CN101769930A - Applying method for fluoric compound in identifying circulatory tumor cells - Google Patents

Abstract

The invention relates to an applying method for a fluoric compound in identifying circulatory tumor cells. The method comprises the following steps of: trapping survival tumor cells in a peripheral blood circulating system, wherein the trapping process comprises a positive enriching method of a monoclonal antibody identifying a micro amount of tumor cells from a large amount of red cells, a negative enriching method of degrading red cells, and a physical separating method of filtration, centrifugation and the like based on the density and size of the tumor cells; forcing the trapped survival tumor cells to remedy a synthesis path to get the fluoric compound under the condition of cell culture; and carrying out electric spraying negative ion tandem mass identification on the fluoric monophosphate product generated by the tumor cell metabolic process and accumulated in the cell media. The invention firstly puts forward a golden identifying method for separating the enriched survival circulatory tumor cell cycles, uses a tandem mass spectrometry to determine the characteristic metabolic compound in the tumor cell proliferative process, and accurately reflects the malignant degree of the tumor cell proliferative cycle and the essential distinction of the periphery blood normal cells.

Description

The application process of fluoric compound in identifying circulating tumor cell

Technical field the present invention relates to the application process of a kind of fluoric compound in identifying circulating tumor cell, particularly " 3 '-deoxidation-3 '-application process of fluoro-thymidine in identifying circulating tumor cell ".

The essence of this method is based on the biological chemistry of the thymidine kinase-1 (TK1) of tumor cell proliferation cycle-S stage-cell medium middle and high concentration, with 3 '-deoxidation-3 '-fluoro-thymidine-5 '-single phosphoric acid derivatives (FLT-5 '-MP) be example, the mass spectrum that the existence circulating tumor cell of catching after the enrichment carries out is identified.

Background technology primary tumo(u)r cell can enter the peripheral blood circulation system after nature, diagnosis and treatment come off.Partly can bear and escape the circulating tumor cell that immune system is killed, invade the profit epithelial tissue and form final fatal metastasis.The catching of peripheral blood circulating tumor cell, identify, count early stage generaI investigation that carry out the clinical stages of tumour, the individual prioritization scheme of operation chemotherapy radiotherapy has unique irreplaceable meaning to sub-health population.But because the extremely low concentration of tumour cell in the peripheral blood circulation system, promptly per 1,000,000,000 normal blood cells only contain and lowly reach several tumour cells, produce thus to the variation tumour cell catch, false male-female problem in the evaluation, counting process.The influence catching, identify and count is a global problem of still not resolving at present to the variation tumour cell to eliminate false male-female problem.

The definition of circulating tumor cell CTCs: 1, TCD is less than 1.077 grams per milliliters, and blood normal cell and granulocytic cell density are greater than 1.077 grams per milliliters.2, cell relative size, tumour cell directly through being generally 6 to 30 microns, maximum leukocytic diameter is 8 to 11 microns in the blood.3, the tumour cell form is irregular circle or oval attitude.4, and nucleus DAPI positive reaction (DAPI:4 ', 6-diamidino-2-phenylidole).5, cytokeratin 8,18,19 grades are positive, and express in pairs in epithelial tissue, and the positive expression of cytokeratin is proved to be the responsive and more special mark of epithelium source property tumour cell at protein level.6, because CTCs does not express leukocyte common antigen (LCA) CD45, CD45 detects negative.

The method that is used for diagnosing tumor at present can be classified as three major types: pathology, iconography and serology.One of Sensitive Detection means that the CellSearch detector of peripheral blood CTCs has been approved by FDA at present.But it is to catching the CTCs identification method after the enrichment, can reduce monoclonal antibody to the identification of tumour expressing protein or based on the mensuration of nucleic acid.CTCs mass spectrum identification method is based on the technical support background of following three different field.

1. catch enrichment and identify the circulating tumor cell technology

CTCs refers to spontaneous or enters peripheral blood round-robin tumour cell because of operation of diagnosis and treatment.Since 1869 found CTCs, its biological behavior was gradually paid attention to for people.Tumour cell has its special growth cycle.All existence cells or be in rest and reorganization G0 attitude perhaps are in the active stage.The propagation of tumour cell is the G1 attitude that is entered the phase one by the G0 attitude, and in the S attitude that this preparation enters synthesis phase, so-called S attitude phalangeal cell nuclear DNA's is synthetic.Enter the G2 stage of lag period subsequently, enter the mitotic M stage of finishing cell then.Being separated into two identical chromatid, whole cells at this DNA is divided into two.In the CDC, the synthetic of DNA not only carries out in cell medium, also can synthesize in mitochondria simultaneously.Two kinds of different isomerases: thymidine kinase TK1 and thymidine kinase-2 (TK2), respectively in cell medium and mitochondria to thymidine (below abbreviation: 5 ' hydroxyl thymidine) carries out phosphorylation reaction.The concentration of TK1 can the G1-S tour increase 10-20 doubly more than, and continues the maintenance high concentration in G2 and M stage.Get back to G0 and G1 after the stage, TK1 then degrades rapidly and gets back to normal level.In contrast, the TK2 concentration in the mitochondria and active not with the influence of cell generation cycle.Because TK1 is in the kinase expression mode of the S in growth of tumour cell cycle phase characteristic, TK1 becomes the target spot of oncotherapy and detects the reliable method of identifying, is acknowledged as the golden standard that characterizes tumour cell at present.The clinical practice of CTCs obtained substantive progress in nearly 5 years, mainly depended on the technical development in following field.

(1-1) immunocytochemistry

Continuous propagation along with tumour cell, 85% epithelium attribute tumour cell can be by expressing a kind of glycoprotein (EpCAM) of epidermal cell adhesion molecule, discern mutually to increase it, diffusivity also makes it to break away from the tumour parent, and then destroy peripheral host's connective tissue, and enter vascular system formation metastasis.In contrast, the cell component of all blood circulations system does not have the biological basis of expressing this epidermal cell adhesion molecule.The mat woven of fine bamboo strips one selectivity that identification is provided, has caught all blood circulation CTCs of system for corresponding antibody thus.

(1-2) catch beneficiation technologies

Data in literature shows that the speed that tumour breaks away from cell is roughly: 1 * 10 ^-5To 3 * 10 ^-6CTCs/ days/gram tumor tissues.Therefore, measure the CTCs counting of all blood circulations system, can be used for the early diagnosis of tumour, the method for tumor operation, chemotherapy assessment.But the concentration of CTCs is extremely low, is approximately whenever 1/1000000000th, or every milliliter contain 5-1281CTCs.Therefore, effectively catch, enrichment CTCs is key problem in technology.CellSearch that have two of market outlook to catch, beneficiation technologies is the immunomagnetic beads fluid and the chip CTC-Chip that forms by the micro-column of 78,000 band antibody.The CellSearch technology of the unique approval of U.S. FDA at present, and catch, the chip technology CTC-Chip of hospital general, masschusetts, u.s.a development that enrichment CTCs potentiality are good is based on the immune antiboidy of tumor cell feature EpCAM.In addition, polycarbonate membrane filtration and separation techniques, and to adopt Ficoll-Paque be the centrifugal separation technology of medium, all CTCs of separable different purity.

(1-3) catch the evaluation of cell

The identification method of the tumour cell of CellSearch and CTC-Chip is divided folding based on cytomorphology and biomarker.The epithelial cell keratin positive, the immunocytochemistry detection is still transcribed polymerase chain reaction,PCR and is all existed certain limitation, can not get rid of false positive.The DAPI positive, promptly blue look fluorescence method in the AT position of identification of dna and CD45 colour developing counting are negative, i.e. CK+/DAPI+/CD45-.This combination identification method is to identify second selectivity of CTCs cellular level at present, and is the FDA approval.And be better than the CTCs that Adams reports and directly count method from the micro-fluidic back diagometer of catching.

(1-4) false positive problem

The false positive problem that cell is caught in evaluation is attributable to following factor: the tumour cell of immunomagnetic beads fluid capture is dead cell substantially, catches at present to render a service to reach 10 ⁶The existence cell purity of the CTC-Chip technology of enrichment is 50%.Genotype-phenotype in the variation is undoubtedly to the immunocytochemistry identification method, and based on the PCR of nucleic acid, the RT-PCR identification method produces false positive reaction.The preparation of relevant monoclonal antibody and fluorescence bonding chemistry, purify, be applied in and have the school mistake between the laboratory.The false positive problem is the matter of utmost importance that low concentration CTCs identifies.

(1-5) the catching of peripheral blood circulating tumor cell, enrichment, the assessment of authenticate technology present situation

The condition precedent of identifying the peripheral blood circulating tumor cell is to catch the CTCs of back existence.At this, whether the application's patent will define the technique effect in current this field with the survival of catching the back tumour cell.The first kind is promptly caught the tumour cell technology of surviving in the back.Its representative technology is the development of hospital general, masschusetts, u.s.a, the CTC-Chip of CELLective Dx company development, the whole blood that contains CTCs is under the laminar flow condition of precision control, flow through in the chip 78, the micro-column of 000 band antibody, make the antibody uniform contact of CTCs and surface chemistry bonding, catch after the immunity identification.The existence tumour cell precision that CTC-Chip catches is 99%, purity 50%.The meaning of catching the existence tumour cell has been to provide the basis of further metabolism group, protein group, genome evaluation.As mensuration to the epidermal growth protein receptor gene mutation of lung cancer.The evaluation of CTC-Chip belongs to the fluorescent combine measured of CD45-/DAPI+/CK+, and promptly the positive normal blood leucocyte of CD45-, DAPI are used for dna content mensuration, the CK+ epidermal cell keratoprotein positive.The survival rate that the two electrophoresis of CTCs are caught cell is 90%, and the evaluation of existence cell is based on the trace fluorometric assay.In addition, also have the cell density gradient centrifugation to separate and the high folate receptor fluorometry of explaining of ovarian neoplasm.Catch rich consor and deposit the CTCs technology and all also locate the laboratory developing stage at present for above-mentioned three.Second class, the tumour cell technology of non-survival after promptly catching.At present unique immunomagnetic beads fluid that is used for clinical CellSearch technical foundation for identification EpCAM.After the processing of a series of semi-automation, separation, cleaning, be captured as and solidify dead CTCs.Its authentication method is also identical with CTC-Chip: the fluorescence combine measured of CD45-/DAPI+/CK+.Be the survival rate of acquisition mode and CTCs with the key distinction of CTC-Chip.The immunomagnetic beads fluid also be used to the epidermal cell keratoprotein identification, catch, join with Ariol scanning authenticate technology, its R﹠D firm Genetix obtains to be better than the result of CellSearch.It is to be noted the RT-PCR detection of the sensitivity of joining by the capture technique based on EpCAM of Adnagen exploitation.The main method of identifying at present CTCs all is confined to the genotype-phenotype in the CTCs variation, and also existing just catching method needs in addition multiple intersection to measure, to fall the false-positive reason of end CTCs.Be called the rich poly-technology of forward with the rich poly-technology of catching of Identification Lists chrotoplast keratoprotein, and erythrocytic selectivity haemolysis technology is called the rich poly-technology of negative sense.

2. be used for positron emission tomography scanning [ ¹⁸Fluorine] for tracer agent (PET)

PET tracer agent technology makes that biochemical metabolism can directly be observed in the cell, but can't distinguish the relative distribution and the relative quantification of tracer agent and specific metabolic product thereof: be used for the 2 '-deoxidation-2 ' of energetic supersession tracer agent-[ ¹⁸Fluorine]-D glucose ([ ¹⁸Fluorine]-FDG), the 3 '-deoxidation-3 ' of DNA metabolism-[ ¹⁸Fluorine] for thymidine ([ ¹⁸Fluorine]-FLT), suppress to participate in damage RNA and the synthetic 5-[of DNA ¹⁸Fluorine] uracil (5-FU).Because the common feature of these three kinds of tracer agents is phosphatase reactions of the metabolism intermediate after the tumour cell absorption, electronegative phosphate radical is then measured for tandem mass spectrometry desirable molecular characterization is provided.The application's patent is only discussed and is entered the label that suppresses further metabolism behind the cell: [ ¹⁸Fluorine]-FDG, [ ¹⁸Fluorine]-FLT and [ ¹⁸Fluorine]-FU.And be example with FLT, carry out the demonstration example of tumour cell mass spectrum identification method.The radioactive isotope of using as PET ¹⁸Fluorine will substitute with the used stable isotope fluorine of mass spectrum.

(2-1) 2 '-deoxidation-2 '-[ ¹⁸Fluorine]-D glucose: the energetic supersession tracer agent

The poor efficiency of tumour cell is given birth to " energy " mechanism and is caused tumour cell to the glucose high requirements.FDG and common glucose chemistry similar performance can produce markd metabolin in human body, and retention time are longer in human body, are convenient to measure.Normal cerebral tissue, cardiac muscular tissue, kidney and bladder body are because high glycometabolic needs thereby more to the FDG picked-up.Some other tissue such as liver, muscle and intestines wall are because its glycometabolism level is low, then less to the intake of FDG, and the FDG activity level that demonstrates is also lower.As a kind of glucalogue, FDG will be absorbed by glucose high usage cell, as brain, kidney and cancer cell.In this type of cell, the phosphorylation process will stop glucose to discharge among cell with original complete form.2 ' oxygen among the glucose is that the institute of glycolysis subsequently is essential.Thereby FDG and 2-deoxy-D-glucose are similar, can't continue metabolism in cell.Therefore, before radioactivity decay, glycolysis will can not take place in the FDG-6-phosphoric acid that is generated by the hexose zymogenesis.[ ¹⁸Fluorine]-distribution situation of FDG well the antimer inner cell to the picked-up of glucose and the overall information of phosphorylation.But, FDG takes place after the radioactivity decay, wherein ¹⁸Fluorine will change into ¹⁸0.And, from environment, obtaining a H ⁺Afterwards, the decay product of FDG has just become G-6-P, and the mark on its 2 ' then becomes harmless on-radiation heavy oxygen.Like this, this decay product just can carry out metabolism according to the mode of common glucose usually.The own approval of FDA [ ¹⁸Fluorine]-FDG is used for PET and holds doubtful tumour patient to assess other technology.[ ¹⁸Fluorine] though-FDG static state accumulates in the tumour cell, based on " energetic supersession " mechanism [ ¹⁸Fluorine]-FDG is used for will causing when low-purity CTCs identifies low pick rate, false positive.The metabolism phosphoric acid derivatives of stable isotope FDG is identified parameter with the mass spectrum that the ratio of corresponding eubolism phosphoric acid derivatives can be made tumour cell.

(2-2) 3 '-deoxidation-3 '-[ ¹⁸Fluorine] for thymidine: the DNA metabolic tracer

[ ¹⁸Fluorine]-FLT carries albumen to enter cell via the cell membrane nucleosides, and then be that TK1 in the cell medium is to 5 ' hydroxyl phosphorylation.With [ ¹⁸Fluorine]-FDG is similar, 3 '-deoxidation-3 '-[ ¹⁸Fluorine] generation-thymidine-5 '-phosphoric acid derivatives only accumulates in the tumour cell, can not participate in the synthetic of DNA.It is to be noted that DNA synthetic relates to two differences by way of picked-up extracellular existing thymidine derivant, promptly remedy synthetic by way of; Or directly absorb intracellular uridine phosphoric acid derivatives, i.e. de novo synthesis.Via extracellular picked-up [ ¹⁸Fluorine]-the FLT level directly reflected the biochemical activity of TK1.Document shows that in the S stage of cell growth cycle, the TK1 biochemical activity of tumour cell is generally Normocellular 10-20 doubly.This just provides the basis of identifying all blood circulation tumour cell biochemical characteristics for the application's patent.

(2-3) 5-[ ¹⁸Fluorine] uracil: DNA and RNA metabolic tracer

Since nineteen fifty-seven, 5-FU is used for the carcinoma of the rectum, breast cancer treatment.Its effect and metabolism mainly contain three approach: one, 5-FU metabolism intermediate 5-fluoro-2 '-BrdU-5 '-single phosphoric acid suppress thymidylate synthase; Two, 5-fluoro uridine-5 '-triphosphoric acid participates in damaging the synthetic of RNA; Three, 5-fluoro-2 '-BrdU-5 '-triphosphoric acid participates in suppressing and damaging the synthetic of DNA.The metabolism phosphoric acid derivatives of stable isotope 5 FU 5 fluorouracil can be made the mass spectrum of tumour cell and identify parameter with the ratio of the corresponding eubolism phosphoric acid derivatives of its structure.

3. receive and rise the electron spray anion source and be used for quadrupole rod flight time tandem mass spectrometer

Be used for positron emission tomography scanning [ ¹⁸Fluorine] radiotracer, promptly the above [ ¹⁸Fluorine]-FDG with [ ¹⁸Fluorine]-FLT, its common ground is that the metabolism intermediate no longer participates in energetic supersession and DNA is synthetic.And 5-[ ¹⁸Fluorine] FU then suppresses, damages the synthetic of DNA and RNA.The accumulation of intracellular phosphorylated derivative is for high-resolution, the evaluation of high-sensitive mass spectrum provide advantage.Identifying for mass spectrum thus provides the extremely similarly tumour relevant information of artificial synthetic compound of native compound and its structure, and the phosphate radical negative charge that helps mass spectroscopy.On the other hand,, adopt advanced accurate mass spectrometer that a minute folding thing is carried out ionization, behind the molecular radical structure adjusting electric polarity for the treatment of branch folding thing, finish mass spectrum and identify with the developing of proteomics.The anionic molecule that produces in the metabolic process imports first order quadrupole rod mass filter, by the matter-He Bi that treats branch folding compound, optionally import quadrupole rod collision disassociation chamber, under high-purity charging argon gas collision, divided the folding compound according to its molecular structure generation fragment cracking.The reflection ad hoc structure fragmention with its matter-He Bi that carries in second-stage flight time quantum detection record.The opinion to identifying CTCs, the key problem in technology of quadrupole rod flight time tandem mass spectrometer Q-TOF is the ionizing efficiency of anion source, and the flight time unit carries out fine adjustment in the scope of low-molecular-weight 100-500Da.Be used for PET [ ¹⁸Fluorine] radiotracer, can not exist in the life, suppress the fluorine of metabolism stable isotope [ ¹⁹Fluorine] substitute.The FLT that relates among the application, FDG, 5-FU all refer to inactive stable fluoric compound.

The one-level mass spectrum is made up of quadrupole rod, the main filtration that plays the selection ion, and second order ms is to divide the folding device flight time, is that the main quality of this instrument is divided the folding unit.Mass ion source and different cascade mass spectrometers also comprise: ionogenic ionizing efficiency of A. and interference impurity; B. the mass resolution of cascade mass spectrometer and sensitivity.These ion guns comprise: matrix is helped laser desorption ionisation source MALDI, ionspray ionization source IonSpray, fast ion bombardment ionization source FIB, fast atom bombardment ionization source FAB.And cascade mass spectrometer can be made up of following: cascade four-electrode bar mass spectrometer, Q3 wherein can be the ion trap mass spectrum and divides folding device Iontrap, tandem flight time-time of-flight mass spectrometer TOF-TOF, the fourier transform ion cyclotron resonance mass spectrometer (ICR) FTICR-MS that resolution is high.

Summary of the invention the objective of the invention is to propose a kind ofly not influenced by the tumour mutation, based on cell growth cycle, novel mass spectrum identification method, it is the gold identification method in tumor cell proliferation cycle, the mass spectrum identification method that is called for short circulating tumor cell is not seen the report that adopts highly sensitive, accurate mass spectrometer to identify circulating tumor cell up to now as yet.

Fluoric compound proposed by the invention application process in identifying circulating tumor cell is characterized in that:

(1) catching existence tumour cell in the periphery blood circulation system, acquisition procedure can be divided into monoclonal antibody is discerned the tumour cell of denier from a large amount of red blood cells positive enrichment process, also can be via the erythrocytic negative enrichment method of degraded, and filtration, the physical partition method based on TCD, size such as centrifugal;

(2) under cell culture condition, force the existence tumour cell of being caught to absorb fluoric compound with salvage route;

(3) the fluoro list phosphoric acid product that the tumour cell metabolism is produced and accumulates in cell medium is carried out the evaluation of electron spray negative ion tandem mass spectrometry.

For obtaining accurate qualification result, at first adopt the known sugars compounds under condition, mass spectrometer to be proofreaied and correct at negative electricity, and then obtain the molecule negative ion and the consequent certain negative fragment ion of fluoro list phosphoric acid product, determine tumour cell metabolism fluoro list phosphoric acid product uniquely by this spectrogram.

The ratio of single phosphoric acid metabolic product of fluoro list phosphoric acid metabolic product and eubolism is eliminated the cumulative errors in the whole qualification process, has characterized the malignant proliferation degree of the tumour cell of catching uniquely.

Fluoric compound, as 3 '-deoxidation-3 '-fluoro-thymidine (FLT), it is synthetic that its phosphorylation product hinders DNA, do not participate in the metabolism of tumour cell, thereby accumulate on the interior high concentration that favourable tumour cell is identified, raising Mass Spectrometer Method reliability, reduction or the elimination false positive of forming of cell.

Fluoro phosphoric acid derivatives and the corresponding phosphoric acid derivatives of eubolism in the tumour cell, the two cellular uptake relative ratio after standardization is eliminated the systematic error in the mensuration process, has reflected the expression and the activity of associated kinase in the cell truly.

The tandem mass spectrometry identification method is an example with quadrupole rod-time flight cascade mass spectrometer.To different accuracy of detection, high flux screening, the cascade mass spectrometer of mass resolution combination also comprises: carbon 18 reversed-phase high-performance liquid chromatography coupling quadrupole rod-quadrupole rod cascade mass spectrometers, the flight time-the flight time cascade mass spectrometer, quadrupole rod-fourier transform ion cyclotron resonance mass spectrometer (ICR).Wherein divide the quadrupole rod of folding device to substitute by ion trap as mass spectrum.

Negative ion mass spectrum is measured endocellular phosphorus acidifying metabolic product, except that using the electron spray anion source, also can help the laser desorption ionisation source by matrix and obtain relevant negative ion and carry out mass spectrum and identify.

With D-(+)-gossypose and fragment product negative ion series thereof, divide the aerial flight distance and the relevant phosphoric acid derivatives measuring scope (100-550Da) of folding device to proofread and correct to the TOF mass spectrum.

The week separation of blood systemic circulation tumour cell can after the gained cell absorbs FLT, be carried out tandem mass spectrometry and identify by the commodity of precision control directly through being that 8 microns membrane separator carries out.

The week separation of blood systemic circulation tumour cell also can be obtained by commodity Ficoll-Paque centrifuging, after the gained cell absorbs FLT, carries out tandem mass spectrometry and identifies.

All blood systemic circulation tumour cell chips are accredited as: after micro-fluidic antibody richness was poly-, the coupling cascade mass spectrometer was introduced electrospray device in micro-fluidic outlet again, formed the intact device that guides mensuration metabolism phosphoric acid derivatives with cascade mass spectrometer ion gun electrode.Micro-fluidic antibody and platinum electrode by Identification Lists chrotoplast characteristic biomarker albumen formed, as the cascade mass spectrometer negative electricity from electric spray ion source.

All blood systemic circulation tumour cells can be obtained by the rich poly-technology of simple negative sense: promptly a large amount of red blood cells is through the degraded of commodity solvent selectivity ground, removing.Tumour cell then is attached to the collagen carrier, after absorbing FLT, carries out tandem mass spectrometry and identifies.

After tumour cell was caught in extraction alone or in combination, the relevant solvent acetonitrile-water (50%) of getting phosphoric acid derivatives of quenching was best high throughput assay solvent.The best that carbon 18 reversed-phase high-performance liquid chromatography are separated is quenched, and to get solvent be acetonitrile-water (2%).Other acetonitrile substitutes organic solvent, as methyl alcohol, also can obtain similar results.

Described fluoric compound is not limited to the synthetic specific FLT of DNA, also comprises other fluoric compound.Comprise inhibition, damage the synthetic 5 FU 5 fluorouracil (5-FU) of DNA and RNA and 2 '-fluoro-the 2 '-deoxyglucose (FDG) of cellular energy metabolism.More than three kinds of fluoric compounds single or be used in combination, all can under zymogenesis, generate the phosphorylation metabolic product, follow the biochemical basis and the basic operation flow process of this patent standard, with its phosphorylation metabolic product of mass spectrometric determination, identify tumour cell.

Described micro-fluidic electric spray ion source, as the rich poly-technology of haemolysis negative sense as described in the claim ten two, the circulating tumor cell diagnostic test box that the biochemical reagents of claim ten four described three kinds of fluoro compounds single or combination, combination accessory constitute can be used for the township, clinic at county level carries out local external cancer screening and sets up individuation knubble information.Catch tumour cell by the assessment diagnosis of tandem mass spectrometry center, result of treatment.

The CTCs that the present invention proposes identifies based on following principle: the A. evaluation of foundation is for catching existence cell malignant proliferation degree, and specifically the synthetic directly related kinases TK1 with DNA in the tumor cell proliferation process is that example describes in detail; 3 ' substituting group fluorine has stable molecule and exempts from degraded in the cell in the B.FLT molecular structure, and the anabolism double action of ending further DNA, causes intracellular accumulation, and improves the reliability that mass spectrum is identified thus, reduces the detection false negative of CTCs.Fluorine has minimum atom half warp in addition, is the desirable non-natural matrix of TK1 therefore; The entrained negative charge of the phosphate radical of C.FLT-5 '-MP provides ionizing group and ion characteristic fragment for negative ion mass spectrum detects; D. (the cellular uptake ratio of derivant of thymidine-5 '-MP) is eliminated the systematic error in the mensuration process, has reflected the expression of TK1 in the CTCs breeding truly with natural thymidine-5 '-single phosphoric acid.C. highly sensitive, the high-resolution of negative ion electrospray ionization source quadrupole rod flight time tandem mass spectrometer provide the uniqueness of structural confirmation, and quantitative to the accurate mass spectrum of TK1 activity.Above-mentioned mass spectrum identification method based on the CTCs malignant proliferation obviously is better than the cell biological label combination identification method of FDA approval, the i.e. fluorescence of CD45-/DAPI+/CK+ combination identification method.

(3-2) advantage of Mass Spectrometer Method FLT-5 '-MP derivant structure

The hydroxyl that the displacement of stable isotope fluorine is 3 ' has following three dot structure advantage: A. to prevent that sugar unit from from the degraded of thymidine unit, causing the high stability of FLT in the cell to the measuring method of mass spectrum of this patent development; The selection of B.FLT pair cell medium T K1 is better than Intramitochondrial TK2, and has only TK1 relevant with cellular proliferative stage; C.3 '-fluoro substituents group stoped the synthetic of DNA, therefore accumulated in the cell medium.

(3-3) the negative ion electrospray spraying tandem mass spectrometer of mensuration CTCs

The institute's branch that remains folding molecule at first needs to carry out ionization at ion gun.For the above-mentioned substance of measuring CTCs for standard absorption ratio, i.e. the absorption ratio of FLT-5 '-MP and corresponding thymidine-5 '-MP.This has the phosphate radical of negative charge to phosphoric acid derivatives, need not any additive, volume ratio is in the 50% acetonitrile-water mixed liquor, directly grant 1,600V voltage is with stable syringe pump, in the flow of 20-100nl/min, via directly through be the electron spray syringe needle of 10 μ m under normal temperature and pressure, form stable pinniform electron spray.Electronegative ion is introduced first order quadrupole rod mass spectrum filtrator through the taper ion inlet of an angle of 90 degrees direction down in 35V voltage.This process is special " Z-electron spray ".Impurity such as neutral molecule, positive ion background signal must give improvement.This literary grace is answered isotope ¹²C=12.0000Da has the single isotopic mass calculating that definite element is formed the m/z of negative ion.

(3-4) correction of low-molecular-weight negative ion mass spectrum instrument

Because the FDG and the FLT of phosphorylation all belong to electronegative micromolecule.The measuring accuracy of flight time tandem mass spectrometer changes with the deposition of temperature and mass spectrometer inner member surface compound.Room temperature changes 1 ℃ of error at measurment that causes 50mDa usually.This just requires to divide the folding device to carry out the aerial flight distance and the low-molecular-weight negative ion dynamic range of 100-500Da is proofreaied and correct to the flight time.The application adopts the anionic molecule on the kapok pool and negative ion fragment thereof to finish above-mentioned binomial correction.Above-mentioned various mass spectrometer is mainly used in the evaluation of XC polymer.As in the proteomics to the evaluation of amino acid sequence of polypeptide, measure at bigger molecular weight ranges from mode with positive ion electrospray usually, as 150-1500Da.And in FLT-5 '-MP and corresponding thymidine-5 '-MP molecular structure and the product fragment negative ion scope 125-323Da micromolecule scope.This scope exists the impurity of more solvent adduct ion to disturb, and measuring accuracy also is subjected to room temperature influence.Therefore very important to the correction of tandem Q-TOF.The application's patent is proofreaied and correct with cubic equation aerial flight distance and the above-mentioned measuring scope of TOF with D-(+)-gossypose and fragment product negative ion yarn row thereof.

The present invention proposes to separate the gold identification method of rich poly-back existence circulating tumor cell first, and this method is not subjected to development in the tumour cell body, shifts operation, the influence of genotype in radiotherapy and the chemotherapy process-phenotype biomarker variation.Measure feature metabolic compounds in the tumor cell proliferation process with cascade mass spectrometer, reflected the grade malignancy and the Normocellular essential distinction of peripheral blood in tumor cell proliferation cycle exactly.

Description of drawings

Fig. 1 is that tandem mass spectrometry is measured tumour cell DNA propagation principle schematic.

Among the figure: enter the high concentration FLT of cell medium by the DNA salvage route, at thymidine kinase (TK1) effect generation FLT-5 '-MP Xing biology down.And further with its DP Xing biology, TP Xing biology, and form mobile equilibrium.But with MP Xing biology is Main Ingredients and Appearance (70%).Because fluorine is in 3 ' replacement of pentose, DNA is synthetic to interrupt thus, causes FLT-5 '-MP Xing bioaccumulation in cell.Synthetic rising can reach 10 to 20 times and thymidine kinase is in G1/S stage of the growth cycle of tumour cell, characterizes the malignant proliferation degree of tumour uniquely as the FLT-5 '-MP Xing biology of enzyme matrix phosphorylation.Electronegative phosphate radical provides highly sensitive structure determination basis for the mensuration of negatron spraying tandem mass spectrometry simultaneously.

Fig. 2 is the specificity structure figure that tumour cell is identified.

Among the figure: S stage high expressed thymidine kinase cell generation cycle (TK1) is to the metabolic product of the phosphorylation reaction of pentose 5 ': FLT-5 '-MP absorbs the propagation attribute that ratio has characterized tumour cell uniquely with respect to the standardization of thymidine-5 '-MP.Biological thymidine-the 5 '-MP Xing biomolecule concentration standard absorption ratio with eubolism of FLT-5 '-MP Xing is eliminated the systematic error that produces in the experiment, reflects the biochemical character of tumor cell proliferation uniquely.

Fig. 3 is used to proofread and correct mass spectrometric D-(+)-gossypose and fragment product negative ion series calculated value figure thereof.

Among the figure: molecule negative ion calculated value, [M-H]=503.1612 are used for the correction that TOF divides the aerial flight distance of folding device according to the experimental observation value.And in experiment 100-550Da measurement range, carry out cubic equation with its fragment negative ion mass spectrometer is proofreaied and correct.

Fig. 4 is the molecular structure anionic cleavage mechanism figure of FLT-5 '-MP.

Among the figure: molecule negative ion m/z=323.04; Thymine negative ion m/z=125.05; 3 '-fluoro-ribodesose-5 '-single phosphoric acid negative ion m/z=197.03; And ribodesose-the 5 '-single phosphoric acid negative ion m/z=177.02 that loses neutral molecule HF (20Da) by m/z=197.03.

Fig. 5 is the mass spectrum experiment value figure of breast cancer cell intracellular metabolite product FLT-5 '-MP and thymidine-5 '-MP.

Among the figure: experiment value m/z 323.00 and m/z 321.16 show of poor quality for 2Da shows the molecule negative ion of FLT-5 '-MP and thymidine-5 '-MP, and this is that this is judged first of molecular structure.The right measuring matter of fragment negative ion 3 '-deoxidation-3 '-fluoro-ribose-the 5 '-single phosphoric acid negative ion that has a structural confirmation and corresponding ribodesose-5 '-single phosphoric acid negative ion is respectively m/z 196.96 and m/z 194.99, and this determines further that to the fragment negative ion ribose-3 ' position goes up substituent difference in quality (2Da): ¹⁹Fluorine (19Da) and hydroxyl (17Da).Fragment negative ion m/z 176.98 loses neutral molecule HF (20Da) and ribodesose-5 '-single phosphoric acid negative ion loses neutral molecule H by 3 '-deoxidation-3-fluoro-ribose-5 '-single phosphoric acid negative ion (m/z 196.96) ₂The common result of O (18Da).Fragment negative ion m/z 125.02 is the common trait of FLT-5 '-MP and thymidine-5 '-MP molecular structure, and promptly the thymine negative ion is irrelevant with ribose 3 ' substituting group.

Fig. 6 is breast cancer cell and the normal human's complete blood cell absorption contrast synoptic diagram to FLT.

Biological thymidine-the 5 '-MP Xing biomolecule with eubolism of Y-axis: FLT-5 '-MP Xing absorbs ratio; X-axis: the concentration determination scope of FLT in the cell culture fluid.The synthetic metabolic product that reaches of remedying of FLT is controlled by the expression of TK1, and is promptly relevant with the cell proliferation grade malignancy.

Fig. 7 is the absorption ratio synoptic diagram of breast cancer cell intracellular metabolite product FLT-5 '-MP and thymidine-5 '-MP.

Among the figure: accumulating on intracellular FLT-5 '-MP and reach 70 with respect to the ratio of thymidine-5 '-MP, is the hundreds of times of normal complete blood cell ratio (about 0.5).Thereby reduce the false positive that circulating tumor cell is identified.

Fig. 8 is that the monoclonal antibody chip docks synoptic diagram with cascade mass spectrometer.

Among the figure: novel structure parts: 1, the 0.22 micron chip outlet in aperture film filter, 2 connects the chip platinum electrode, it is zero T type polytetrafluoroethylene tee, 3 that PicoTip spraying emission needle and chip outlet kapillary adopt dead volume, PicoTip spraying emission needle internal diameter is 10 microns, receives liter/minute flow at 20-75, room temperature, 1, under 600 volts of voltage conditions, acetonitrile-water (50%) forms the pinniform electron spray, and it is auxiliary to need not nitrogen.

Embodiment further specifies advantage of the present invention and good effect below in conjunction with accompanying drawing.

Embodiment 1: cellular incubation and mass spectrometry

The breast cancer cell MCF-7 that ATCC is obtained places and contains 10% ox tire serum RPMI1640 nutrient solution.The content of incubator air carbon dioxide is 5%, 37 ℃ of temperature.Changed a nutrient solution in per two days, reach 70% unicellular degrees of fusion when cultured cell after, cellular incubation is replaced by that to contain FLT concentration respectively be 0.01,0.05,0.1,0.5,1, the nutrient solution of 1.5mM.Cell absorbs FLT, 1 hour time down for 37 ℃ in temperature.After cleaning cell with PBS, add 0.05% trypsase and remain in 37 ℃ about 10 minutes.Reclaimed cast-off cells in centrifugal 2 minutes in 5000g, and cell little cake in bottom is resuspended among the PBS of 1ml, slight concussion is cleaned and is placed in the erppendorf centrifuge tube of 1.5ml.Repeat PBS cleaning, the centrifugal recovery of 5000rpm three times.Add volume ratio and be 50% acetonitrile-water cytolysate, 100 microlitres in the little cake of cell, fierce concussion placed ultrasound wave 2 minutes after 5 seconds.Cell through three times-78 ℃ dark freeze thaw with 4 ℃ after, in 14, centrifugal 2 minutes of 000rpm reclaims that the upper strata is cleaned and via hole diameter is that 0.22 micron membranes filters.On the other hand, be used for the control experiment of above variable concentrations FLT in whole blood 100 microlitres of the healthy premenopausal volunteers of EDTA collection tube collection.Treated breast cancer cell and whole blood FLT picked-up are directly used in tandem Q-TOF qualitative, quantitative and measure.

FLT to the inhibition mechanism of DNA building-up process referring to figure-1.As the synthetic elementary cell of DNA, thymidine-5 '-MP derivant can be obtained under the thymidylate synthase effect by BrdU list phosphoric acid in the cell, i.e. de novo synthesis in the cell; Perhaps absorb the salvage route of extracellular thymidine.As phosphate group alms giver's ATP, the physiological concentration of the mM level that the interior tool of its cell is higher, therefore, this patent employing phosphate group acceptor density is that the FLT conduct of 1mM is measured CTCs.In the hope of obtaining minute quantity CTCs is reached sensitiveer detection limit.Figure-2 shows that cell generation cycle S stage high expressed kinases TK1 is to the metabolic product of the phosphorylation reaction of pentose 5 ': the Theoretical Calculation of FLT-5 '-MP is m/z=323.04, and the Theoretical Calculation of thymidine-5 '-MP is m/z=321.05.This standardization to compound absorbs the propagation attribute that ratio has characterized tumour cell uniquely.Obtain in micromolecule negative ion scope, to proofread and correct cascade mass spectrometer with D-(+)-gossypose before interior this accurate mensuration of pair cell to compound.The negative electricity of D-(+)-gossypose from the single isotope calculated value of fragment product referring to figure-3, wherein negative molion m/z=503.1612, fragment negative ion C ₆H ₁₁O ₆M/z=179.0556, fragment negative ion C ₈H ₁₃O ₇M/z=221.0661, fragment negative ion C ₁₀H ₁₉O ₉M/z=281.0873, fragment negative ion C ₁₂H ₂₀O ₁₀M/z=323.0978 is as mark in proofreading and correct.When the concentration of D-(+)-gossypose is 5ng/ μ l, the aerial flight of TOF apart from measured value be 503.161+/-0.05Da.Micromolecule measuring scope is distributed less than 10mDa as the standard by proofreading and correct by the molecule negative ion of D-(+)-gossypose and the measured value and the residual error between the calculated value of the above-mentioned product yarn row of ionization fragment.

Calibrated mass spectrometer can be implemented the mensuration of above-mentioned associated metabolic thing.Quadrupole rod mass spectrum filtrator imports the quadrupole rod collision cell according to the matter-charge ratio of negative ion FLT-5 '-MP with corresponding thymidine-5 '-MP.This by high-purity argon gas under 22V voltage, bump with negative ion m/z=323.04 and m/z=321.05, form the fragment product ion.The signal intensity of fragment product ion divides the folding device accurately to measure by the second-stage flight time mass spectrum.The negative ion series calculated value of FLT 5 '-MP is referring to scheming-4: molecule negative ion m/z=323.04; Thymine negative ion m/z=125.05; 3 '-fluoro-ribodesose-5 '-single phosphoric acid negative ion m/z=197.03; And lose the HF that the neutral molecule molecular weight is 20Da by m/z=197.03, i.e. ribodesose-5 '-single phosphoric acid negative ion m/z=177.02.Thymidine-5 '-the MP structure is ribodesose-5 '-single phosphoric acid negative ion m/z=195.03 to negative ion series accordingly therewith.It is to be noted that 3 '-fluoro-ribodesose-5 '-single phosphoric acid negative ion m/z=197.03 and ribodesose-5 '-single phosphoric acid negative ion m/z=195.03's is of poor quality for 2Da.The of poor quality of this 2Da is due to quality 19Da fluorine atom replaces by 3 ' quality 17Da of ribose hydroxyl.Figure-5 shows the mass spectrum measured value of experiment of FLT-5 '-MP and thymidine-5 '-MP.M/z 323.00 and m/z 321.16 mass spectrum his-and-hers watches bright of poor quality be the FLT-5 '-MP of 2Da and the molecule negative ion of thymidine-5 '-MP, this is that first of structural confirmation is judged.The right measuring matter of fragment negative ion 3 '-deoxidation-3 '-fluoro-ribose-the 5 '-single phosphoric acid negative ion that has a structural confirmation and corresponding ribodesose-5 '-single phosphoric acid negative ion is respectively m/z 196.96 and m/z 194.99, and this determines further that to the fragment negative ion it is the 2Da difference that substituent quality is gone up in ribose-3 ' position: ¹⁹Fluorine (19Da) is poor with hydroxyl (17Da).Fragment negative ion m/z 176.98 loses neutral molecule HF (20Da) and ribodesose-5 '-single phosphoric acid negative ion loses neutral molecule H by 3 '-deoxidation-3 '-fluoro-ribose-5 '-single phosphoric acid negative ion (m/z 196.96) ₂The common result of O (18Da).At last, fragment negative ion m/z 125.02 is the common trait of FLT-5 '-MP and thymidine-5 '-MP molecular structure, and promptly the thymine negative ion is irrelevant with ribose 3 ' substituting group.All mass spectral characteristics that observe confirm that undoubtedly FLT is that TK1 is at ribose 5 ' hydroxyl generation phosphorylation reaction in tumour cell.Table 1 is Q-TOF calculated value and the measured value comparing result of FLT-5 '-MP and thymidine-5 '-MP.

The Q-TOF tandem mass spectrometry of table 1.FLT-5 '-MP and thymidine-5 '-MP molecular structure is identified

FLT-5 '-MP thymidine-5 '-MP

	Calculated value	Measured value	Calculated value	Measured value
					The molecule negative ion	??323.04	??323.00	??321.04	??321.16
The thymine negative ion	??125.05	??125.02	??125.05	??125.02
					Ribodesose-5 '-single phosphoric acid negative ion	??197.03	??196.99	??195.03	??194.99
Ribodesose-5 '-single phosphoric acid negative ion-HF	??177.02	??176.98	??177.02	??196.98

Embodiment 2: cellular incubation and mass spectrum are quantitative

The healthy human body vein whole blood of above-mentioned breast cancer cell MCF-7 and fresh collection is undertaken by embodiment 1 condition the picked-up contrast experiment of FLT.Identify that CTCs to the selection basis of the picked-up condition of FLT is: A, because high concentration in the cell of ATP mM, so the nutrient solution upper limit of concentration of FLT is made as 1.5mM.B, the metabolism of FLT studies show that FLT absorbs in 60 minutes peakings, and continues to keep 2 hours.Distribute based on FLT-5 '-MP in the C, the cell of FLT phosphoric acid Xing biology, account for 70%.D, the high dense FLT of nutrient solution helps " salvage route ", i.e. the TK1 of breast cancer cell middle and high concentration.E, FLT-5 '-MP are not the good matrix compounds of deoxyribonuclease, and promptly 5 ' dephosphorylation of ribose is slow relatively.F, the phosphate radical negative charge of FLT-5 '-MP hinder its by in the cell to extracellular diffusion.Standardization absorbs ratio m/z 323.04:m/z 321.05, i.e. FLT-5 '-MP: the ratio of thymidine-5 '-MP characterize TK1 to this to the selectivity of compound and the phosphorylation reaction of 5 ' hydroxyl of ribose.Having expressed the two concerns at intracellular relative quantification: FLT-5 '-MP ends metabolism in cell, and accumulation forms high concentration, and thymidine-5 '-MP is in eubolism, keeps dynamic low concentration.The footpath of FLT is by the clean absorbed dose relation of salvage route, and this kind absorption difference is determined by TK1 is unique with the propagation of tumour cell fully.Therefore, standardization absorb ratio characterized tumour cell the propagation degree.The tumour cell fluorescence combination identification method that the CTCs of this law and FDA approval is current popular is completely different.The standardization of breast cancer cell absorb ratio with predose be 0.6 of 0.001mM be increased to 1mM dosage 70, referring to figure-6.The i.e. increase of two orders of magnitude.In contrast, the healthy complete blood cell of fresh extraction is under the condition of same FLT nutrient solution, and its standardization absorbs ratio＜1.Be that normal complete blood cell is almost nil by salvage route active effect under experiment condition of TK1 domination.Figure-7 shows that it is FLT-5 '-MP that the standardization of mass spectroscopy absorbs ratio m/z 322.99:m/z 320.98: the ratio of thymidine-5 '-MP has accurately reflected the grade malignancy of tumour cell delicately.

Embodiment 3: the oppositely rich poly-tandem mass spectrometry identification method of circulating tumor cell haemolysis

10 milliliters of venous blood of fresh collection first phase breast cancer patient are collected in and contain in the EDTA test tube and enforcement erythrocyte hemolysis degraded in 8 hours.Commodity ammonium chloride erythrocyte hemolysis degraded damping fluid is formed: 155mM ammonium chloride, 10mM KHCO ₃, 0.1mM EDTA, pH=7.4,50 milliliters.The haemolysis damping fluid of degrading is preheated to room temperature and places 100 milliliters of centrifuge tubes, 2 milliliters of venous blood collecting are added, the loam cake sealing.Go up slim and graceful haemolysis about 10 minutes in shaking dish.Centrifuge tube under room temperature, in 300g centrifuging 5 minutes, is gone to fall the upper strata red suspension.Should obtain leucocyte and tumour cell precipitum in the centrifuge tube bottom, if still have layering can repeat above-mentioned steps.Precipitum heavily is dissolved in 0.5 milliliter of PBS/EDTA solution, and the concentration of EDTA is 2mM.The adding inside surface scribbles 96 porocyte double dish of collagen, spends the night in 37 ℃ and adheres to.After PBS/EDTA washing, add 0.5 milliliter of the RPMI1640 nutrient solution that contains 1mM FLT, be statically placed in 37 ℃ of incubators 1 hour.Through the PBS washing, adding volume ratio is that 50% acetonitrile-water cytolysate, 100 microlitres extract relevant phosphoric acid derivatives.And abide by the embodiment 1 described circulating tumor cell tandem mass spectrometry that carries out and identify.This law is optionally carried out the haemolysis degraded to red blood cell, keeps complete leucocyte and tumour cell, promptly red blood cell is carried out the negative sense richness and gathers.The standardization of accurate mass spectroscopy absorb ratio m/z 323.04:m/z 321.05 greater than 5 for making a definite diagnosis.

Embodiment 4: circulating tumor cell isolated by filtration tandem mass spectrometry identification method

Polycarbonate (polycarbonate) membrane filtration separating cycle tumour cell is based on precision machined film hole diameter.The epidermis tumour cell that volume is bigger in the week blood circulation yarn system can separate with the less blood normal cell of volume.Fresh acquired volume is 2 milliliters of first phase breast cancer patient whole bloods after equal volume PBS/EDTA dilution, crosses Millipore Isopore product film separation vessel.The film diameter: 13 millimeters, 8 microns of bore dias, 8-20% aperture error.The dilution whole blood can pass through separation vessel in about 5 minutes, and circulating tumor cell stays in separation vessel.After twice volume is 7.5 milliliters of PBS/EDTA washings, add and contain 1 milliliter of the RPMI RPMI-1640 that concentration is 1mM FLT, be statically placed in 37 ℃ of incubators 1 hour.After PBS washing and centrifuging, adding 1 ml volumes ratio is 50% acetonitrile-water, and ultrasound wave was placed on-80 ℃ in 2 minutes.-80 ℃ in cytolysate footpath is dark to be frozen 4 ℃ and is recovered in the eppendorf centrifuge tube after thawing, footpath 14, and centrifugal 2 minutes of 000rpm, the upper strata is cleaned and is changed another eppendorf centrifuge tube over to.After placing-80 ℃ of dark freezing, Speed-Vac is to doing under room temperature.After heavily being dissolved in 50 microlitre volume ratios and being 50% acetonitrile-water, in Q-TOF bioassay standard absorption ratio.The standardization of accurate mass spectroscopy absorb ratio m/z 323.04:m/z 321.05 greater than 5 for making a definite diagnosis.

Embodiment 5: circulating tumor cell centrifuging tandem mass spectrometry identification method

This method is based on the product RosetteSep of commercial separation of C TCs.It is 7.5 milliliters of first phase breast cancer patient whole bloods that 350 microlitre RosetteSep centrifugates are added fresh acquired volume, and mixing placed room temperature 20 minutes.After 7.5 milliliters of PBS dilutions, light even.To dilute the back whole blood and evenly place 50 milliliters of centrifuge tube tops of Ficoll-Paque, and carefully avoid both to mix mutually as far as possible.The 1200g centrifuging is 20 minutes under the room temperature.Close hydro-extractor stop brake before centrifugal.Reclaim rich tumour cell after poly-at Ficoll-Paque and serum interface, and clean twice in the enppendorf centrifuge tube with 1 milliliter of PBS.Add and to contain 1 milliliter of the RPMI RPMI-1640 that concentration is 1mM FLT, be statically placed in 37 ℃ of incubators 1 hour.After 7.5 milliliters of washings of PBS.Adding 1 ml volumes ratio is 50% acetonitrile-water, and ultrasound wave was placed on-80 ℃ in 2 minutes.-80 ℃ in cytolysate footpath is dark to be frozen 4 ℃ and is recovered in the eppendorf centrifuge tube after thawing, footpath 14, and centrifugal 2 minutes of 000rpm, the upper strata is cleaned and is changed another eppendorf centrifuge tube over to.After placing-80 ℃ of dark freezing, Speed-Vac is to doing under room temperature.After heavily being dissolved in 50 microlitre volume ratios and being 50% acetonitrile-water, in Q-TOF bioassay standard absorption ratio.The standardization of accurate mass spectroscopy absorb ratio m/z323.04:m/z 321.05 greater than 5 for making a definite diagnosis.

Embodiment 6: the micro-fluidic richness of circulating tumor cell is gathered the tandem mass spectrometry identification method

Micro-fluidic chip after the improvement and the subsidiary RPMI RPMI-1640 reagent that contains 1mM FLT, can be used for township, the health at county level method of hospital's lesion detection in one's power, chip is after treatment posted to the mass spectrum center, carries out the mensuration that standardization absorbs ratio in the disposable electrospray device of chip outlet assembling.The facture of microchip structure is improved Adams device, and advantage is that 1 milliliter of undressed first phase breast cancer patient whole blood was finished catching of CTCs after 37 minutes, and this device adopts the embedding platinum electrode to can be used as the mass spectrometer ion source electrode.Fig. 8 docks synoptic diagram for identification EpCAM chip with cascade mass spectrometer.Critical component after wherein improving comprises: 1, the chip outlet aperture is that 0.22 micron membranes filtrator, 2 connects the chip external platinum electrode, it is zero T type threeway, 3 that PicoTip spraying emission needle and chip export dead volume capillaceous, PicoTip spraying emission needle mouth internal diameter is 10 microns, form the pinniform electron spray under the liter/minute flow room temperature condition receiving, it is auxiliary to need not nitrogen.Reclaim the tumour cell after richness is gathered, clean twice with PBS (0.5 milliliter).Feeding contains 16400.5 milliliters of nutrient solutions of RPMI 1 hour of 1mM FLT.After 0.5 milliliter PBS washing, feeding 0.5 ml volumes ratio is 50% acetonitrile-water displacement PBS, and chip can carry out the cascade mass spectrometer butt joint through ultrasound wave after 2 minutes.This method on the basis of Adams device, the outlet of its micro-fluidic chip introduce disposable electrospray device (PicoTip emitter, part number FS360-20-10-D-20-C7, New Objective, Woburn, MA.USA).Electrospray device is 20 microns of external diameters, and emission hollow needle internal diameter is 10 microns.The micro-fluidic chip outlet is by the sealing of insulation plastic cement.Platinum electrode and Q-TOF ion gun electrode form the pinniform electron spray under 1,600 volt of voltage, thus under highly sensitive state to electronegative phosphorylation-standardization of FLT-5 '-MP and thymidine-5 '-MP absorbs ratio and carries out relative quantification mensuration.The standardization of accurate mass spectroscopy absorb ratio m/z 323.04:m/z 321.05 greater than 5 for making a definite diagnosis.

Claims (14)

1. the fluoric compound application process in identifying circulating tumor cell is characterized in that:

(1) catching existence tumour cell in the periphery blood circulation system, acquisition procedure is divided into monoclonal antibody is discerned the tumour cell of denier from a large amount of red blood cells positive enrichment process, via the erythrocytic negative enrichment method of degraded, and filtration, the physical partition method based on TCD, size such as centrifugal;

(2) under cell culture condition, force the existence tumour cell of being caught to absorb fluoric compound with salvage route;

(3) the fluoro list phosphoric acid product that the tumour cell metabolism is produced and accumulates in cell medium is carried out the evaluation of electron spray negative ion tandem mass spectrometry.

2. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that for obtaining accurate qualification result, at first adopt the known sugars compounds under condition, mass spectrometer to be proofreaied and correct at negative electricity, and then obtain the molecule negative ion and the consequent certain negative fragment ion of fluoro list phosphoric acid product, determine tumour cell metabolism fluoro list phosphoric acid product uniquely by this spectrogram.

3. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, the ratio that it is characterized in that single phosphoric acid metabolic product of fluoro list phosphoric acid metabolic product and eubolism is eliminated the cumulative errors in the whole qualification process, characterizes the malignant proliferation degree of the tumour cell of catching uniquely.

4. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that fluoric compound, as 3 '-deoxidation-3 '-fluoro-thymidine (FLT), it is synthetic that its phosphorylation product hinders DNA, do not participate in the metabolism of tumour cell, thereby accumulate on the interior high concentration that favourable tumour cell is identified, raising Mass Spectrometer Method reliability, reduction or the elimination false positive of forming of cell.

5. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that fluoro list phosphoric acid derivatives and the corresponding single phosphoric acid derivatives of eubolism in the tumour cell, the two cellular uptake relative ratio after standardization, eliminate the systematic error in the mensuration process, the expression and the activity of associated kinase in the reflection cell.

6. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that the tandem mass spectrometry identification method is an example with quadrupole rod-time flight cascade mass spectrometer, to different accuracy of detection, high flux screening, the cascade mass spectrometer of mass resolution combination also comprises: carbon 18 reversed-phase high-performance liquid chromatography coupling quadrupole rod-quadrupole rod cascade mass spectrometers, the flight time-the flight time cascade mass spectrometer, quadrupole rod-fourier transform ion cyclotron resonance mass spectrometer (ICR) wherein divides the quadrupole rod of folding device to be substituted by ion trap as mass spectrum.

7. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that negative ion mass spectrum mensuration endocellular phosphorus acidifying metabolic product, except that using the electron spray anion source, also can help the laser desorption ionisation source and obtain relevant negative ion and carry out mass spectrum and identify by matrix.

8. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that with D-(+)-gossypose and fragment product negative ion series thereof, divide the aerial flight distance and the relevant phosphoric acid derivatives measuring scope (100-550Da) of folding device to proofread and correct the TOF mass spectrum.

9. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, the separation that it is characterized in that all blood systemic circulation tumour cells can be by the commodity of precision control directly through being that 8 microns membrane separator carries out, after the gained cell absorbs FLT, carry out tandem mass spectrometry and identify.

10. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, the separation that it is characterized in that all blood systemic circulation tumour cells also can be obtained by commodity Ficoll-Paque centrifuging, after the gained cell absorbs FLT, carry out tandem mass spectrometry and identify.

11. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that all blood systemic circulation tumour cell chips are accredited as: after micro-fluidic antibody richness is poly-, coupling cascade mass spectrometer again, introduce electrospray device in micro-fluidic outlet, form the intact device that the metabolism phosphoric acid derivatives is measured in guiding with cascade mass spectrometer ion gun electrode.Micro-fluidic antibody and platinum electrode by Identification Lists chrotoplast characteristic biomarker albumen formed, as the cascade mass spectrometer negative electricity from electric spray ion source.

12. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that all blood systemic circulation tumour cells can be obtained by the rich poly-technology of simple negative sense: promptly a large amount of red blood cells is degraded, is removed through commodity solvent selectivity ground.Tumour cell then is attached to the collagen carrier, after absorbing FLT, carries out tandem mass spectrometry and identifies.

13. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that extracting alone or in combination catch tumour cell after, relevant solvent acetonitrile-the water (50%) of getting phosphoric acid derivatives of quenching be best high throughput assay solvent, and the best of carbon 18 reversed-phase high-performance liquid chromatography separation is quenched, and to get solvent be acetonitrile-water (2%).

14. the application process of fluoric compound according to claim 1 in identifying circulating tumor cell, it is characterized in that fluoric compound is not limited to the synthetic specific FLT of DNA, also comprise other fluoric compound, comprise inhibition, damage the synthetic 5 FU 5 fluorouracil (5-FU) of DNA and RNA and 2 '-fluoro-the 2 '-deoxyglucose (FDG) of cellular energy metabolism, more than three kinds of fluoric compounds single or be used in combination, all under zymogenesis, generate the phosphorylation metabolic product, with its phosphorylation metabolic product of mass spectrometric determination, identify tumour cell.

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