CN107796864A - A kind of application of nano particle coupling probe system in the highly sensitive detections of ctDNA - Google Patents

A kind of application of nano particle coupling probe system in the highly sensitive detections of ctDNA Download PDF

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CN107796864A
CN107796864A CN201710961877.0A CN201710961877A CN107796864A CN 107796864 A CN107796864 A CN 107796864A CN 201710961877 A CN201710961877 A CN 201710961877A CN 107796864 A CN107796864 A CN 107796864A
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dna
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ctdna
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CN107796864B (en
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胡萍
施剑林
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Shanghai Institute of Ceramics of CAS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The present invention relates to a kind of application of nano particle coupling probe system in the highly sensitive detections of ctDNA, nano particle coupling probe material system is made up of the first probe and the second probe, and the expression formula of the first probe is MNP X DNA1, wherein MNP is magnetic nanoparticle kernel, and X is the hydrophily shell for being coated on magnetic nanoparticle kernel, DNA1To modify the first complementary nucleic acid chain that can be complementary with the Part I of target circulation Tumour DNA on hydrophily shell, the expression formula of the second probe is Rep DNA2, wherein Rep is that can use the report element kernel of the report element formation of ICP MS detections, DNA2To modify the second complementary nucleic acid chain that can be complementary with the Part II of target circulation Tumour DNA on report element kernel.The double nano probe MNP@X DNA of the present invention1With Rep DNA2Prepare simple, controllable, easy repetition.

Description

A kind of application of nano particle coupling probe system in the highly sensitive detections of ctDNA
Technical field
The invention belongs to medical bio technical field of nano material, and in particular to nano particle coupling is used in a kind of ctDNA detections Close probe material and its preparation method and application.
Background technology
The first step caused by tumour is gene mutation, and cancer is a kind of genopathy after all.With cancer research not Disconnected to go deep into, people detect the mutator consistent with tumour cell, i.e. Circulating tumor DNA in the peripheral blood of cancer patient (circulating tumor DNA, ctDNA).CtDNA is that the gene being discharged into by tumour cell in blood circulation system is broken Piece, it is " information password " that cancer is scattered in blood.By stages directly related to the ctDNA concentration in blood, the middle evening of cancer Phase patient ctDNA concentration is significantly higher than early stage or extreme early patient.CtDNA concentration raises with tumor development, because swollen Knurl is controlled and reduced, and is the tumor blood label of a kind of hypersensitivity, high specific.The change of ctDNA concentration is remote Earlier than the change that the means such as iconography detect tumor tissues.With traditional imaging diagnosis, endoscopy and pathology Diagnosis is compared, and the accurate analysis to ctDNA can monitor the multidate information of tumor load in patient body in real time, can more sensitively send out The change of existing disease, more science, the effect for rapidly evaluating a certain therapeutic scheme, advantage are notable.Most importantly, it is right CtDNA monitoring is only needed to extract a small amount of peripheral blood of patient, and patient is free from side effects, and high frequency monitoring in real time can be achieved repeatedly. Therefore, ctDNA detection and its research of biological indicator would be possible to the early diagnosis for clinical tumor, Index for diagnosis and with Track follow-up provides a series of convenient, fast, special, noninvasive detection means.CtDNA detections are a kind of Novel noninvasive " liquid Biopsy " mode, it is expected to make up the deficiency of existing methods for clinical diagnosis, there is provided the analysis from tumor cell levels to molecular level is put down Platform, reference is provided for individualized treatment.Noninvasive " liquid biopsy " technology based on ctDNA detections is expected to substitute invasive " tissue biopsy " is used for the monitoring of oncotherapy progress, and the basic research in the field has very important scientific meaning and clinic Value.
CtDNA detections are the excellent modes of tumour early screening, but in tumorigenic early stage, extreme early even cell Stage, the extremely low (10~180ng.ml of ctDNA concentration in blood-1), how to detect extremely low in earlier stage cancer patients' blood contain The ctDNA of amount is primary technological difficulties.This sensitivity to detection method proposes very high requirement.In addition, the serum of patient Composition complex, ctDNA only account for the 0.01%-1% in total nucleic acid in hematological system, therefore to realize that ctDNA's is highly sensitive Degree detection, another technological difficulties are how to avoid serious background genes from disturbing.In recent years, increasing scholar is directed to The research of ctDNA detections.It has been reported that detection method be mainly based upon two thinkings, first, passing through PCR (PCR) ctDNA is significantly expanded, makes up to the concentration that can be detected.Including digital pcr and BEAMing technologies.Digital pcr Available for the specific mutations for assessing and detecting the ctDNA in blood plasma, available for absolute quantitation, sensitivity is up to single nucleic acid point Son, test limit as little as 0.001%.BEAMing technology combination digital pcrs determine catastrophe with streaming technology, and sensitivity reaches To 0.005%.Another existing detection thinking is sequenced again after direct progress genome sequencing or PCR expand, It is complete outer including mark amplification deep sequencing (TAM-Seq), cancer individuation deep sequencing (CAPP-Seq), genome sequencing Aobvious son sequencing etc..TAM-Seq is that the target area circulated first with specific primer expands, and recycles connecing for different qualities Leader label carry out secondary amplification, then carry out two-way repetition and are sequenced.CAPP-Seq is used as " sieve by the use of the mutational site storehouse customized Select device ", ultra-deep sequencing is carried out again after target capture is carried out to sample, and detection sensitivity is high, high specificity.
The research in the world to cancer early stage ctDNA detections is in starting stage, the detection side of PCR-based amplification at present The problem of method is present is that test result is largely dependent upon gene magnification, and false positive probability is high, and pretreatment is cumbersome, depends on The gene mutation information that biopsy provides, detection device and reagent are completely dependent on import.Detection method based on gene sequencing although The rate that reports an error is low, but the cost researched and developed and tested is all very high, and detection cycle is up to more than one week, it is difficult to be widely used in clinic Detection.In general, existing detection method also fails to because of sensitivity, test limit, the detection limitation such as price and detection cycle Promoted on a large scale in clinical medicine.Probe boldly, make great efforts convenient, fast, special, the sensitive ctDNA detection methods one of exploitation It is directly the target that researcher pursues.In recent years, the foundation of nanometer diagnosis and treatment medical science and the serious medical conditions such as cancer are developed into Early diagnosis and situ treatment provide a brand-new multi-functional diagnosis and treatment platform, are expected to increase substantially cancer early stage diagnosis and treatment Efficiency.Nano material has the features such as synthesis is controllable, appearance structure, surface characteristics easy-regulating, the biology biography based on nano material The detection sensitivity of biomolecule can be greatly improved by properties such as the unique physics of nano material, chemistry for sensor, have choosing Selecting property is high, analyze speed is fast, operates the features such as simple and instrument price is cheap, is one important point of nano biological medical research Branch, progressively as a focus of field of biomedical research.
In summary, develop the ctDNA nanometer detection new strategies based on nanometer biotechnology, research it is a kind of it is convenient, fast, Specifically, noninvasive ctDNA nanometer detections method, application of the liquid biopsy in tumour diagnosis and treatment field is promoted, it is significant And value.
The content of the invention
In view of the shortcomings of the prior art, can high sensitivity, specific recognition ctDNA it is an object of the invention to provide one kind Nano particle coupling probe material system, and using the system detection method.
Herein, on the one hand, the present invention provides a kind of nano particle coupling probe material system, and the nano particle coupling is visited Needle material system is made up of the first probe and the second probe, and the expression formula of first probe is MNP X-DNA1, wherein MNP is Magnetic nanoparticle kernel, X are the hydrophily shell for being coated on the magnetic nanoparticle kernel, DNA1To modify in the parent The first complementary nucleic acid chain that can be complementary with the Part I of target circulation Tumour DNA on water-based shell, second probe Expression formula be Rep-DNA2, wherein Rep is that can use the report element kernel for the report element formation that ICP-MS is detected, DNA2For Second complementary nucleic acid that can with the Part II of target circulation Tumour DNA complementary of the modification on the report element kernel Chain.Wherein, DNA1For the complementary strand (such as half base of ctDNA complementary strands) of a target ctDNA part, DNA2For target The complementary strand (such as ctDNA complementary strands second half base) of ctDNA another part.In aftermentioned detection process, target ctDNA can By DNA1And DNA2Connect so as to by two kinds of nano particle bridgings.
It is preferred that the MNP is formed by paramagnetic nanoparticle, preferably by Fe3O4And/or amorphous Fe is formed.
It is preferred that the Rep is formed by the heavy metal element that can be delicately detected with ICP-MS, preferably by Au, Ag, Pt At least one of formed, ICP-MS Monitoring lower-cuts as little as 0.01ppb.
It is preferred that first probe is repaiied by will carry out amino after magnetic nanoparticle Surface coating hydrophily shell Decorations, and and DNA1Generation condensation reaction obtains.In one example, can be by preparing particle diameter 30-50nm magnetic Nano Grain, Surface coating hydrophily shell (such as SiO2) hydrophilic modifying is carried out, obtain MNP@X (such as MNP@SiO2), MNP@X are entered Row is amido modified, and using amino with catching DNA1Condensation reaction occurs for the carboxyl of upper modification, will catch DNA1Success is modified MNP@X surfaces, prepare MNP@X-DNA1。
It is preferred that the X is SiO2And/or hydrophilic organics hydrophilicity.X is SiO2In the case of, the first probe can be expressed as MNP@SiO2-DNA1。
It is preferred that second probe is by by DNA2(the DNA containing multiple base A2) and Rep nano particles (such as Gold nano grain) stable dispersion obtains in aqueous.In one example, it is right according to adenine deoxyribonucleotide (A) Golden nanometer particle shows strong affinity, can effectively stablize gold nano grain, utilize golden nanometer particle and adenine alkali The special affinity interaction of base will be connected with the primed DNA of multiple adenines2Modify and prepare probe Rep- in golden nanometer particle surface DNA2。
On the other hand, the present invention also provides a kind of application method using above-mentioned nano particle coupling probe material system, Including：The DNA in test serum is extracted with dissociative DNA extracts kit, is dispersed in anaerobic water, heating makes double-stranded DNA solution Chain is single stranded DNA；Add first probe and the second probe；Obtaining magnetic nanoparticle surface by Magnetic Isolation has report The nano particle of element and/or magnetic nanoparticle surface do not have the nano particle of report element, specifically, when target circulation swells In the presence of knurl DNA, first probe and second probe are coupled by target circulation Tumour DNA, and Magnetic Isolation obtains magnetic Property nano grain surface has the nano particle of report element；When target circulation Tumour DNA is not present, Magnetic Isolation obtains magnetic Nano grain surface does not have the nano particle of report element；Report element is quantified, and according to target circulation Tumour DNA with The linear relationship of report element concentration, target circulation Tumour DNA is quantified.The detection target ctDNA of the present invention includes institute There is oncogene known to base sequence, include the KRAS of mutation, NRAS, BRAF, EGFR, PIK3CA etc..
In the application method of the present invention, the complementary base sequence of the ctDNA genes of mutation is divided into two sections of primed DNAs1, DNA2Prepare, then catch DNA in MNP@X magnetic nanoparticles surface modification1, prepare magnetic probe MNP@X-DNA1, Rep Grain (such as Au nano particles) surface modification catches DNA2, prepare probe Rep-DNA2.In the presence of target ctDNA, ctDNA with Two sections of primer strand (DNA1、DNA2) complementary pairing, ctDNA links together MNP@X nanos particles and Rep nano particles, passes through There are Rep nano particles on Magnetic Isolation, MNP@X surfaces；In the presence of without target ctDNA, MNP@X surfaces that Magnetic Isolation obtains without Rep nano particles.With reference to Lorentz transmission electron microscopy, target ctDNA can directly be judged whether by visually observing.Pass through The content of report element (Rep elements) in ICP-MS detection architectures, according to Rep contents in standard liquid and ctDNA contents Linear relationship, realize the accurate quantitative analysis to ctDNA.Compared with existing ctDNA detection techniques, the present invention has special excellent Gesture：Double nano probe (MNP@X-DNA1With Rep-DNA2) prepare simple, controllable, easy repetition；Utilize excellent magnetic response characteristic Magnetic nanoparticle carries out quick, high magnetic separation and enrichment to ctDNA, avoids cumbersome PCR processes and complicated gene Sequencing, substantially reduces detection cycle.The DNA primer and target ctDNA selectivity complementary pairings of double nano particle surface modification, The high degree of specificity of detection can be ensured；It need not be expected to be greatly reduced using PCR amplification ctDNA, false positive probability；MNP@X with The unique physical and chemical properties of nano material are applied to the liquid biopsy skill for ctDNA detections by Rep nanometers Fourier Series expansion technique Art, the early diagnosis being expected to as cancer provide a quick, reliable new way, promote liquid biopsy in tumour diagnosis and treatment field In application value it is great.
It is preferred that magnetic screening agent is added before the first probe and the second probe is added.Thus, it is possible to pass through Magnetic Isolation Remove wild type gene (wild type ctDNA；Such as wild type Kras genes).For actual cancer plasma's sample, except Saltant type ctDNA (target ctDNA), also in the presence of a large amount of wild type ctDNA, in order to shelter interference of the wild type ctDNA to detection, Need to prepare the magnetic nanoparticle for sheltering DNA modification, the magnetic nanoparticle of input masking DNA modification, is sheltered before detection DNA and wild type ctDNA complementary pairings, then Magnetic Isolation remove magnetic screening agent and wild type ctDNA.That is, this In invention, magnetic screening agent can added and magnetic screening agent is removed with being detected after wild type ctDNA by Magnetic Isolation Magnetic probe MNP@X-DNA are added in system1With probe Rep-DNA2Carry out saltant type ctDNA detection.
It is preferred that the expression formula of the magnetic screening agent is MNP@X-DNA3, wherein MNP is magnetic nanoparticle kernel, X To be coated on the hydrophily shell of the magnetic nanoparticle kernel, DNA3Be modification on the hydrophily shell can be with The complementary nucleic acid chains of wild type Circulating tumor DNA.
It is preferred that the magnetic screening agent is repaiied by will carry out amino after magnetic nanoparticle Surface coating hydrophily shell Decorations, and with sheltering DNA3Generation condensation reaction obtains.In one example, repaiied in the complementary series end of wild type Kras genes Adorn carboxyl and prepare masking DNA3, pass through MNP@X (such as AFe@SiO2) surface amino groups and DNA3Carboxyl occur EDC and NHS mediation Amidation process by DNA3MNP@X surfaces are grafted onto, prepare magnetic screening agent MNP@X-DNA3。
According to the application method of the present invention, the DNA primer and target ctDNA selectivitys of double nano particle surface modification are complementary Pairing, it is ensured that the high degree of specificity of detection；It need not be expected to be greatly reduced using PCR amplification ctDNA, false positive probability；Blood In clear sample analysis, magnetic screening agent is added, wild type free nucleic acid is removed by Magneto separate, avoids background nucleic to ctDNA The interference of detection, it is expected to realize the high-sensitivity detection under zero background, is expected to overcome existing ctDNA detection techniques to face dense Spend low, two serious hang-ups of ambient interferences, develop the ctDNA nanometer detection new strategies based on nanometer biotechnology.
Brief description of the drawings
Fig. 1 is transmission electron microscope (TEM) photo that the AFe nano particles obtained by the embodiment of the present invention 1 are dispersed in water；
Fig. 2 is the XRD spectrum of the AFe nano particles obtained by the embodiment of the present invention 1；
Fig. 3 is power spectrum (EDS) figure of the AFe nano particles obtained by the embodiment of the present invention 1；
Fig. 4 is the AFe nano particle coated Sis O obtained by the embodiment of the present invention 12Transmission electron microscope (TEM) photo of shell；
Fig. 5 is after the AFe nano particles obtained by the embodiment of the present invention 1 are dispersed in water, to be separated in externally-applied magnetic field magnetic Design sketch (left figure is photo in kind, and right figure is by the weight/mass percentage composition of the ferro element of attraction)；
Fig. 6 is transmission electron microscope (TEM) photo that the Au particles obtained by the embodiment of the present invention 1 are dispersed in water；
Fig. 7 is the Au particles and Au-DNA obtained by the embodiment of the present invention 12Ultraviolet-visible absorption spectroscopy figure；
Fig. 8 is with AFe@SiO2-DNA1With Au-DNA2When probe system detects a series of ctDNA standard liquids of concentration gradients, The TEM photos of Magnetic Isolation product；
Fig. 9 is with AFe@SiO2-DNA1With Au-DNA2When probe system detects a series of ctDNA standard liquids of concentration gradients, The linear relationship of Au concentration and ctDNA concentration；
Figure 10 is with AFe@SiO2-DNA1With Au-DNA2Probe system isolates the ESI-TOF-MS of mutation KRAS genes (135T) Figure；
Figure 11 is to design the AFe@SiO prepared for KRAS genes2-DNA1With Au-DNA2Probe system is for other ctDNA The recall rate evaluation of (NRAS, BRAF, EGFR, PIK3CA)；
Figure 12 is to design the AFe@SiO prepared for KRAS genes2-DNA1With Au-DNA2Probe system is for various cancers cell The detection performance evaluation of KRAS genes is mutated in system；
Figure 13 is AFe@SiO2-DNA1, Au-DNA2The recall rate that probe system is mutated KRAS genetic fragments for different length is commented Valency；
Figure 14 is AFe@SiO2-DNA1With Au-DNA2Probe body ties up in human blood sample sheet the ESI- of the ctDNA fragments captured TOF-MS spectrograms；
Figure 15 is AFe-DNA1With Au-DNA2(a is without place for detection performance evaluation of the probe system for tumor-bearing mice ctDNA Manage tumor-bearing mice gross tumor volume and the change curve of ctDNA concentration；B is tumor-bearing mice gross tumor volume and ctDNA concentration after chemotherapy Change curve, c is the change curve of gross tumor volume and ctDNA concentration after tumor-bearing mice Operation in early stage, and d is tumor-bearing mice Gross tumor volume and the change curve of ctDNA concentration after late surgery treatment).
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and following embodiments, it should be appreciated that following embodiments are only used for Illustrate the present invention, be not intended to limit the present invention.
The present invention relates to one kind can high sensitivity, specific recognition Circulating tumor DNA (ctDNA) double nano particle coupling Close probe material system.Coupling probe system is made up of magnetic Nano probe and reporter probe, and it can pass through in use CtDNA is coupled to form.By hydrophobic magnetic nano grain surface cladding hydrophily shell X (SiO2And/or other hydrophilies are organic Thing) carry out hydrophilic modifying, carry out it is amido modified, and by the amidation process of amino and carboxyl by alkali complementary target ctDNA Substrate segment DNA1Magnetic nanoparticle (magnetite nanoparticle, be abbreviated as MNP) surface is arrived in primer strand modification, is designated as Probe MNP@X-DNA1, MNP includes Fe3O4, the paramagnetic nano particle such as amorphous Fe；By the complementary base pieces of target ctDNA Segment DNA2The nano grain surface modified in report element prepares Reporter-DNA2(it is abbreviated as Rep-DNA2), report element refers to The element that can be detected with ICP-MS, such as the heavy metal element with extremely low Monitoring lower-cut, including Au, Ag and Pt etc..MNP@X- DNA1And Rep-DNA2The nano particle coupling probe material system of composition has high sensitive for ctDNA quantitative detection High and extremely low Monitoring lower-cut is spent, to developing the ctDNA nanometer detection new strategies based on nanometer biotechnology, propulsion is based on Application of the liquid Biopsy of ctDNA detections in early diagnosis of cancer has great importance.
Hereinafter, nano particle coupling probe material, the nano particle coupling probe material system of the present invention is illustrated Preparation method and the detection method using the system.The detection target ctDNA of the present invention is that the generation with tumour is closely related, and And oncogene known to mutant nucleotide sequence (base sequence), such as EGF-R ELISA (EGFR) passage downstream key gene The mutation status such as KRAS, BRAF, PIK3CA, NRAS.
In the present invention, the complementary base sequence of the ctDNA genes of mutation is divided into two sections of primed DNAs1,DNA2Prepare, in magnetic Property nano grain surface modification catch DNA1, magnetic probe (the first probe) is prepared, the modification of Rep particle surfaces catches DNA2, prepare Reporter probe Rep-DNA2(the second probe).
(magnetic Nano probe)
The magnetic Nano probe of the present invention is functional DNA1The magnetic Nano probe of modification, expression formula are MNP@X-DNA1, its Middle MNP is magnetic nanoparticle, preferably paramagnetic nanoparticle, more preferably amorphous Fe nano particle, and X is hydrophily shell, It is preferred that SiO2And/or hydrophilic organics hydrophilicity, hydrophilic organics hydrophilicity include polyvinylpyrrolidone (PVP) etc., DNA1For target CtDNA partially complementary nucleic acid chain.Specifically, DNA1For the half base of target ctDNA complementary strands, in aftermentioned reporter probe DNA2For second half base of target ctDNA complementary strands.In aftermentioned detection process, in the presence of target ctDNA, target CtDNA is by DNA1And DNA2Complementary pairing, two kinds of nano particle bridgings are got up.
On preparing magnetic Nano probe, first, magnetic nanoparticle is prepared, magnetic nanoparticle of the invention can use Commercially available or self-control.
As an example, in the case that MNP is amorphous Fe, the preparation of magnetic nanoparticle can for example include：Press According to (1~1.5):(16~18):The mol ratio of (0.5~1) is by source of iron ferric citrate, organic matter polyvinylpyrrolidone (PVP, mean molecule quantity 55000), surfactant F127 (mean molecule quantity 9000) are dissolved in appropriate anaerobic water, argon atmospher 80-100 DEG C is warming up under enclosing, mechanical agitation removes the oxygen in reaction system；According to ferric citrate:Reducing agent mol ratio (1 ~1.5):The ratio of (20~30) is by reducing agent (NaBH4) be dissolved in the water, it is slowly added dropwise in reaction system and produces largely Bubble；Ethanol is added after 3~4 hours and removes bubble removing, Magnetic Isolation, the amorphous iron nano-particle (AFe nano particles) of collection.This The magnetic nanoparticle particle size range of invention is 30-50nm.
Then, hydrophilic modifying is carried out to magnetic nanoparticle.Specifically, can be in magnetic nanoparticle Surface coating SiO2 And/or other hydrophilic organics hydrophilicities etc..In magnetic nanoparticle Surface coating SiO2In the case of, alkalescence condition can be utilized Lower tetraethyl orthosilicate (TEOS) hydrolysis (Method), in magnetic nanoparticle Surface coating SiO2Shell, improvement are received The stability of rice grain, dispersiveness and biocompatibility in water.
As an example, SiO is used2Carrying out the hydrophilic modifying of amorphous iron nano-particle can for example include：Appropriate AFe Be added in the mixed solution (pH value 7.8-8) of ethanol, water and ammoniacal liquor, ultrasonic disperse for a period of time after, be added dropwise to appropriate Tetraethyl orthosilicate (TEOS), room temperature mechanical stirring, Magnetic Isolation, obtains solid product i.e. SiO2The amorphous iron nano-particle of cladding (AFe@SiO2).The mol ratio of wherein AFe and tetraethyl orthosilicate can be 1~1.8.In the present invention, hydrophily shell thickness is 5 ~10nm.The MNP@X of the present invention possess efficient magnetic responsiveness and good biocompatibility.Specifically, efficient magnetic rings Performance is answered to derive from kernel (such as amorphous iron), good biocompatibility derives from nano grain surface hydrophily shell (example Such as SiO2) cladding.
Then in magnetic Nano (core shell structure MNP@X) surface modification DNA by hydrophilic modifying1.In the present invention, The progress of MNP@X surfaces is amido modified, utilizes amino with catching DNA1Condensation reaction occurs for the carboxyl of upper modification, will catch DNA1Into Work(is modified on MNP@X surfaces.In magnetic nanoparticle Surface coating SiO2In the case of, can be by SiO2The silicone hydroxyl of shell with APTES (APTES) carries out Silanization reaction, in AFe@SiO2Surface modification amino, by amino with The amidation process of carboxyl is by base fragment DNA complementary target ctDNA1MNP@X surfaces are arrived in primer strand modification.
As an example, MNP@X surface modifications DNA1Such as it can include：Appropriate (such as 50mg) the MNP@X of general (such as AFe@SiO2) be dispersed in (such as 100mL) ethanol, 78~85 DEG C are heated under mechanical agitation, add (such as 100 μ L) APTES, return stirring for a period of time after, centrifugation, solid product is washed with water；According to (3~5)：(10~15)：(20~25) rub You compare DNA1, EDC, NHS be added in (such as pH=7.4) PBS aqueous solution, being stirred at room temperature makes DNA1On carboxyl it is whole Activation, adds appropriate MNP@X-NH2PBS solution, stirring, Magnetic Isolation obtains magnetic Nano probe MNP@X-DNA1。
(reporter probe)
The reporter probe of the present invention is functional DNA2The reporter probe of modification, expression formula Rep-DNA2, wherein Rep is report Element, DNA2For the partially complementary nucleic acid chain of target circulation Tumour DNA.In the present invention, report element, which refers to, to be detected with ICP-MS, And the heavy metal element with extremely low Monitoring lower-cut, including Au, Ag and Pt etc..
In the present invention, by by base fragment DNA complementary target ctDNA2Modify the nano particle table in report element Face prepares reporter probe.Specifically, preparing reporter probe can include：By 5~10nm Rep nano particles (such as Au nanometers Particle) and DNA2By (1~1.5)：The ratio mixing of (40~80), adds appropriate citrate buffer agent, with liquid-transfering gun gently Piping and druming so that Rep nano particles and DNA2It is well mixed, deionized water is added, centrifuges, obtains Rep-DNA2.As one Example, using Au as report element in the case of, golden nanometer particle is shown according to adenine deoxyribonucleotide (A) It strong affinity, can effectively stablize gold nano grain, utilize golden nanometer particle and the special affinity interaction of adenine base The primed DNA of multiple adenines will be connected with2Modify and prepare probe Au-DNA in golden nanometer particle surface2。
(ctDNA detections)
In the present invention, magnetic Nano probe and reporter probe can be utilized to form nano particle coupling probe material system, be used for CtDNA is detected.
First, the DNA in test serum is extracted with dissociative DNA extracts kit, is dispersed in anaerobic water, heating makes double Chain DNA unwinds as single stranded DNA.
Then, magnetic Nano probe is added in detection architecture and reporter probe carries out saltant type ctDNA detection.
In the present invention, magnetic screening agent can be added before magnetic Nano probe and reporter probe is added.For actual cancer Disease patient blood sample, except saltant type ctDNA, also in the presence of a large amount of wild type ctDNA, in order to shelter wild type ctDNA to inspection Survey interference, it is necessary to prepare masking DNA modification magnetic nanoparticle, can put into before testing masking DNA modification magnetic Property nano particle, masking DNA and wild type ctDNA complementary pairings, then by Magnetic Isolation remove magnetic screening agent with it is wild Type ctDNA, thus, it is possible to the interference that masking wild type gene detects to mutator completely.
The expression formula of the magnetic screening agent of the present invention is MNP@X-DNA3, wherein MNP is magnetic nanoparticle, and X is hydrophilic Property shell, preferably SiO2And/or hydrophilic organics hydrophilicity, DNA3For wild type target ctDNA complementary nucleic acid chain.
, can be by by the complementary nucleic acid chain DNA of wild type gene on the preparation of the magnetic screening agent3Modification is in magnetic Nano grain surface, prepare magnetic screening agent MNP@X-DNA3.In one example, such as the complementary sequence in wild type ctDNA Arrange end modified carboxyl and prepare masking DNA3, pass through MNP@X surface amino groups and DNA3Carboxyl occur EDC and NHS mediation acid amides Change reaction by DNA3MNP@X surfaces are grafted onto, prepare magnetic screening agent MNP@X-DNA3。
Then, after saltant type ctDNA with after the magnetic Nano probe in addition detection architecture and reporter probe hybridization, carrying out Magnetic Isolation.In the present invention, in the presence of target ctDNA, magnetic Nano probe and reporter probe are coupled by ctDNA, magnetic point There is the nano particle of report element from magnetic nanoparticle surface is obtained, when target ctDNA is not present, Magneto separate obtains magnetic Nano grain surface does not have the nano particle of report element.With reference to Lorentz transmission electron microscopy, visually observing directly to sentence It is disconnected to whether there is target ctDNA.
Then, report element is quantified, the concentration of report element can use ICP absolute quantitations.In the present invention, due to covering DNA is covered to be removed in Magnetic Isolation with wild type ctDNA with magnetic screening agent after wild type gene complementary pairing, can be complete The interference that masking wild type gene detects to mutator, report element concentration and ctDNA concentration meet linear relationship, pass through Au constituent contents in ICP-MS detection architectures, according to Au contents in standard liquid and the linear relationship of ctDNA contents, Neng Goushi Now to ctDNA accurate quantitative analysis.
Advantages of the present invention：
Double nano probe (the MNP@X-DNA of the present invention1With Rep-DNA2) prepare simple, controllable, easy repetition；Utilize excellent magnetic The magnetic nanoparticle of response characteristic carries out quick, high magnetic separation and enrichment to ctDNA, avoid cumbersome PCR processes and Complicated gene sequencing, substantially reduces detection cycle；
The DNA primer and target ctDNA selectivity complementary pairings of double nano particle surface modification, it is ensured that the height of detection is special The opposite sex；
It need not be expected to be greatly reduced using PCR amplification ctDNA, false positive probability；
In serum sample analysis, magnetic screening agent is added, wild type free nucleic acid is removed by Magneto separate, background core can be avoided Interference of the acid to ctDNA detections, is expected to realize the high-sensitivity detection under zero background, is expected to overcome existing ctDNA detections skill The concentration that art faces is low, two serious hang-ups of ambient interferences, develops the new plan of ctDNA nanometer detections based on nanometer biotechnology Slightly；
The unique physical and chemical properties of nano material are applied to for ctDNA detections by MNP@X with Rep nanometers Fourier Series expansion technique Liquid Biopsy, the early diagnosis being expected to as cancer provide a quick, reliable new way, promote liquid biopsy swollen Application value in knurl diagnosis and treatment field is great.CtDNA novel nanos detection method proposed by the present invention is for clinical or clinical turn Change the liquid Biopsy based on ctDNA detections in medical science to anticipate for the application during tumour early detection is assessed with tumor load Justice is great.
Embodiment is enumerated further below to describe the present invention in detail.It will similarly be understood that following examples are served only for this Invention is further described, it is impossible to is interpreted as limiting the scope of the invention, those skilled in the art is according to this hair Some nonessential modifications and adaptations that bright the above is made belong to protection scope of the present invention.Following examples are specific Technological parameter etc. is also only an example in OK range, i.e. those skilled in the art can be done properly by this paper explanation In the range of select, and do not really want to be defined in the concrete numerical value of hereafter example.
Embodiment 1
One .AFe@SiO2-DNA1, Au-DNA2The preparation of probe.
1. prepare amorphous iron (AFe) nano particle：0.36mmol (175mg) ferric citrate, 9mmol PVP are weighed respectively (Average Mw=55000) and F127 (1.0g) are dissolved in 40mL anaerobic waters, 70 DEG C are warming up under argon atmosphere, machinery stirs (rotating speed 600rpm) 60min is mixed, removes the oxygen in reaction system.7.5mmol(283g)NaBH4It is dissolved in 6mL water, delays Slowly (0.1mL/min), which is added dropwise in reaction system, produces a large amount of bubbles.After 4 hours, add 5mL ethanol and go bubble removing, magnetic Separation, the amorphous iron nano-particle of collection carry out supersound washing three times with ethanol, scattered to preserve in ethanol.
Fig. 1 is the TEM collection of illustrative plates that amorphous iron (AFe) nano particle obtained by the present embodiment is dispersed in water, can by Fig. 1 See：Obtained nano particle is spherical, and good dispersion, particle size range is in 30-50nm.
Fig. 2 is the XRD spectrum of amorphous iron (AFe) nano particle obtained by the present embodiment, from Figure 2 it can be seen that obtained Nano particle is amorphous state.
Fig. 3 is power spectrum (EDS) figure of the AFe nano particles obtained by the embodiment of the present invention 1, as seen from Figure 3, obtained Nano particle main component is fe.
2. prepare SiO2Amorphous iron nano-particle (the AFe SiO of cladding2)：50mg AFe are added in ethanol (14mL), water In the mixed solution of (6mL) and ammoniacal liquor (0.2mL).After ultrasonic disperse 30min, the positive silicic acid second of 0.2mL (0.1mL/min) is added dropwise to Ester (TEOS), after room temperature mechanical stirs 8h, Magnetic Isolation, solid product is washed three times with ethanol successively, is washed three times, freeze-drying It is stand-by afterwards.
Fig. 4 is the AFe@SiO obtained by the present embodiment2The TEM collection of illustrative plates being dispersed in water, as seen from Figure 3 SiO2Shell success Parcel, shell thickness about 10nm, core shell structure is high-visible, and nano particle good dispersion, particle size range is in 50-80nm.
Fig. 5 is the AFe@SiO obtained by the embodiment of the present invention 12After nano particle is dispersed in water, under externally-applied magnetic field Magnetic Isolation design sketch, 2.5 minutes Magneto separate efficiency is up to 100% Magneto separate after 78%, 15 minute as seen from Figure 5, it was demonstrated that AFe@ SiO2Nano particle has excellent magnetic responsiveness energy.
3.AFe@SiO2Surface modification DNA1:First by the SiO of nano particle2Shell amination.By 50mgAFe@SiO2Point It is dispersed in 100mL ethanol, 80 DEG C is heated under mechanical agitation, adds 100 μ L APTES.After return stirring 4 hours, centrifugation, Gu Body product is washed with water three times.DNA1(35 μm of ol), EDC (0.1mmol) and NHS (0.2mmol) are added to pH=7.4 PBS In the aqueous solution.After being stirred at room temperature 15 minutes, DNA1On carboxyl all activate, add 4mL AFe@SiO2-NH2(20mg) PBS solution, stir 12h after, Magnetic Isolation obtains solid AFe@SiO2-DNA1It is washed with water three times, is finally dispersed in pH=7.4 Water in it is stand-by.
4.Au-DNA2The preparation of probe：Au nano particles (~5nm) and DNA2(mol ratio AuNPs/DNA by a certain percentage2 =1:50) mix, add 20 μ L citrate buffer agents, gently blown and beaten with liquid-transfering gun so that Au nano particles and DNA2Mixing Uniformly.Deionized water is added, adjusts Au-DNA2Ultimate density be 10mM.Centrifuge, solid product is washed with water 3 times, obtained To product Au-DNA2Probe is dispersed in stand-by in high purity water.
Fig. 6 is the TEM collection of illustrative plates that the Au particles obtained by the embodiment of the present invention 1 are dispersed in water, as seen from Figure 6：It is obtained Nano particle to be spherical, good dispersion, particle size range is in 5-10nm.
Fig. 7 is the Au particles and Au-DNA obtained by the embodiment of the present invention 12Ultraviolet-visible absorption spectroscopy figure, modifying DNA2 The change of ultraviolet-visible absorption spectroscopy proves DNA afterwards2Successfully modify in Au particle surfaces.
2nd, the test experience of KRAS genes is mutated in standard liquid：
1. draw standard curve：The PBS for preparing a series of mutation Kras genes of concentration gradients (0.1pg/mL-10ng/mL) is molten Liquid, respectively take 1mL to be added in 1.5mL sterile centrifugation tube, be separately added into 5 μ L AFe@SiO2-DNA1(0.2mg/mL) and 5 μ L Au-DNA2The PBS solution of (0.2mg/mL), it is placed in shaking table at 37 DEG C and reacts 30 minutes.After carry out Magnetic Isolation, isolate Magnetic nanoparticle is washed with deionized water three times, is finally dispersed in again in 2mL high purity waters.With Lorentz transmission electron microscope observing, If there is gold nano grain on amorphous iron nano-particle surface, illustrate target ctDNA be present.Quantitative analysis further is carried out to ctDNA Research.Test the AFe@SiO that every group of Magneto separate goes out respectively with ICP-MS2Au contents in the-ctDNA-Au aqueous solution.Formulate Au The standard curve that constituent content changes with KRAS mrna concentrations.
Fig. 8 is with AFe@SiO2-DNA1With Au-DNA2Probe system detects a series of concentration gradients (0.3ng/mL (upper rows Left figure), 0.12ng/mL (above arranges right figure), 0.06ng/mL (lower row left figure), 0ng/mL (lower row's right figure)) ctDNA standards it is molten During liquid, the TEM photos of Magnetic Isolation product.
Fig. 9 is with AFe@SiO2-DNA1With Au-DNA2Probe system detects a series of ctDNA standard liquids of concentration gradients When, the linear relationship of Au concentration and ctDNA concentration.In the test experience of Fig. 9 description standards solution mutation KRAS genes, report member Plain Au concentration and the concentration of KRAS genes meet linear relationship：Y=209.07x, (R2=0.997, Y:Au concentration, x:KRAS bases The concentration of cause).
2. the extraction and molecular weight detection that are mutated KRAS are tested：The AFe@SiO that Magnetic Isolation is obtained2- ctDNA-Au couplings Zoarium system, which is heated to 95 DEG C, makes double-stranded DNA unwind for single stranded DNA, Fourier Series expansion technique dissociation, Magneto separate removing AFe@SiO2-DNA1, add Enter 1mL NaCl (50mM) solution, treat Au-DNA2Sedimentation of reuniting centrifuges afterwards to be removed.Only surplus detection target in last solution It is mutated KRAS genes.Using ESI-TOF-MS, its molecular weight is determined.Located in advance using the supporting system sample of nucleic acid of flight mass spectrometer Kit fragmented nucleic acids point target is managed, micro liquid is layouted on the target of flight mass spectrometer, runs Mass Spectrometer Method, collects data Analysis.
Figure 10 is AFe@SiO2-DNA1With Au-DNA2Probe system isolates the ESI-TOF- of mutation KRAS genes (135T) MS schemes.The theoretical molecular of 135T genetic fragments is 5930.9, and the molecular weight detected is 5930.0, test value and theoretical value one Cause.
3.AFe@SiO2-DNA1With Au-DNA2The selectivity of probe in detecting system assesses experiment：Using above-mentioned same inspection Survey method, influence of the presence of other 4 kinds of genes (NRAS, BRAF, EGFR, PIK3CA) to testing result is tested, this is assessed and receives The selectivity of rice detection method.Six kinds of typical tumor cell lines such as U87 are chosen, are extracted with zooblast DNA extraction kit Various tumor cell line genomic DNAs, and DNA is purified.Six kinds of differences are detected using above-mentioned same detection method to swell The mutation KRAS genes of oncocyte system genomic DNA.
Figure 11 is to design the AFe@SiO prepared for KRAS genes2-DNA1With Au-DNA2Probe system is for other CtDNA (NRAS, BRAF, EGFR, PIK3CA) recall rate evaluation.The isogenic presence pair of NRAS, BRAF, EGFR, PIK3CA Interference is not present in the detection of KRAS genes, illustrates the AFe@SiO prepared for the design of KRAS genes2-DNA1With Au-DNA2Probe System has good selectivity to KRAS genes.
Figure 12 is to design the AFe@SiO prepared for KRAS genes2-DNA1With Au-DNA2Probe system is for various cancers The detection performance evaluation of KRAS genes is mutated in cell line.AFe@SiO2-DNA1With Au-DNA2Probe in detecting system is for existing The A2780 of KRAS genes, LOVO are mutated, A549 cell lines detected positive findings.
4.AFe@SiO2-DNA1With Au-DNA2Probe in detecting system is mutated KRAS genetic fragments detection property for different length Experiment can be assessed：Synthesize a series of mutation KRAS genes 134A of different lengths Single-stranded DNA fragments (19bp, 30bp, 60bp, 70bp,80bp,100bp,150bp).With the AFe@SiO designed for 134A2-DNA1With Au-DNA2Probe in detecting system is assessed The detection performance of the KRAS fragments of different length, while compared with the ddPCR means of testing of classics.
Testing result in Figure 13 shows, double nano probe couple detection architecture for bases longs 19-150bp base Cause can 100% detection.And ddPCR method of testings only have 30% recall rate for 60bp genetic fragment, less than 30bp's Short-movie section ddPCR methods can hardly measure positive findings.This explanation double nano probe coupling detection architecture can avoid short-movie Section ctDNA missing inspection.
3rd, in serum sample Kras genes test experience
1st, magnetic screening agent is prepared:Masking DNA is prepared in the end modified carboxyl of the complementary series of wild type Kras genes3, pass through AFe@SiO2Surface amino groups and DNA3Carboxyl occur EDC and NHS mediation amidation process, by DNA3It is grafted onto AFe@SiO2 Surface, prepare magnetic screening agent AFe@SiO2-DNA3。
2nd, patients with lung adenocarcinoma serum sample Kras genetic tests:Randomly select different (I, II, III, IV) adenocarcinomas of lung by stages The serum sample of patient is studied for 23 totally.The new blood of patient is mounted in anticoagulant sterile centrifugation tube, centrifugation, taken Upper serum.The all DNA in serum sample is extracted with dissociative DNA extracts kit, is dispersed in anaerobic water, heating makes double Chain DNA unwinds as single stranded DNA.Magnetic screening agent corresponding to adding wild type Kras genes hybridizes therewith, and Magnetic Isolation removes wild Raw type Kras genes.AFe@SiO are added into surplus solution2-DNA1And Au-DNA2, after Kras genes to be mutated hybridize therewith Carry out Magnetic Isolation, obtained AFe@SiO2- ctDNA-Au Fourier Series expansion techniques are dispersed in water again, with ICP-MS Accurate Determinings Au The content of element, the standard curve changed according to Au constituent contents with KRAS mrna concentrations quantitatively calculate the concentration of KRAS genes. CtDNA concentration and the relation of cancer staging are obtained according to testing result.Pay special attention to asymptomatic I phases Sera of Lung Cancer Kras genes Recall rate.
Table 1 is AFe@SiO2-DNA1With Au-DNA2Inspection of the probe system for mutation KRAS genes in 23 patient bloods Survey result.Table 1 shows, double nano probe Fourier Series expansion technique is 72.7%, II for the positive rate of I phase blood samples, III, the IV phases The positive rate of blood sample is 100%.Compared with ddPCR testing results, blood sample four, ddPCR does not detect positive findings, And double nano probe Fourier Series expansion technique detected positive findings.Point of KRAS fragments is further isolated with ESI-TOF-MS detections Son amount, Figure 14 demonstrate mutation KRAS genes 134A presence, eliminate the possibility of false positive.
Table 1：AFe@SiO2-DNA1With Au-DNA2Detection of the probe system for mutation KRAS genes in 23 patient bloods Performance evaluation
aTumor tissues DNA mutation state is detected by digital pcr and nano particle coupling process；
bThe concentration of report element is detected by ICP-MS；
cPass through the concentration and linear relationship (y=209.07x, the x of mutation KRAS mrna concentrations of report element:It is mutated KRAS genes Concentration, y:The concentration of report element), the dense of mutation KRAS genes is calculated by the concentration of the ICP-MS report elements detected Degree.
In summary, double nano probe coupling detection architecture avoids cumbersome biological amplification procedure, the magnetic of rational design Property screening agent eliminates the interference of wild type free nucleic acid, realizes quick, the special, high-sensitivity detection under zero background.
3rd, the Kras gene levels real-time tracking monitoring in tumor-bearing mice serum：Human lung adenocarcinoma cell (A549) is taken to plant in naked Mouse is subcutaneous (the naked female mices of Balb/c, average weight 20g), starts to test when tumour longest diameter grows to about 5-6mm, Mei Geyi Fix time and take nude mice eyes-affinity venous blood to detect.The venous blood of tumor-bearing mice is placed in anticoagulant centrifuge tube, centrifuges, takes blood Clearly.The all DNA in serum sample is extracted with dissociative DNA extracts kit, heating makes double-stranded DNA unwind for single stranded DNA.Add Magnetic screening agent corresponding to entering wild type Kras genes hybridizes therewith, and Magnetic Isolation removes wild type Kras genes.It is molten to residue AFe@SiO are added in liquid2-DNA1And Au-DNA2, progress Magnetic Isolation, is obtained after Kras genes to be mutated hybridize therewith AFe@SiO2- ctDNA-Au Fourier Series expansion techniques are dispersed in water again.It is first according to Au with the content of ICP-MS Accurate Determining Au elements The standard curve that cellulose content changes with KRAS mrna concentrations quantitatively calculates the concentration of KRAS genes.Tumour is drawn according to testing result The changing rule that the Kras mrna concentrations being discharged into blood circulation are presented with tumor development.Tumor-bearing mice intratumor injection cis-platinum Solution, the change of Kras mrna concentrations in certain time point blood after chemotherapy is monitored in real time.Ablation of tumors hand is carried out to mouse Art, the change of Kras mrna concentrations in certain time point blood after performing the operation is monitored in real time.
Figure 15 is AFe-DNA1With Au-DNA2Probe system is evaluated for tumor-bearing mice ctDNA detection performance.(a is not Through handling tumor-bearing mice gross tumor volume and the change curve of ctDNA concentration；B is tumor-bearing mice gross tumor volume and ctDNA after chemotherapy The change curve of concentration, c are gross tumor volume and the change curve of ctDNA concentration after tumor-bearing mice Operation in early stage, and d is lotus knurl The change curve of gross tumor volume and ctDNA concentration after the treatment of mouse late surgery) Figure 15 shows, ctDNA concentration and gross tumor volume Growth trend it is consistent.After injecting cisplatin medicine chemotherapy, the presentation downward trend of ctDNA concentration, but ctDNA concentration after seven days Bottom out again.Test the 7th day, implement tumor resection, postoperative ctDNA concentration is remarkably decreased, still without increase after 49 days. Test the 21st day and implement tumor resection, postoperative ctDNA concentration is also remarkably decreased, but ctDNA concentration starts again after one week Rise.This explanation double nano probe Fourier Series expansion technique can be used for the real-time monitoring to tumor load, be provided for follow-up clinical research Experimental basis.
In summary, it is seen then that the invention provides a kind of convenient, economic, overdelicate ctDNA nanometer detection new methods, The high sensitivity specific recognition of ctDNA in cancer plasma's sample can be realized.This is based on nanometer biotechnology to development CtDNA nanometer detection new strategies, promote based on ctDNA detection liquid Biopsy in early diagnosis of cancer application tool There is important meaning.

Claims (10)

  1. A kind of 1. nano particle coupling probe material system, it is characterised in that the nano particle coupling probe material system by First probe and the second probe composition, the expression formula of first probe is MNP X-DNA1, wherein MNP is magnetic nanoparticle Kernel, X are the hydrophily shell for being coated on the magnetic nanoparticle kernel, DNA1To modify on the hydrophily shell Can be with the first complementary nucleic acid chain of the Part I complementation of target circulation Tumour DNA, the expression formula of second probe Rep-DNA2, wherein Rep is that can use the report element kernel for the report element formation that ICP-MS is detected, DNA2To modify described The second complementary nucleic acid chain that can be complementary with the Part II of target circulation Tumour DNA on report element kernel.
  2. 2. nano particle coupling probe material system according to claim 1, it is characterised in that the MNP is by paramagnetism Nano particle is formed, and preferably amorphous state fe is formed.
  3. 3. nano particle coupling probe material system according to claim 1 or 2, it is characterised in that the Rep is by that can use The heavy metal element that ICP-MS is detected is formed, and is preferably formed by least one of Au, Ag, Pt.
  4. 4. nano particle coupling probe material system according to any one of claim 1 to 3, it is characterised in that described First probe is amido modified by will be carried out after magnetic nanoparticle Surface coating hydrophily shell, and and DNA1Generation condensation is anti- It should obtain.
  5. 5. nano particle coupling probe material system according to any one of claim 1 to 4, it is characterised in that the X For SiO2And/or hydrophilic organics hydrophilicity.
  6. 6. nano particle coupling probe material system according to any one of claim 1 to 5, it is characterised in that described Second probe is by by DNA2Disperse to obtain in aqueous with Rep nanoparticles stables.
  7. 7. a kind of application method of the nano particle coupling probe material system any one of usage right requirement 1 to 6, its It is characterised by, including：The DNA in test serum is extracted with dissociative DNA extracts kit, is dispersed in anaerobic water, heating makes Double-stranded DNA unwinds as single stranded DNA；Add first probe and the second probe；Magnetic nanoparticle is obtained by Magnetic Isolation Surface has the nano particle of report element and/or magnetic nanoparticle surface not to have the nano particle of report element；To report member Element is quantified, and according to target circulation Tumour DNA and the linear relationship of report element concentration, target circulation Tumour DNA is entered Row is quantitative.
  8. 8. application method according to claim 7, it is characterised in that add magnetic before the first probe and the second probe is added Property screening agent remove wild type Circulating tumor DNA.
  9. 9. application method according to claim 8, it is characterised in that the expression formula of the magnetic screening agent is MNP@X- DNA3, wherein MNP is magnetic nanoparticle kernel, and X is the hydrophily shell for being coated on the magnetic nanoparticle kernel, DNA3 To modify the nucleic acid chains that can be complementary with wild type Circulating tumor DNA on the hydrophily shell.
  10. 10. application method according to claim 8 or claim 9, it is characterised in that the magnetic screening agent is by by magnetic Nano Particle surface cladding hydrophily shell after carry out it is amido modified, and with shelter DNA3Generation condensation reaction obtains.
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