Only when the target DNA, nanoprobe and magnetic beads connected with capture DNA are all available can an obvious lanthanide element signal be detected, which can be used for the detection of HPV-DNA. Therefore, this example also proves the conditions required for the formation of the sandwich sandwich structure and the feasibility of the analysis method of the present invention.

[0111] Example 3 Explore the specific detection of HPV-DNA by the analysis method of the present invention

[0112] In this example, taking HPV-DNA as an example, several mismatch types of HPV-DNA and random sequences were detected using an analytical method based on the detection of lanthanide nanoparticles to explore the specificity of this method.

[0113] Using the above steps, six kinds of nanoparticles were used as probes to detect six kinds of HPV-DNA (HPV-16, HPV-18, HPV-31, HPV-39, HPV-56, and HPV-58, 1 nM), mis-1 (single-base mismatch, 1 nM) of six kinds of HPV-DNA, and mis-3 (three-base mismatch, 1 nM) of six kinds of HPV-DNA. Keeping other conditions exactly the same, the target DNA was mixed with 5 μL of magnetic bead mixture and 10 μL of nanoprobe mixture solution and reacted at 25 °C for 30 min, washed 3 times with TBS buffer. After magnetic separation, 50 μL of aqua regia was added for digestion, and the signals of lanthanide elements 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho, and 169 Tm were detected using ICP-MS.

[0114] The results are as Figure 4 shown. Except that the corresponding HPV-DNA can cause a significant increase in the signals of the corresponding lanthanide elements, other several mismatch types and non-corresponding HPV sequences cannot cause significant changes in the signals, proving that the specific recognition of base complementary pairing between DNAs in the present invention can accurately perform multiplex detection of HPV-DNA.

[0115] Example 4 explores the sensitivity detection of the analytical method of the present invention for HPV-DNA

[0116] In this example, the sensitivity and linearity of the analytical method based on the detection of six kinds of lanthanide nanoparticles for the detection of six kinds of HPV-DNA were explored.

[0117] In the experiment, 5 μL of magnetic bead solution and 10 μL of nanoprobe solution were added to HPV-DNA solutions with different concentrations, reacted at 25 °C for 30 min, washed 3 times with TBS buffer. After magnetic separation, 50 μL of aqua regia was added for digestion, and the signals of lanthanide elements 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho, and 169 Tm were detected using ICP-MS.

[0118] From Figure 5It can be seen that according to the relationship that the concentration is proportional to the response signal, the linear relationships between the concentrations of six kinds of HPV-DNA and 89 Y, 141 Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm response signals are obtained. When the concentration of HPV-16 is within 10 - 2500 pM 159 the Tb response signal shows a linear relationship, the linear correlation coefficient is 0.990, and the calculated detection limit is 1.14 pM (n = 12, 3σ). When the concentration of HPV-18 is within 1 - 2500 pM 165 the Ho response signal shows a linear relationship, the linear correlation coefficient is 0.984, and the calculated detection limit is 0.38 pM (n = 12, 3σ). When the concentration of HPV-31 is within 1 - 2500 pM 153 the Eu response signal shows a linear relationship, the linear correlation coefficient is 0.992, and the calculated detection limit is 0.25 pM (n = 12, 3σ). When the concentration of HPV-39 is within 1 - 5000 pM 141 the Pr response signal shows a linear relationship, the linear correlation coefficient is 0.983, and the calculated detection limit is 0.15 pM (n = 12, 3σ). When the concentration of HPV-56 is within 100 - 20000 pM 89 the Y response signal shows a linear relationship, the linear correlation coefficient is 0.933, and the calculated detection limit is 7.29 pM (n = 12, 3σ). When the concentration of HPV-58 is within 0.1 - 2000 pM 169 the Tm response signal shows a linear relationship, the linear correlation coefficient is 0.992, and the calculated detection limit is 0.07 pM (n = 12, 3σ). It is proved that this analysis method has good sensitivity.

[0119] Example 5 explores the detection performance of the analysis method of the present invention in serum

[0120] Sample collection: Human serum samples are collected from the Seventh People's Hospital of Chengdu.

[0121] Sample pretreatment: In view of the complex composition and viscosity of serum samples, 1% bovine serum albumin (BSA) TBS buffer is used to dilute the serum samples.

[0122] Detection of human serum samples by the analysis method based on lanthanide nanoparticles detection:

[0123] Add 5 μL of magnetic bead solution and 10 μL of nanoprobe solution into the human serum sample, react at 25 °C for 30 min, wash 3 times with TBS buffer, after magnetic separation, add 50 μL of aqua regia for digestion, and use ICP-MS to detect lanthanide elements 89 Y, 141Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm signals are detected.

[0124] Table 1 Recovery rates of HPV-DNA in human serum samples

[0125]

[0126] The detection results are shown in Table 1. It can be seen from Table 1 that the recovery rates of HPV-DNA in serum samples are all concentrated in the range of 92.85 - 112.3%, showing good recovery rates. This indicates that this analytical method can adapt to the complex environment of actual samples and ensure accuracy.

[0127] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A multiplex sensitive detection and analysis method based on the combination of lanthanide nanoprobes and ICP-MS, characterized in that: The multiplex sensitive detection analysis method comprises the following steps: (1) Synthesis of oleic acid-coated lanthanide nanoparticles; (2) synthesizing lanthanide nanobioprobes using the oleic acid-coated lanthanide nanoparticles prepared in step (1); (3) Modifying the magnetic beads as capture units to obtain modified magnetic beads; (4) mixing the target DNA with the lanthanide nanobioprobe prepared in step (2) and the modified magnetic beads prepared in step (3), incubating and digesting the mixture to obtain a digested solution; (5) The digested solution is subjected to ICP-MS detection.

2. The multiplex sensitive detection and analysis method according to claim 1, characterized in that: In step (1), the specific steps of synthesizing oleic acid-coated lanthanide nanoparticles are: (1.1) Weigh 0.5-2 mmol of lanthanum chloride salt, 3-12 mL of oleic acid and 7.5-30 mL of 1-octadecene, mix the three in an oxygen-free and water-free environment and heat them to 140-180 °C to ensure that the lanthanum chloride salt is completely dissolved in the organic solvent; (1.2) After the solution is cooled to room temperature, slowly add 5-20 ml of methanol containing 50-200 mg of NaOH and 74-296 mg of NH4F and stir at room temperature for 50 min. (1.3) Raise the temperature until excess methanol and water in the solution are removed, and continue to raise the temperature to 280-290°C under nitrogen protection for 0.5-1h, and then allow the solution to cool naturally to room temperature; (1.4) The reaction solution and acetone were mixed in a ratio of 1:1, and the mixture was centrifuged at 10,000 rpm for 30 min. The precipitate was then redispersed in a mixture of ethanol and cyclohexane and washed twice under the same centrifugal conditions to obtain oleic acid-coated lanthanide nanoparticles.

3. The multiplex sensitive detection and analysis method according to claim 1, characterized in that: In step (2), the specific steps of synthesizing the lanthanide nanobioprobe are: (2.1) Mix 1-2 ml of the oleic acid-coated lanthanide nanoparticle solution with 1-2 ml of hydrochloric acid solution and sonicate for 30 minutes; (2.2) Centrifuge at 14000-15000 rpm for 20-30 min, redissolve in 1-2 ml hydrochloric acid solution, and sonicate for 30 min; (2.3) Centrifuge at 14000-15000 rpm for 20-30 min, redissolve in ethanol solution, and wash twice under the same centrifugation conditions to obtain nanoparticles dispersed in deionized water; (2.4) Mix 40-56 μL of DNA solution with 50-70 μL of nanoparticles and react in an incubator at 37 °C for 10-15 h; (2.5) Centrifuge at 14000 rpm for 5 min, redissolve in TBS solution, wash once with TBS buffer under the same centrifugation condition, and then disperse in 10% BSA TBS buffer. The blocking procedure is completed by incubating at 25°C for 1 h, and then wash once with TBS buffer and disperse in 10% BSA TBS buffer to obtain the lanthanide nanobioprobe.

4. The multiplex sensitive detection and analysis method according to claim 1, characterized in that: In step (3), the specific steps of modification are: (3.1) Take 2-4 μL of magnetic bead suspension and wash it three times with TBS buffer, and remove the washing solution by magnetic separation; (3.2) Incubate the magnetic beads with 2-4 μL of captured DNA solution at room temperature for 1 h, and label the captured DNA with biotin-streptavidin reaction; (3.3) Remove excess captured DNA by magnetic separation, disperse the magnetic beads in TBS buffer containing 10% BSA for blocking, wash with TBS 4 times, and then redissolve the captured DNA-labeled magnetic beads in TBS buffer to obtain the modified magnetic beads.

5. The multiplex sensitive detection and analysis method according to claim 4, characterized in that: In step (3.3), the blocking step is incubation at 25°C for 0.5-1.5h.

6. The multiplex sensitive detection and analysis method according to claim 1, characterized in that: In step (4), the specific steps of incubation and digestion are: (4.1) 5-10 μL of modified magnetic beads were used as capture units and incubated with HPV-DNA sample solutions of different concentrations and 10-20 μL of lanthanide nanobioprobes at 25 °C for 30 min; (4.2) Wash the magnetic beads three times with 200 μL TBS buffer; (4.3) After magnetic separation, remove the supernatant and digest the magnetic beads and lanthanide nanoparticles with 50-100 μL aqua regia at room temperature.

7. The multiplex sensitive detection and analysis method according to claim 1, characterized in that: In step (5), the specific steps of the ICP-MS detection are: (5.1) Take 50 μL of the digested solution, dilute it to 5-10 mL, and transfer it to an EP tube; (5.2) In ICP-MS 89 Y. 141 Pr, 153 Eu, 159 Tb, 165 Ho and 169 Tm was tested, SRD mode; (5.3) 89 Y. 141 Pr, 153Eu, 159 Tb, 165 Ho and 169 Tm was used as the standard, and the measured intensity was substituted into the linear equation to calculate the concentration.

8. The multiplex sensitive detection and analysis method according to claim 1, characterized in that: The multiplex sensitive detection and analysis method can be used for the detection of high-risk HPV subtypes.

9. A lanthanide nanobiological probe, characterized in that: The lanthanide nano-bioprobe is synthesized by the relevant steps in the multiple sensitive detection and analysis method according to any one of claims 1 to 8.

10. Use of the lanthanide nanobiological probe as claimed in claim 9 in the preparation of a product for detecting high-risk HPV subtypes.

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