CN109374890A - Breast cancer detection system and method based on the extracellular vesica detection of thermophoresis - Google Patents

Breast cancer detection system and method based on the extracellular vesica detection of thermophoresis Download PDF

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CN109374890A
CN109374890A CN201811321129.7A CN201811321129A CN109374890A CN 109374890 A CN109374890 A CN 109374890A CN 201811321129 A CN201811321129 A CN 201811321129A CN 109374890 A CN109374890 A CN 109374890A
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expression
extracellular
extracellular vesica
vesica
albumen
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孙佳姝
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National Center for Nanosccience and Technology China
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National Center for Nanosccience and Technology China
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads

Abstract

A kind of breast cancer detection system and method based on the extracellular vesica detection of thermophoresis, comprising: to the heating unit heated to vesica extracellular in person under test's blood；The sample bin chamber unit of the heating unit side is set；The signal processing unit of sample bin chamber unit side is set, the signal processing unit obtains at least one optical signal parameter, and by quantization and using without weighting and/or thering is weighted model to calculate corresponding optical parameter, single kind protein expression intensity is obtained.By the present invention in that carrying out signal to the extracellular vesica in blood samples of patients cell with aptamer or antibody, and the optical parameter in signal is detected and handled using detection unit, by quantization optical parameter and by using without weighting and/or there is weighted model to calculate corresponding optical parameter, it can fast and accurately show that the expression albumen intensity of extracellular vesica, detection accuracy are high.

Description

Breast cancer detection system and method based on the extracellular vesica detection of thermophoresis
Technical field
The present invention relates to cancer diagnosis technology field more particularly to a kind of breast cancer based on the extracellular vesica detection of thermophoresis Detection system and method.
Background technique
Tyrosine kinase-ErbB-2 (Her-2 also known as Her2/neu, c-erbB-2) is in 20%- There are the amplification of gene and the overexpression of albumen in 30% patient with breast cancer.The Infiltration of the Her-2 positive is strong, disease-free Life cycle is short, poor prognosis.Herceptin (Trastuzumab) is Humanized monoclonal antibodies derived from a kind of recombinant DNA, is suitable for The breast cancer of Her-2 overexpression.Due to there was only patient with breast cancer's Herceptin of Her-2 overexpression and gene magnification Treatment is just effective, therefore the Her-2 state of correct detection and evaluation breast cancer is most important.
There are two types of Her-2 detection methods usually used at present.One is immunohistochemical methods, by detecting mammary gland Her-2 protein expression situation in cancerous tissue sample determines Her-2 state.This method is considered as the goldstandard of Her-2 detection, But there is research to confirm that this method cannot be distinguished the patient that Her-2 albumen is moderate positive and whether there is the amplification of Her-2 gene.Separately A kind of method is the light hybridization in situ (FISH) based on detection Her-2 gene magnification level, and specific higher, current clinic is answered With also more and more.Both detection methods are to be particularly suitable for early-stage breast cancer row using breast cancer tissue as test object The patient of surgical resection therapy.But part shortage operation is accused of or there are patient with breast cancer's (such as advanced stages of surgical contraindication Patient with breast cancer), since tumor tissues can not be cut off, fine needle puncture tissue be easy to cause tumour spread, Her-2 status assessment again It becomes difficult.
Circulating tumor cell (Circulating tumor cell, CTC) is to fall off from aptamer tumour and enter peripheral blood Liquid circulation tumour cell, it is present in the Several Kinds of Malignancy such as breast cancer, prostate cancer, colorectal cancer, detection for The prognosis and the suitable individualized treatment of selection for assessing tumor patient especially late tumor patient have important clinical meaning Justice.Because CTC detection have the characteristics that it is minimally invasive, real-time, referred to as " liquid biopsy ".
The main method of CTCs detection at present includes relying on the immunology detection technology and base of tumor related marker analyte detection In the membrane filtration technique (ISET) of morphology enrichment.The former is to express different antigen markers based on cell surface, will be directed to tumour Specific antibody EpCAM, CK of cell surface antigen etc. are coated in magnetic bead surfaces to complete the specific recognition of tumour cell, borrow Help externally-applied magnetic field screening enrichment CTCs.The advantage of the technology is that specificity is higher, has the semi-automatic detection device of commercialization (CellSearch)；But the missing of tumour specific antigen expression will cause false negative result.The latter is according to cell size The biggish tumour cell screening of volume is enriched with by difference using the method for filtering.The advantage of the technology is that method is simple, price Cheap, the CTCs for separating acquisition active can be used for follow-up study；But due to lacking Morphologic Diagnosis goldstandard, the enrichment skill Art specificity is relatively poor.
China Patent Publication No.: CN201711042462.X discloses one kind and is used for three kinds of breast cancer tumour markers simultaneously The method of the light detection kit and detection of detection is related to measurement or the method for inspection comprising enzyme or microorganism, specifically relates to And a kind of method of light detection kit and detection for three kinds of breast cancer tumour markers while detection, three kinds of mammary gland Cancerous swelling tumor markers are respectively CEA, CA153, CA125, and the kit includes 10 kinds of solution, respectively G1 liquid, G2 liquid, G3 Liquid, Q1 liquid, Q2 liquid, Q3 liquid.It can be seen that the detection method has the following problems:
The first, the tumor markers being directed in the described detection method are only three kinds, and above-mentioned three kinds of markers are in other cancers Also there is higher expression in disease, therefore can not accurately show whether person under test suffers from the conclusion of breast cancer when being detected.
The second, the regression equation in the described detection method needs to carry out data a large amount of and cumbersome operations, causes described The detection cycle of detection method is long.
Summary of the invention
For this purpose, the present invention provides a kind of breast cancer survey detection system and method based on the extracellular vesica detection of thermophoresis, use To overcome the problems, such as that detection method is since the limitation of detection parameter causes detection accuracy low in the prior art.
On the one hand, the present invention provides a kind of breast cancer detection system based on the extracellular vesica detection of thermophoresis, comprising:
To the heating unit heated to the extracellular vesica in person under test's blood；
It is arranged in the heating unit side, to load the sample bin chamber unit of extracellular vesica, the sample warehouse In the outer vesica of unit inner cell to breast cancer have highly expressed expression albumen can occur to specifically bind with aptamer or antibody and Optical signal is marked, after the heating unit is to sample bin chamber unit heating, thermophoresis is generated in the sample bin chamber unit Extracellular vesica is converged in the lower side of temperature in the sample bin chamber unit by effect and convection current；
The side of the sample bin chamber unit far from the heating unit is set, to amplify the letter with reflected light signal Optical signal can be reflexed to designated position by number amplifying unit, the signal amplification unit；
Setting is in sample bin chamber unit side, at signal that amplified optical signal is acquired and is calculated Unit is managed, the signal processing unit obtains at least one optical signal parameter, and by quantization optical parameter and uses nothing It weights and/or there is weighted model to determine corresponding optical signal parameter, obtaining in the extracellular vesica of singleton has high table to breast cancer The expression intensity of the expression albumen reached.
Further, the chemiluminescent process are as follows: first by the aptamer or antibody with the catalytic materials that shine and extracellularly Vesica is incubated for together, by specifically binding by luminous catalysis substance markers on extracellular vesicle surface, and is added to it luminous Substrate, by shining, catalytic materials catalytic luminescence substrate reaches excited state, and light is discharged during being converted into ground state It can be so that extracellular vesicle surface marks optical signal.
Further, the weighted sum method packet of extracellular vesica expression albumen intensity is calculated in the signal processing unit It includes:
Step a: the weighting expression intensity of albumen is set as dependent variable Y, the extracellular vesica that signal processing unit is measured Marker optical parameter is set as independent variable X, then the optical parameter of unlike signal object is set as according to measuring sequence on extracellular vesica: X1, X2..., Xk；
Step b: it since weighting expression intensity Y and optical parameter X is in a linear relationship, is calculated as follows at this time:
Y=β0+β1X1+β2X2+...+βkXk+ε (1)
Wherein, β0, β1, β2..., βkFor regression parameter, ε is stochastic error；
Step c: in the step b formula (1) and optical parameter X make basic assumption to guarantee to be weighted to data Parameter Estimation, the validity of statistical check and Estimating Confidence Interval when summation；
Step d: when the formula (1) and optical parameter X meets the hypothesis, expectation is taken to the formula (1) both sides, is obtained:
E(Y|X1,X2...Xk)=β0+β1X1+β2X2+...+βkXk (2)
Wherein, E (Y | X1, X2..., Xk) indicate in given optical parameter XiUnder conditions of albumen weight expression intensity Y Conditional mean；
Step e: after the completion of taking expectation to the formula (1), regression parameter β is provided according to optical parameter X0, β1, β2..., βkPhase The estimated value answeredThe corresponding estimated value of albumen weight expression intensity Y is obtained at this time:
Above-mentioned formula (3) be F (Y | X1, X2..., Xk) point estimate；
Step f: when the formula (1) and optical parameter X meet the hypothesis in the step c, joined by least square Number estimation, it is assumed that
It is right respectively in the formula (4)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, is obtained:
Equation group in above-mentioned formula (5) is solved, regression parameter β is obtained0, β1, β2..., βkEstimated valueWith albumen weight expression intensity Y.
Further, the optical parameter in the step a is one in brightness L, luminous intensity C, absorbance A or light frequency λ Kind is a variety of.
Further, the basic assumption made in the step c to the formula (1) and optical parameter X includes:
It is assumed that c1: the probability distribution of stochastic error ε has zero-mean, E (ε)=0；
It is assumed that c2: the probability distribution of stochastic error ε has same variance, the variance of ε for different argument list present worths Not with XijVariation and change, D (ε)=σ2；
It is assumed that c3: auto-correlation, cov (ε is not present in stochastic error εi, εj)=0；
It is assumed that c4: εiWith any explanatory variable XiIt is uncorrelated, cov (εi, Xi)=0；
It is assumed that c5: perfect collinearity being not present between independent variable X；
Wherein, above-mentioned hypothesis c1-c4 is identical as the hypothesis of simple regression analysis, and the hypothesis c5 for explanation to become Amount.
Further, the weighted sum method of the extracellular vesica expression total abundance of albumen is calculated in the signal processing unit Include:
Step a: the extracellular vesica expression total abundance of albumen is set as dependent variable M, the optical parameter of extracellular vesica marker It is set as independent variable D, then is respectively set as the optical parameter measured according to the expression protein order of detection: D1,D2,...,Dk；
Step b:, need to be according to different types of since abundance of the variety classes expression albumen between different patients is all different It expresses albumen and corresponding weight coefficient α is set1,α2,...αk, then under the extracellular vesica expression total abundance M of albumen can pass through Formula acquires:
M=α1D1+α2D2+...+αkDk (6)
Step c: determining the total quantity N of measurement cancer species, and determines various types of expression albumen in the cancer species number There is highly expressed number n in amount1,n2,...nk, then respectively expression albumen has highly expressed ratio in cancer are as follows:
Step d: it is averaged to opalescence parameter D is respectively expressed in the step aAnd to light Parameter D seeks variance:
Step f: it is determined according to the data obtained in the step c and step d to weight coefficient α:
After step g: weight coefficient α determines, extracellular vesica is acquired according to formula in step b and expresses the total abundance of albumen:
Further, the sample bin chamber unit is arranged in the heating unit side, in the sample bin chamber unit Portion is equipped with sample liquids to load the extracellular vesica and aptamer, comprising:
Setting is in the heating unit side and is transparent material, and first to absorb the heating unit heat is thermally conductive Face；
It is arranged below first thermal conductive surface and is transparent material, second to absorb the heating unit heat leads Hot face, wherein the thermal conductivity for being more thermally conductive than the first thermal conductive surface of the second thermal conductive surface；
It is arranged between first thermal conductive surface and the second thermal conductive surface and offers through-hole at center, loads the sample The gasket of product liquid.
Further, one of the sample bin chamber unit far from the heating unit is arranged in the signal amplification unit Side, the optical signal to vesicle surface outside magnocell, comprising:
The side of second thermal conductive surface far from the heating unit is set, to observe the object lens of optical signal；
Side of the object lens far from the heating unit and in a certain angle with the object lens is set, to reflected light The acquisition reflective mirror of label；
Side of the object lens far from the heating unit and in a certain angle with the object lens is set, to reflected light The amplification reflective mirror in source；
Setting is in the amplification reflective mirror side, to provide the observation light source of amplification light source for signal.
On the other hand, the present invention provides a kind of breast cancer detection method based on the extracellular vesica detection of thermophoresis, feature It is, comprising:
Blood sample of patient is obtained as sample liquids, and by extracellular vesica therein and with the aptamer or antibody of signal It is incubated for together, there is highly expressed expression albumen to carry out specific knot breast cancer by aptamer or antibody and extracellular vesicle surface It closes, extracellular vesicle surface is marked into upper optical signal；
Extracellular vesica after incubation is put into sample bin chamber unit, and sample bin chamber unit is heated to generate thermophoresis effect Should and convection current, extracellular vesica is converged in into the low temperature side in the sample bin chamber unit, on vesica outside magnocell Optical signal converts optical signals into corresponding specific single kind number by being calculated at least one optical signal parameter Value；
It after the completion of detection, repeats the above steps, using a variety of in vesica outside different types of aptamer or antibody on cell There is highly expressed expression albumen to be marked and detect respectively breast cancer, obtains the number of a variety of expression albumen in extracellular vesica Value group；
Bring the above-mentioned correspondence numerical value group found out to different optical parameters into weighted model and/or without weighted model to corresponding light Parameter is calculated, and obtains the weighting expression intensity of extracellular vesicle protein and/or without weighting expression intensity and expression albumen Total abundance expresses figure to patient person under test by obtaining the SUM expression figure of extracellular vesica in conjunction with three kinds of numerical value, and according to SUM Whether determined with breast cancer.
On the other hand, the present invention is provided a kind of detected based on the extracellular vesica of thermophoresis and utilizes chemiluminescent breast cancer detection Method, comprising:
Blood sample of patient is obtained as sample liquids, and by extracellular vesica therein with luminous catalytic materials aptamer or Antibody is incubated for culture together, has highly expressed expression albumen to carry out breast cancer by aptamer or antibody and extracellular vesicle surface Specific binding, by the catalytic materials that shine on extracellular vesicle surface label；
Extracellular vesica after incubation is put into sample storehouse chamber unit, and to luminous substrate is added in sample storehouse chamber unit, So that it is reached excited state with the luminous catalytic materials catalysis of extracellular vesicle surface, and discharges luminous energy during being converted into ground state So that extracellular vesicle surface marks optical signal；
Sample storehouse chamber unit is heated to generate thermophoretic effect and convection current, extracellular vesica is converged in into the sample warehouse Low temperature side in unit, with the optical signal on vesica outside magnocell；After amplification, optical signal is carried out using signal processing unit Acquisition and analysis, corresponding specific single kind numerical value is converted optical signals by being calculated different optical parameters；
It after the completion of detection, repeats the above steps, using a variety of in vesica outside different types of aptamer or antibody on cell Expression albumen is marked and detects respectively, obtains the numerical value group of a variety of expression albumen in extracellular vesica；
Bring the above-mentioned correspondence numerical value group found out to different optical parameters into weighted model and/or without weighted model to corresponding light Parameter is calculated, and obtains the weighting expression intensity of extracellular vesicle protein and/or without weighting expression intensity and expression albumen Whether total abundance by obtaining the SUM expression figure of extracellular vesica in conjunction with three kinds of numerical value, and is suffered from according to SUM expression figure person under test There is breast cancer to determine.
Compared with prior art, the beneficial effects of the present invention are by using aptamer or antibody to blood samples of patients cell Outer vesicle surface has highly expressed expression albumen to carry out signal breast cancer, and using detection unit to the object of light in signal Reason parameter is detected and is handled, and can fast and accurately show that extracellular vesica has high table to breast cancer by analyzing optical parameter The expression albumen weighting expression intensity reached, detection accuracy are high.Especially, the present invention is examined by using the accumulation of a variety of physical parameters Survey, and it is indirect detected by biological respinse, detection process is easier and easy to operate, and sample dosage is small, also, passes through physics Whether the accumulation of parameter detects, and detects compared to biological respinse, and the expression of detection limit is easier to quantify, can suffer to person under test Breast cancer is clearly determined.In particular, the present invention for extracellular vesica to breast cancer have it is highly expressed expression albumen detection, And the parametrization of luminosity, the calculation based on weighted model and/or without weighted sum model obtains, then according to the egg of standard The canonical function relationship of white matter marker concentration and certain parameter of light, to determine canceration degree.Such as, by luminous intensity C, light The light characteristics detection physical quantity such as concentration of specimens under degree L, light frequency λ, specific wavelength absorbance A is determined.Also, it is being based on In the detection process of same antibody or aptamer, by the detection and calculating to a variety of physical parameters, it is compared to each other, chooses optimal The expression of physical parameter is to determine final canceration result.
In particular, the light that the present invention is combined using chemiluminescence and thermophoretic effect, convection current builds up mode, by with it is extracellular Vesica in conjunction with aptamer or antibody in the catalysis of luminous catalytic materials and reach excited state, and discharged during being converted into ground state Luminous energy is so that extracellular vesicle surface marks optical signal, and when detecting, it is luminous that the luminous catalytic materials are able to maintain the long period.
In particular, calculation of the present invention due to passing through weighted model and/or without weighted sum model, by a variety of thin The quantization accurate calculation of extracellular vesica expression albumen, can accurately determine canceration degree.
Further, sample bin chamber unit of the present invention is equipped with the first thermal conductive surface and the second thermal conductive surface of transparent material, passes through Extracellular vesica is set to generate thermophoretic effect the heating of the sample liquids of its inside and to mobile at low temperature, meanwhile, after temperature increases Sample liquids can generate thermal convection and extracellular vesica is made to accumulate in designated position, with the optical signal of vesica outside this magnocell, In this way, the specific value of extracellular vesica optical parameter can be more accurately observed when detecting to optical parameter, Further improve the detection accuracy of the detection system.In particular, a variety of optical physics ginsengs can be acquired simultaneously by this kind of mode Amount, e.g., the detection to parameters such as luminous intensity C, brightness L, light frequency λ, specific wavelength absorbance As.
Further, detection system of the present invention uses thermophoretic effect and the outer vesica of thermal convection effect Cumulative cell, because This, the detection system is not specifically limited the size of the sample bin chamber unit.Sample in the sample bin chamber unit Liquid is for loading extracellular vesica and aptamer or antibody and it can be made to generate thermophoretic effect and convection current, therefore this detection system It is not specifically limited in the selection of sample liquids, as long as the sample liquids can drive cell external capsule under thermal convection effect Bubble is mobile and builds up.Further, the extracellular vesica is connect by way of specific binding with aptamer or antibody Together, in this way, being linked on extracellular vesica of can consolidating of signal also can be more when extracellular vesica is built up Accurately the optical parameter of extracellular vesica is observed to come, further improves the detection accuracy of the detection system.Extracellularly The power that vesica is subject under thermophoretic effect is directly proportional to its diameter square, and unrelated with extracellular vesica quantity, therefore, is detecting When only need a small amount of blood sample can be completed, only need 0.1 microlitre of sample dosage for extracellular vesica, and without to sample Carry out preposition processing.
Further, the sample bin chamber unit is when being heated, if first thermal conductive surface and the second thermal conductive surface it Between there are the temperature difference, it will be able to make to generate thermophoretic effect and thermal convection in sample bin chamber unit and the cell external capsule of signal will be had Bubble is built up to designated position.It only needs to detect the extracellular vesica optical parameter of accumulation in the detection system, without using Other specific apparatus have saved the cost of detection device in the case where not influencing detection system detection accuracy.
Further, data acquisition unit is equipped in the detection system, it can be from collected protein expression profile It is middle to extract specified optical parameter, and carry it into weighted model and/or without in weighted sum model to calculate cell external capsule Bubble expresses the weighting expression intensity of albumen and/or without weighting expression intensity, by the way that intuitive image is converted to specific number, Further improve the detection accuracy of the detection system.
Further, the detection system can select corresponding aptamer or antibody for expression albumen high in various cancers It is marked, the abundance map of corresponding expression albumen is measured with this and calculates expression intensity, in this way, the detection system Breast cancers can not only be detected, while other cancers can also quickly and accurately be detected, such as: lung cancer, pancreas Gland cancer, colorectal cancer, gastric cancer, prostate cancer, head-neck carcinoma, cutaneum carcinoma, kidney, carcinoma of testis, thyroid cancer, bladder cancer, uterine cancer, Carcinoma of vagina, carcinoma of endometrium, oophoroma, cancer of the esophagus, carcinoma of mouth, salivary-gland carcinoma, laryngocarcinoma, peritoneal cancer, rhinocarcinoma, laryngocarcinoma, fallopian tubal Cancer, kidney mother cell cancer, lymph cancer, cholangiocarcinoma and swing sarcoma, synovial sarcoma, medulloblastoma, trophocyte's tumor, glue Matter tumor, spongioblastoma, cholesteatoma, chondrosarcoma, ependymoma, neurinoma, neuroma, rhabdomyosarcoma.
Detailed description of the invention
Fig. 1 is that the present invention is based on the structure charts of the breast cancer detection system of the extracellular vesica detection of thermophoresis；
Fig. 2 is the structure the present invention is based on the extracellular vesica detection of thermophoresis using chemiluminescent breast cancer detection system Figure；
Fig. 3 is the schematic diagram that the present invention detects extracellular vesica sample absorbance using monochromator；
Fig. 4 is that the present invention is based on the extracellular vesica detections of thermophoresis to utilize chemiluminescent breast cancer detection system detection mammary gland The excretion body surface of cancer patient reaches protein abundance map.
Specific embodiment
In order to which objects and advantages of the present invention are more clearly understood, the present invention is further retouched below with reference to embodiment It states；It should be appreciated that specific embodiment described herein is used only for explaining the present invention, it is not intended to limit the present invention.
Below in conjunction with attached drawing, the forgoing and additional technical features and advantages are described in more detail.
The preferred embodiment of the present invention described with reference to the accompanying drawings.It will be apparent to a skilled person that this A little embodiments are used only for explaining technical principle of the invention, are not limiting the scope of the invention.
It should be noted that in the description of the present invention, the instruction such as term " on ", "lower", "left", "right", "inner", "outside" Direction or the term of positional relationship be direction based on the figure or positional relationship, this is intended merely to facilitate description, and It is not that indication or suggestion described device or element must have a particular orientation, be constructed and operated in a specific orientation, therefore not It can be interpreted as limitation of the present invention.
In addition it is also necessary to explanation, in the description of the present invention unless specifically defined or limited otherwise, term " peace Dress ", " connected ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integrally Connection；It can be mechanical connection, be also possible to be electrically connected；Can be directly connected, can also indirectly connected through an intermediary, It can be the connection inside two elements.To those skilled in the art, it can understand that above-mentioned term exists as the case may be Concrete meaning in the present invention.
System embodiment one
The embodiment of the present invention is the breast cancer detection system based on the extracellular vesica of thermophoresis, refering to Figure 1, it is this The structural schematic diagram of breast cancers detection system of the invention based on the extracellular vesica detection of thermophoresis, the system of the present embodiment includes adding Hot cell 1, sample bin chamber unit 2, signal amplification unit 3 and signal processing unit 4, wherein the heating unit 1 is arranged in institute The top for stating sample bin chamber unit 2, to be heated to the sample in the sample bin chamber unit 2；In the sample bin chamber unit 2 Equipped with sample liquids, to load extracellular vesica and with the aptamer of fluorescent marker；The signal amplification unit 3 is arranged in institute 2 lower section of sample bin chamber unit is stated, to amplify the fluorescence signal of the fluorescent marker, the signal processing unit 4 is arranged in institute State 3 side of signal amplification unit, to acquire and record the amplified fluorescence signal, and to the brightness of fluorescence signal, One or more of luminous intensity and optical wavelength parameter are carried out or are obtained, while using weighted sum without weighted sum to sample to be tested Extracellular vesica carries out the detection of cancerous lesion degree.
Specifically, before the breast cancers detection system work based on the extracellular vesica detection of thermophoresis, it first will be extracellular Vesica and aptamer with fluorescent marker are put into sample bin chamber unit 2, by one kind of aptamer and extracellular vesicle surface to cream Gland cancer has highly expressed expression albumen to be specifically bound, by fluorescent marker on extracellular vesica.After the completion of label, institute It states heating unit 1 to start to heat sample bin chamber unit 2, after sample bin chamber unit 2 is heated, internal sample liquids start to produce Simultaneously convection current occurs for raw thermophoretic effect, and by labeled extracellular vesicular aggregates in designated position；After the completion of aggregation, signal amplification Unit 3 can emit comparison light source to the position of extracellular vesicular aggregates, and signal processing unit 4 can acquire the extracellular vesica of aggregation Relevant information, and it is analyzed accordingly, passes through one of brightness L, luminous intensity C, wavelength lambda parameter of light or several Kind obtains, and takes weighted sum to carry out cancerous lesion degree without the mode of weighted sum and is judged.Those skilled in the art can With understanding, the present embodiment be cannot be only used for based on the breast cancer detection system of the extracellular vesica detection of thermophoresis to cell external capsule Bubble is assembled and is detected, and can also detect to extracellular vesica or other kinds of micro-nano biomone, as long as meeting institute Its specified working condition can be reached by stating the breast cancers detection system based on the extracellular vesica detection of thermophoresis.
Please continue to refer to shown in Fig. 1, heating unit of the embodiment of the present invention 1 is a laser heater, is arranged in the sample 2 top of product warehouse unit, to be heated to the sample liquids inside the sample bin chamber unit 2, to generate inside it Circular heating region.After the completion of extracellular vesica is labeled, the heating unit 1 to the sample bin chamber unit 2 inside Sample liquids heated, extracellular vesicular aggregates are got up.It is understood that the heating side of the heating unit 1 Formula is not limited in laser irradiation, and selection the present embodiment of the direction of laser irradiation and power is not specifically limited, as long as Meeting the heating unit 1 can make to generate the temperature difference inside the sample bin chamber unit 2 to converge extracellular vesica.
Please continue to refer to shown in Fig. 1, sample bin chamber unit 2 described in the embodiment of the present invention is arranged under the heating unit 1 Side, to contain the sample liquids containing extracellular vesica and aptamer, including the first thermal conductive surface 21, the second thermal conductive surface 22 and gasket 23；Wherein the gasket 23 is arranged below first thermal conductive surface 21 and contacts, described to contain sample liquids Second thermal conductive surface 22 is arranged below the gasket 23, to together will be inside the gasket 23 with first thermal conductive surface 21 Sample liquids sealing.When the heating unit 1 heats the sample bin chamber unit 2, laser can sequentially pass through described First thermal conductive surface 21 and the second thermal conductive surface 22 simultaneously heat it, described thin after the heating due to the heated heating of sample liquids Extracellular vesica temperature can also increase, and extracellular vesica can generate thermophoretic effect and to lower first thermal conductive surface, 21 He of temperature at this time Second thermal conductive surface, 22 direction is mobile, since first thermal conductive surface 21 is different from the thermal conductivity of the second thermal conductive surface 22, after the heating Temperature at first thermal conductive surface, 21 hot spot can be higher than the temperature at 22 hot spot of the second thermal conductive surface and produce sample liquids Raw temperature difference, sample liquids along low temperature in sample bin chamber unit 2 to high temperature prescription to thermal convection is generated to make in sample Extracellular vesica is migrated and is accumulated at second thermal conductive surface 22.It is understood that sample warehouse list described in the present embodiment Member 2 can be set in lower section, top, left or the right of the heating unit 1, as long as the heating unit 1 can be to described Sample bin chamber unit 2 heats and the sample liquids inside it is made to increase temperature.
Specifically, the first thermal conductive surface 21 of the present invention is sheet glass, it is arranged above the gasket 23, to close It seals the sample liquids inside the gasket 23 and sample liquids is heated together with the heating unit 1.When the heating unit 1 Laser pass through first thermal conductive surface 21 when, can be heated at the center to first thermal conductive surface 21, and improve plus Temperature at heat, after temperature improves, first thermal conductive surface 21 can transfer heat to the sample liquids in the gasket 23, and Sample liquids are made to generate convection current, with vesica outside Cumulative cell.It is understood that the material of first thermal conductive surface 21 can be Glass, polymethyl methacrylate (PMMA), dimethyl silicone polymer (PDMS), sapphire or other kinds of transparent material, As long as meeting first thermal conductive surface 21 can be heated heating.
Specifically, the second thermal conductive surface 22 of the present invention is the sheet glass for being more thermally conductive than first thermal conductive surface 21, It is arranged below the gasket 23, to seal sample liquids inside the gasket 23 and with the heating unit 1 together Sample liquids are heated.It, can be thermally conductive to described second when the laser of the heating unit 1 passes through second thermal conductive surface 22 It is heated at the center in face 22, and improves the temperature at heating, after temperature improves, second thermal conductive surface 22 can pass heat The sample liquids being handed in the gasket 23 are more thermally conductive than first thermal conductive surface 21 due to second thermal conductive surface 22, After the completion of the heating unit 1 heating, the temperature of second thermal conductive surface 22 can be less than the temperature of first thermal conductive surface 21, The temperature difference is generated in the gasket 23, and sample liquids is made to generate convection current, with vesica outside Cumulative cell.It is understood that institute The material for stating the second thermal conductive surface 22 can be glass, polymethyl methacrylate (PMMA), dimethyl silicone polymer (PDMS), indigo plant Jewel or other kinds of transparent material heat up and as long as meeting second thermal conductive surface 22 and can be heated at a temperature below sample Liquid center temperature.
Specifically, the gasket 23 is a disk for being equipped with through-hole, it is arranged in first thermal conductive surface 21 and second Between thermal conductive surface 22, to load vesica outside sample liquids and Cumulative cell.When the heating unit 1 is to the sample warehouse list When member 2 is heated, the Focus Club of heating laser is located at the sample liquids inside the gasket, by heating to sample liquids Extracellular vesica inside sample liquids is set to generate thermophoretic effect and assemble to second thermal conductive surface 22, simultaneously as described the The temperature difference between one thermal conductive surface 21 and the second thermal conductive surface 22, sample liquids start to generate convection current and by extracellular vesicular aggregates in institute At the laser irradiation for stating the second thermal conductive surface 22.It is understood that sample liquids material in the gasket 23 can for blood plasma, The derivative sample of serum or any form of blood or its processing, as long as meeting the sample liquids can load extracellularly Vesica and aptamer simultaneously can make it generate thermophoretic effect and convection current.
Please continue to refer to shown in Fig. 1, signal amplification unit 3 described in the embodiment of the present invention is arranged in the sample bin chamber unit 2 lower sections, to irradiate the extracellular vesica assembled in second thermal conductive surface 22 and believe the fluorescence in the extracellular vesica Number amplification, including object lens 31, acquisition reflective mirror 32, amplification reflective mirror 33 and observation light source 34.Wherein, the setting of object lens 31 exists Second thermal conductive surface, 22 lower section, to collect the fluorescence signal with vesica outside fluorescence labeled cell, the acquisition is anti- Light microscopic 32 is arranged below the eyepiece 31, described to put the fluorescence signal of amplification is reflected to the signal processing unit 4 Big reflective mirror 33 is arranged below the acquisition reflective mirror 32, the light in the observation light source 34 is reflexed to the object In mirror 31, the setting of observation light source 34 is on 33 right side of magnifying reflecting mirror, to provide the light of amplification fluorescence signal.When When the signal amplification unit is started to work, the observation light source 34 emits light, is reflexed to by the amplification reflective mirror 33 The object lens 31, the object lens 31 expose to light at the extracellular vesicular aggregates in second thermal conductive surface 22, and with this The fluorescence signal for amplifying the outer extracellular vesica, after the completion of amplification, it is reflective that the signal processing unit 4 will use the acquisition Mirror 32 acquires the amplified fluorescence signal, and the acquisition and processing of fluorescence signal are completed with this.It is understood that the letter Number amplifying unit 3 can be set in top, lower section, left or the right of the sample bin chamber unit 2, can be right as long as meeting it The indoor fluorescence signal of sample bin is acquired.
Specifically, under object lens 31 of the present invention are arranged at the extracellular vesicular aggregates of second thermal conductive surface 22 Side, to collect the fluorescence signal in the extracellular vesica, when the light of the observation light source 34 exposes to object lens 31, object Mirror 31 can expose to light on second thermal conductive surface 22, amplify the fluorescence signal on the extracellular vesica with this.It can be with Understand, this embodiment is not specifically limited for the type of the object lens 31, refers to as long as meeting the object lens 31 and can reach it Fixed working condition.
Specifically, acquisition reflective mirror 32 described in the embodiment of the present invention is a flat surface mirror, it is arranged under the object lens 31 Fang Bingyu object lens 31 are in 45 ° of angles, to reflect the amplified fluorescence signal.When the fluorescence signal of the extracellular vesica After being amplified, fluorescent marker is reflexed to the signal processing unit 4 by the acquisition reflective mirror 32, to complete adopting for fluorescence signal Collection.It is understood that this embodiment is not specifically limited for the size of the acquisition reflective mirror 32, as long as it is anti-to meet the acquisition Fluorescence signal can completely be reflexed to the signal acquisition unit by light microscopic 32.
Please continue to refer to shown in Fig. 1, signal processing unit 4 described in the embodiment of the present invention includes a CCD camera, and setting exists 32 right side of acquisition reflective mirror, to acquire the fluorescence signal of the extracellular vesica.After the fluorescence signal is amplified, The acquisition reflective mirror 32 can reflex to amplified fluorescence signal in the signal processing unit 4, the signal processing list First 4 pairs of fluorescence signals are acquired and arrange, and form the map of single detection, it is to be understood that signal processing unit 4 can be with Including CDD camera, or any instrument that can detect fluorescence signal, as long as the signal processing unit 4 can pass through The signal amplification unit 3 takes pictures to the extracellular vesica with fluorescent marker, obtains information.Certainly, the letter Number processing unit 4 can be located at left side, right side, upside or the downside of the signal amplification unit 3, as long as meeting at the signal Reason unit 4 can be acquired and be handled fluorescence signal by signal amplification unit 3.
Whether this system embodiment detection system suffers from when breast cancer detects to person under test by first by fluorescence mark Note is connected with aptamer, then aptamer is incubated for together with the extracellular vesica of sample to be tested extracellular vesica is put on fluorescent marker, Easy to operate, easy to carry out, when being detected using this system to multiple persons under test, patient only needs provide a small amount of blood sample, Can the state of an illness to patient quickly diagnosed.
System embodiment two
The embodiment of the present invention is to utilize chemiluminescent breast cancer detection system based on the extracellular vesica detection of thermophoresis, as The preferred embodiment of the present invention please refers to shown in Fig. 2, is based on the extracellular vesica of thermophoresis for the embodiment of the present invention and detects utilizationization The structural schematic diagram of luminous breast cancer detection system is learned, the system of the present embodiment includes heating unit 1, sample bin chamber unit 2 With signal processing unit 4, the said units are identical as above-described embodiment one.
Unlike above-described embodiment one, signal of the invention uses chemiluminescent labeling method, is based in use Before the breast cancer detection system of thermophoresis extracellular vesica detection, first by extracellular vesica sample to be tested and it is marked with luminous catalytic materials Antibody be incubated for together, luminous catalytic materials can be enzyme, by will be thin in conjunction with antibody and extracellular vesica expression protein-specific Extracellular vesica marker enzyme after the completion of label, will be incubated for the sample completed and be put into the sample bin chamber unit 2, and to the sample Luminous substrate is added inside warehouse unit, enzymatic luminous substrate simultaneously reaches excited state, and when it is converted into ground state Issue optical signal.
When the vesica detection chemiluminescent breast cancer detection system starts of utilization extracellular based on thermophoresis, institute It states heating unit 1 to heat the sample bin chamber unit 2, the extracellular vesica for keeping it internal generates thermophoretic effect and starts The one side low to temperature is mobile, while the two sides temperature difference of the sample bin chamber unit makes inside the sample bin chamber unit 2 Start to generate convection current, and extracellular vesica is accumulated in into designated position, after the completion of accumulation, the signal acquisition unit 3 starts pair The optical signal of extracellular vesica is acquired, and obtains the abundance map of the extracellular vesicle surface expression albumen.Continuing with As shown in fig.2, signal processing unit 4 described in the embodiment of the present invention is arranged below the sample bin chamber unit 2, to institute The extracellular vesica for stating aggregation is observed and acquires.
Specifically, in the present embodiment using in chemiluminescent extracellular vesica detection architecture, the marker enzyme can be with For horseradish peroxidase (HRP), alkaline phosphatase (ALP) or other kinds of marker enzyme；The luminous substrate is luminol (32 amino phthalyl hydrazine), different luminol (42 amino phthalyl hydrazine) or other kinds of derivative, as long as meeting The marker enzyme can react with extracellular vesica and be sticked to extracellular vesicle surface, and the luminous substrate can be with institute Marker enzyme is stated to react and issue light.
The present embodiment is compared with above-mentioned real detection system applies example one, heating unit 1, the structure of sample bin chamber unit 2, principle It is all the same with work functions, but due to the present embodiment using chemical reaction generate light extracellular vesica is marked, right Breast cancer, which has highly expressed expression albumen that rear extracellular vesicle surface has been marked, can maintain highlighted optical signal for a long time, Therefore amplify without using the amplification reflective mirror 33 and observation light source 34 to optical signal can also be to cell external capsule for the present embodiment The optical signal of bubble is accurately observed and is acquired.
Antibody is first incubated for together with extracellular vesica when detecting and is made its interconnection by the present embodiment detection system, is incubated Extracellular vesica is put into the sample bin chamber unit 2 together with luminous substrate after the completion of educating, passes through the catalysis that shines in antibody Object catalytic luminescence substrate reaches excitation state, and discharges luminous energy when it is converted to ground state with this in extracellular vesicle surface mark Remember optical signal, meanwhile, chemical reaction described in the present embodiment is catalysis reaction, and the catalytic materials that shine can urge always as catalyst Change luminous substrate to react and continue to generate light, in this way, when detecting, the extracellular vesica is able to maintain to be sent out for a long time Light.
Further, since optical signal can maintain for a long time, so acquired without carrying out secondary amplification to optical signal When optical signal, as soon as compared to the system embodiment, the present embodiment only needs once carry out optical signal clear and accurate adopt Collection, has saved the detection time of system, has improved detection efficiency.
Further, when detecting, the extracellular vesica that label is completed is transferred to from incubation container equipped with sample liquid In the sample bin chamber unit 2 of body, so, just eliminates luminous catalytic materials and the luminous substrate catalysis in Excess antibody and send out The raw phenomenon reacted and shine together, so that described existed based on the extracellular vesica of thermophoresis using the cancer detection system of chemical detection When being detected to extracellular vesica, in the base with the cancer detection system advantage based on the extracellular vesica detection of thermophoresis On plinth, possess the accuracy of height.
For above two embodiment, detection and parametrization for the luminosity of extracellular vesica are based on weighted sum The calculation of no weighted sum obtains, then according to the canonical function of the protein markers concentration of standard and certain parameter of light Relationship, to determine canceration degree.Such as, by the concentration of specimens under luminous intensity, brightness, light frequency, specific wavelength absorbance Equal light characteristics detection physical quantity is determined.
It is illustrated below by embodiment.
As a preferably embodiment, the optical parameter X in the present embodiment uses brightness L, at this time in the detection system Collector select CCD camera, when being measured to brightness, by using CCD camera to being sent out after extracellular vesica set Light out is shot, to obtain the single map of extracellular vesica optical signal, after being measured, to single expression albumen Multiple measurement results can be used the brightness table of comparisons for the brightness L in each map and be converted into specific value L from image1, L2...Lk, and bring into the weighted sum model in this, as independent variable X to a certain albuminoid weight expression intensity Y It is calculated, the weight expression intensity Y of a certain albuminoid is obtained with this.
At this point, the albumen of the extracellular vesica weights expression intensity YLAre as follows:
YL=β0+β1L1+β2L2+...+βkLk+ε (10)
After assuming to above-mentioned formula (10), expectation is taken to both sides, can be obtained:
E(YL|L1,L2...Lk)=β0+β1L1+β2L2+...+βkLk (11)
After the completion of taking expectation to the formula (10), regression parameter β is provided according to brightness L0, β1, β2..., βkAccordingly Estimated valueAvailable albumen weights expression intensity Y at this timeLCorresponding estimated value:
Parameter Estimation is obtained using least square at this time:
It is right respectively in the formula (13)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, is obtained:
Equation group in above-mentioned formula (14) is solved, regression parameter β can be obtained0, β1, β2..., βkEstimated valueExpression intensity Y is weighted with albumenL。
Find out the estimated value of parameterExpression intensity Y is weighted with albumenLAfterwards, according to expression protein classes With the weighting expression intensity Y of each albuminoidL。
By calculating, it can be concluded that, when using brightness L to be calculated as optical parameter X, the light map measured can The abundance of the outer vesica expression albumen of more intuitive expression cell, also easier when calculating, measurement period is short.
As a preferably embodiment, in the present embodiment, is measured using luminous intensity C rather than brightness L, detected at this time Collector used in system selects illumination photometer (or lux meter) to measure the luminous intensity C in extracellular vesica optical signal.Its In, illumination photometer is the photoelectric cell for luminous energy being directly changed into electric energy.When light is mapped to selenium cell surface, incident light is penetrated Metallic film reaches on the interface of semiconductor selenium layer and metallic film, and photoelectric effect is generated on interface.The photoproduction electricity of generation The size and the illumination on photocell light receiving surface of stream have certain proportionate relationship.At this moment if connecting external circuit, electricity is just had Stream passes through, current value from lux (Lx) be scale microampere meter on indicate.
It is surveyed by using luminous intensity of the illumination photometer to type a certain in the extracellular vesica of many cases expression albumen signal After amount, can obtain the brightness C that albumen is expressed a certain type1,C2...Ck, and the weighting is brought into this, as independent variable X Expression intensity Y is weighted to a certain albuminoid in summation modelCIt is calculated, the weighting expression of a certain albuminoid is obtained with this Intensity YC。
At this point, the albumen of the extracellular vesica weights expression intensity YCAre as follows:
YC=β0+β1C1+β2C2+...+βkCk+ε (15)
After assuming to above-mentioned formula (15), expectation is taken to both sides, can be obtained:
E(YC|C1,C2...Ck)=β0+β1C1+β2C2+...+βkCk (16)
After the completion of taking expectation to the formula (15), regression parameter β is provided according to luminous intensity C0, β1, β2..., βkAccordingly Estimated valueAvailable albumen weights expression intensity Y at this timeCCorresponding estimated value:
Parameter Estimation is obtained using least square at this time:
It is right respectively in the formula (18)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, is obtained:
Equation group in above-mentioned formula (19) is solved, regression parameter β can be obtained0, β1, β2..., βkEstimated valueExpression intensity Y is weighted with albumenC。
Find out the estimated value of parameterExpression intensity Y is weighted with albumenCAfterwards, according to expression protein classes With the weighting expression intensity Y of each albuminoidC。
When using luminous intensity C to be calculated as optical parameter X, the anti-interference with higher when measuring light map, The extracellular vesica weighting expression intensity Y acquiredCIt is relatively accurate；
As preferably another embodiment, in the present embodiment, surveyed using light frequency ν rather than brightness L, luminous intensity C Amount, the collector selected in the detection system at this time are that spectrometer passes through when measuring to extracellular vesica optical signal Wavelength X of the spectrometer to every extracellular vesica optical signal in a certain type expression albumen1,λ2...λk, obtain optical wavelength Data after, light frequency value ν corresponding to each optical signal is calculated according to formula λ ν=c=299792458 (m/s)1,ν2... νk, brought into the weighted sum model as independent variable X to a certain albuminoid weighting expression after the completion of calculating Intensity YνIt is calculated, the weighting expression intensity Y of a certain albuminoid is obtained with thisν。
At this point, the albumen of the extracellular vesica weights expression intensity YνAre as follows:
Yν=β0+β1ν1+β2ν2+...+βkνk+ε (20)
After assuming to above-mentioned formula (20), expectation is taken to both sides, can be obtained:
E(Yν|ν1,ν2...νk)=β0+β1ν1+β2ν2+...+βkνk (21)
After the completion of taking expectation to the formula (20), regression parameter β is provided according to luminous intensity ν0, β1, β2..., βkAccordingly Estimated valueAvailable albumen weights expression intensity Y at this timeνCorresponding estimated value:
Parameter Estimation is obtained using least square at this time:
It is right respectively in the formula (23)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, is obtained:
Equation group in above-mentioned formula (24) is solved, regression parameter β can be obtained0, β1, β2..., βkEstimated valueExpression intensity Y is weighted with albumenν。
When using light frequency ν to be calculated as optical parameter X, the light map measured can be carried out to accurately number and turned It changes, the extracellular vesica expression intensity Y acquiredνIt is worth accuracy highest.
As preferably another embodiment, in the present embodiment, using extracellular vesica concentration of specimens H rather than brightness L, light Intensity C or light frequency ν are measured, and the collector selected in the detection system at this time is monochromator, wherein utilizing the list Color device measures the principle of absorbance as shown in figure 3, the monochromator includes light source 5, monochromator 6, adjusting hole 7, glass tube 8, light Quick resistance 9, amplifier 10 and cutout screen 11；When light source 5 issues the light of specified luminous intensity and exposes to the monochromator 6, The light of optical signal can be dispersed into the monochromatic light of different wave length by monochromator 6；The adjusting hole 7 is adjusted at this time, is made The monochromatic light of 450nm wavelength passes through and by the monochromatic photo-electric switch of other wavelength；Equipped with to be detected extracellular in the glass tube 8 Vesica sample, when the monochromatic light of the 450nm wavelength passes through glass tube 8, extracellular vesica sample can absorb 450nm wavelength list The part luminous intensity of coloured light；Monochromatic light after being absorbed can expose in photo resistance 9, and luminous intensity is converted into resistance；Institute It states photo resistance 9 to amplify by the amplifier 10, be transported on cutout screen 11, monochromatic luminous intensity is shown after absorbing Show on the screen.Luminous intensity after being absorbed, and combine and absorb preceding luminous intensity, the extinction of extracellular vesica sample can be calculated Spend A.
Before being measured to extracellular vesica optical signal, by a series of protein markers item for measuring known concentrations Absorbance under part under 450nm wavelength obtains the specific function relationship between concentration and absorbance as reference, at this point, logical It crosses the monochromator and absorbance A of the optical signal at 450nm wavelength is measured to every extracellular vesica of unknown concentration respectively1, A2...Ak, after obtaining the data of optical signal absorbance, according to the functional relation calculate each example absorbance corresponding to concentration H1,H2...Hk, brought into the weighted sum model as independent variable X to a certain albuminoid weighting table after the completion of calculating Up to intensity YHIt is calculated, the weighting expression intensity Y of a certain albuminoid is obtained with thisH。
At this point, the albumen of the extracellular vesica weights expression intensity YHAre as follows:
YH=β0+β1H1+β2H2+...+βkHk+ε (25)
After assuming to above-mentioned formula (25), expectation is taken to both sides, can be obtained:
E(YH|H1,H2...Hk)=β0+β1H1+β2H2+...+βkHk (26)
After the completion of taking expectation to the formula (25), regression parameter β is provided according to luminous intensity ν0, β1, β2..., βkAccordingly Estimated valueAvailable albumen weights expression intensity Y at this timeνCorresponding estimated value:
Parameter Estimation is obtained using least square at this time:
It is right respectively in the formula (28)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, is obtained:
Equation group in above-mentioned formula (29) is solved, regression parameter β can be obtained0, β1, β2..., βkEstimated valueExpression intensity Y is weighted with albumenH。
When using extracellular vesica concentration of specimens H to be calculated as optical parameter X, the light map measured can be carried out Accurately number conversion, the extracellular vesica weighting expression intensity Y acquiredHAccuracy highest.
10 patient with breast cancers are chosen below, are first passed through common detection methods and patient are detected to obtain each patient's body The practical weighting expression intensity Y of interior each expression albumen0, and respectively using above-mentioned four kinds of measurement methods respectively in each patient table Weighting expression intensity up to albumen measures, and obtains expression protein measurement intensity YL,YC,Yν,YH, found out by following formula Each expression protein measurement intensity YL,YC,Yν,YHWith expression albumen actual strength Y0Deviation з, to judge above-mentioned four kinds of methods Accuracy in detection:
Wherein, in the extracellular vesica marker of the present embodiment detection, tri- kinds of CD9, CD63 and CD81 extracellular vesicas are general All over having more highly expressed albumen, so selecting above-mentioned three kinds of expression albumen quasi- with the detection to above-mentioned four kinds of methods when detecting Exactness compares, and comparing result is as shown in table 1:
Table 1
It can be concluded that, relative to other two methods, made using the extracellular vesica concentration of specimens H of luminous intensity C according to table 1 For independent variable X to vesicle protein weight expression intensity Y is detected and is calculated outside Patient cells when, the deviation phase that obtains To higher, measurement precision is low；And brightness L and light frequency ν is being selected to add as independent variable X to vesicle protein outside Patient cells When power weighting expression intensity Y is detected and calculated, the deviation obtained is significantly lower than luminous intensity deviation зC, so, relatively In luminous intensity C and extracellular vesica concentration of specimens H, the present embodiment can select a kind of parameter conduct from brightness L and light frequency ν Independent variable X in weighted sum model.
However, the value data used is very huge when using light frequency ν to be calculated as the independent variable X, this Sample can consume a large amount of time before measuring albumen weight expression intensity Y, while after the completion of data preparation, using adding It is equally huge due to arranging the light frequency ν numerical value obtained when power summation model carries out operation to the light frequency data sorted out, because This, which is also required to a large amount of operation just, can obtain the extracellular vesicle protein weight expression intensity Y, and whole process can expend A large amount of time and operation, therefore, in the similar situation of deviation, the present embodiment is selected relatively simple in data processing Just independent variable X of the brightness L as weighted sum model.
Specifically, including: to the weighted sum method of extracellular vesica expression protein abundance in this detection system
Step a: the extracellular vesica expression total abundance of albumen is set as dependent variable M, the optical parameter of certain marker is set as The optical parameter measured is then set as according to the expression protein order of detection: D by independent variable D respectively1,D2,...,Dk；
Step b: it since abundance of the variety classes expression albumen between different patients is all different, needs according to not of the same race Corresponding weight coefficient α is arranged in the expression albumen of class1,α2,...αk, then the extracellular vesica expression total abundance M of albumen can lead to Following formula is crossed to acquire:
M=α1D1+α2D2+...+αkDk (6)
Step c: determining the total quantity N of measurement cancer species, and determines various types of expression albumen in the cancer species number There is highly expressed number n in amount1,n2,...nk, then respectively expression albumen has highly expressed ratio in cancer are as follows:
Step d: it is averaged to opalescence parameter D is respectively expressed in the step aAnd to light Parameter D seeks variance:
Step f: it is determined according to the data obtained in the step c and step d to weight coefficient α:
After step g: weight coefficient α determines, it is total that extracellular vesica expression albumen can be acquired according to formula in step b Abundance:
Wherein, the optical parameter selects brightness L.
Embodiment 1
Studies have shown that the cell of the nearly all species outer vesica of energy secretory cell, according to source difference, extracellular vesica It can be divided into three classes: excretion body, microvesicle and apoptotic body, wherein excretion body contains complex lipids, RNA and protein.
Excretion body is rich in cholesterol and sphingomyelins, and its mRNA ingredient carried can enter in cytoplasm and be translated into Protein, not only mRNA, the microRNA that excretion body is shifted equally have bioactivity, can be with after entering target cell Targeting adjusts the level of mRNA in cell.
In conclusion the present embodiment selects excretion body to examine as detection sample to whether person under test suffers from breast cancer It surveys, comprising the following steps:
Step 1: obtaining blood sample of patient as sample liquids, and by excretion body therein and with the aptamer one of fluorescent marker With culture is incubated for, specifically bind the expression albumen of aptamer and excretion body surface face with fluorescent marker, it will be outer with this Secrete body surface markers glazing；
Step 2: the excretion body that completion is incubated in the step 1 is put into the sample bin chamber unit 2；
Step 3: after the completion of excretion body addition, the sample bin chamber unit 2 being added using heating unit 1 Heat, and the focus of laser is arranged on the sample liquids inside sample bin chamber unit 2, the excretion after heating, in sample liquids Know from experience and generate thermophoretic effect, and is mobile to low-temperature region；Meanwhile the sample liquids expanded by heating generates buoyancy, thus in institute It states and generates convection current in sample bin chamber unit 2, the heating region of sample bin chamber unit 2 is directed toward in the direction of convection current from surrounding, by surrounding Excretion body converge in the low temperature side of sample bin chamber unit 2；
Step 4: when the excretion body accumulation after the completion of, using 4 pairs of the signal acquisition unit build up excretion body it is glimmering Optical signal is acquired, and carries out carry out illumination, light to the accumulation excretion body using the signal amplification unit 3 after the completion of acquisition According to after the completion using the signal acquisition unit 4 to the fluorescence signal progress secondary acquisition for building up excretion body；
Step 5: after the completion of acquisition, by the fluorescence signal parametrization acquired before and after irradiating and subtracting each other, to obtain excretion The abundance of albumen is individually expressed in body；
Step 6: after the completion of detection, step 1- step 5 is repeated, using different types of aptamer to more in the excretion body Kind expression albumen is marked and detects, and obtains the abundance maps of a variety of expression albumen in the excretion body；
Step 7: turning luminosity each in protein graphical spectrum up to the table of comparisons in protein graphical spectrum in conjunction with excretion body surface in the step 6 It changes data into and finds out the weighting expression intensity Y that excretion body surface in sample to be tested reaches albumen without weighted sum using weighted sumL, table Up to protein abundance M and without weighting expression intensity ∑ L, and excretion body SUM expression figure is obtained according to three kinds of numerical value；
Step 8: the excretion body SUM in protein graphical spectrum and the step 7 is reached according to the excretion body surface obtained in the step 6 Expression figure determines the seriously ill degree of patient.
Wherein, aptamer described in the present embodiment selection be through in-vitro screening technology SELEX (index concentration Fas lignand system into Change) oligonucleotide fragment of energy specific binding protein or other small-molecule substances that filters out.
The basic thought of the SELEX technology is that iii vitro chemical synthesizes a single-stranded oligonucleotide library, with it and target substance It mixes, there are the compound of target substance and nucleic acid in mixed liquor, washes off the nucleic acid not in conjunction with target substance, separation and target substance knot The nucleic acid molecules of conjunction carry out PCR amplification by template of this nucleic acid molecules, carry out the screening process of next round.Pass through duplicate sieve Choosing and amplification, it is some not combined with target substance or there is low-affinity, the DNA of middle affinity or RNA molecule to be washed away with target substance, And aptamer (Adaptorprotein) has the DNA or RNA of high-affinity to divide from very big random library with target substance It separates out and, and purity increases with the progress of SELEX process, from P moles to n moles, finally occupy most of (> the 90% of library Left and right)., adaptation range big with storage capacity extensively, high-resolution, high-affinity, screening process relative ease, quickly, it is economical, Practicability and aptamer feature small in size.
Specifically, the fluorescent marker aptamer of the present embodiment is the single stranded DNA of 40 bases, the ball of string in sample liquids is straight Diameter is less than 5 nanometers, and excretion body diameter is 30-150 nanometers；
Specifically, the excretion body sample of the present embodiment is cell culture medium supernatant, the incubation conditions of sample are equal are as follows: 2 is small When incubation time, 0.1 every liter of micromole of aptamer concentrations, incubation temperature room temperature.
Specifically, heating unit described in the present embodiment is heated using the infrared laser of 1480nm wavelength for sample, function Rate is 200 milliwatts, and focus goes out about 200 microns of laser spot diameter.In the present embodiment, laser irradiates from top to bottom, sample bin The upper thermal conductive surface of chamber unit uses bright material, and such as glass, PMMA, PDMS, lower thermal conductive surface uses the better sapphire of thermal conductivity, Bottom surface, which forms low-temperature space, makes the swimming of excretion body heat converge at bottom surface.Upper thermal conductive surface with a thickness of 1mm, lower thermal conductive surface with a thickness of 1mm, The height of intermediate washer and sample bin chamber unit is 240mm.
When aptamer identification excretion body surface up to albumen and it is in combination when, the fluorescent marker on aptamer follows excretion body to be accumulated Sample bin chamber unit bottom section below laser spot, and generate enhancing fluorescence signal；When the unidentified excretion body surface of aptamer When up to albumen, free aptamer cannot be converged since size is small, and signal does not enhance.
In the present embodiment, luminophore Cy5 excitation/emission wavelength is 649/666nm, and fluorescence signal is connected with light microscope The CCD record connect.By CCD recording laser irradiation front and back fluorescence signal, by analysis laser irradiation before and after fluorescence signal, Obtain the abundance of excretion body surface expressed proteins.
The marker of the present embodiment detection includes excretion body marker: tri- kinds of excretion bodies of CD9, CD63 and CD81 generally have More highly expressed albumen；And cancer is related and protein marker in the expression of excretion body surface face: CEA, EGFR, EpCAM, HER2 And MUC-1.Excretion physical examination for above-mentioned marker is surveyed, its expression and distribution is analyzed.
It selects 15 patient with breast cancers and 19 healthy persons under test and it is detected respectively, it is suitable using 8 kinds of different nucleic acid Body detects the abundance of 16 kinds of excretion body expression albumen in blood serum sample.
However, since patient's excretion body surface face marker expression measurer has height heterogeneity, it is difficult to pass through single kind mark Will object effectively distinguishes patient with breast cancer and non-cancer patient.
But the expression quantity by will test marker be weighted and without weighted sum after, good diagnosis effect can be obtained Power.The fluorescent brightness of unlike signal object on the present embodiment excretion body is set as L according to measuring sequence1,L2,...LkAs independent variable It brings into the weighted sum model of a certain expression albumen intensity in calculating excretion body surface face, as shown in formula (10), at this point, to upper It states after formula (10) assumed, expectation is taken to both sides, can be obtained formula (11), and regression parameter β is provided according to fluorescent brightness L0, β1, β2..., βkCorresponding estimated valueExpression intensity Y is weighted by the available albumen of formula (12)LAccordingly Estimated value；Parameter Estimation is carried out to formula (12) using least square at this time, can show that formula (13) is right respectively in the formula (13)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, formula (14) are obtained, after solving to formula (14) Obtain regression parameter β0, β1, β2..., βkEstimated valueExpression intensity Y is weighted with albumenL.Find out parameter Estimated valueExpression intensity Y is weighted with albumenLAfterwards, according to the weighting of expression protein classes and each albuminoid Expression intensity YL。
The fluorescent brightness of unlike signal object on the present embodiment excretion body is set as L according to measuring sequence1,L2,...LkAs Independent variable is brought the calculating excretion body surface into and is reached in the weighted sum model of protein abundance, as shown in formula (6), meanwhile, use formula (7) to the fluorescent brightness L1,L2,...LkSeek variances sigma2, and will test cancer sum N and a certain type expression albumen described There is highly expressed number n in cancer species quantity1,n2,...nkWith the variances sigma2It brings into formula (8) and is respectively added with determining together Weight coefficient α1,α2,...αk, a certain type expression total abundance M of albumen of excretion body is found out using formula (6) after determination.
The fluorescent brightness of unlike signal object on the present embodiment excretion body is set as L according to measuring sequence1,L2,...LkAs Independent variable is brought into no weighted sum, and acquiring using fluorescent brightness L is a certain type expression albumen of the excretion body of parameter without weighting Expression intensity ∑ L=L1+L2+...+Lk。
In conjunction with the weighting expression intensity Y of each albuminoidL, the total abundance M of excretion body a certain type expression albumen and without weighting Expression intensity ∑ L makes the excretion body SUM expression figure of sample to be tested.
Figure is expressed to SUM and draws person under test's performance curve (ROC), area (Auc) and is assessed with this under calculated curve The diagnosis effect of the present embodiment detection system.
It is computed, in the patient of the present embodiment, the AUC using ROC Diagnosis of Breast cancer is 0.9521, thus, this Check system described in embodiment possesses good diagnosis effect.
Embodiment 2
The present embodiment selects excretion body to detect as detection sample to whether person under test suffers from breast cancer, including following Step:
Step 1: obtaining blood sample of patient as sample liquids, and by excretion body therein and the antibody with luminous catalytic materials It is incubated for culture together, by antibody label on excretion body；
Step 2: the excretion body sample after incubation being put into the sample bin chamber unit 2, and luminous substrate is added, makes to shine Luminous catalytic materials on substrate and antibody are catalyzed and reach excited state, and discharge luminous energy so that outer during being converted into ground state Secrete body surface markers optical signal；
Step 3: the sample bin chamber unit 2 being heated using heating unit 1, and the focus of laser is arranged in sample On sample liquids inside product warehouse unit 2, after heating, excretion in sample liquids, which is known from experience, generates thermophoretic effect, and to low temperature Region is mobile；Meanwhile the sample liquids expanded by heating generates buoyancy, so that convection current is generated in the sample bin chamber unit 2, The heating region of sample bin chamber unit 2 is directed toward in the direction of convection current from surrounding, and the excretion body of surrounding is converged in sample bin chamber unit 2 Low temperature side；
Step 4: obtaining the optical signal amplified after excretion body is built up using the signal processing unit 4, pass through analysis light letter Number, show that excretion body individually expresses the abundance figure of albumen；
Step 5: after the completion of detection, step 1- step 4 is repeated, using different types of antibody to more in the excretion body Kind expression albumen is marked and detects, and obtains the abundance maps of a variety of expression albumen in the excretion body；
Step 6: turning luminosity each in protein graphical spectrum up to the table of comparisons in protein graphical spectrum in conjunction with excretion body surface in the step 5 It changes data into and finds out the weighting expression intensity Y that excretion body surface in sample to be tested reaches albumen without weighted sum using weighted sumL, table Up to protein abundance M and without weighting expression intensity ∑ L, and excretion body SUM expression figure is obtained according to three kinds of numerical value；
Step 7: the excretion body SUM in protein graphical spectrum and the step 6 is reached according to the excretion body surface obtained in the step 5 Expression figure decisions making to the seriously ill degree of patient.
Chemiluminescence immune assay includes two parts, i.e. immune response system and chemiluminescence analysis system.Chemistry hair Light analysis system is the oxidation using catalysis and oxidant of the chemiluminescence catalytic materials through catalyst, is formed in an excitation state Mesosome when this excitation state intermediate returns to stable ground state, while launching photon (hM), utilizes luminous signal measuring instrument Device measures quantum yield of luminscence.Immune response system be luminous catalytic materials are marked directly on antigen or antibody, or enzyme effect in Luminous substrate.
To current thermophoresis system, the present embodiment selects on excretion body marker to be detected as antigen, the effect of antibody by Aptamer realizes that enzyme is then linked on aptamer, and luminous substrate is added before detection to sample.In conclusion this Luminous catalytic materials on antibody described in embodiment select luminol, and luminous substrate selects hydrogen peroxide.
Specifically, the selection of each component material and the heating are single in the incubation conditions of the present embodiment sample, system The heating parameters of member 1 and direction are identical as the condition in the embodiment 1.
The marker of the present embodiment detection includes extracellular vesica marker: tri- kinds of excretion bodies of CD9, CD63 and CD81 are universal With more highly expressed albumen；And cancer is related and protein marker in the expression of excretion body surface face: CEA, EGFR, EpCAM, HER2 and MUC-1.Excretion physical examination for above-mentioned marker is surveyed, its expression and distribution is analyzed.
It selects 15 patient with breast cancers and 19 healthy persons under test and it is detected respectively, it is suitable using 8 kinds of different nucleic acid Body detects the abundance of 16 kinds of vesica expression albumen extracellular in blood serum sample.Testing result is as shown in Figure 4.
As can be seen from FIG. 4, the extracellular vesica of all blood serum of patients with human breast carcinoma contains CD9, CD63, CD81 protein expression, And cancer Research of predicting markers CEA, EGFR, EpCAM, HER2 and MUC-1 and extracellular vesica marker CD9, CD63, CD81 albumen Expression between different patients also has difference.
However, due to outside Patient cells vesica surface marker expression quantity have height heterogeneity, it is difficult to pass through single kind Class marker effectively distinguishes patient with breast cancer and non-cancer patient.
But it after the expression quantity by will test marker is carried out without weighted sum (SUM) and having weighted sum, can obtain Obtain good diagnosis effect.
The brightness of unlike signal object on the present embodiment excretion body is set as L according to measuring sequence1,L2,...LkAdded Power sums and without weighted sum, and combines the weighting expression intensity Y of each albuminoidL, excretion body a certain type expression albumen it is total Abundance M and the excretion body SUM expression figure that sample to be tested is made without weighting expression intensity ∑ L.Wherein, the weighting of the present embodiment is asked It is identical as the calculation method in the embodiment 1 with calculation method and without weighted sum calculation method.
Person under test's performance curve is drawn to SUM and the total abundance M of extracellular vesica single kind expression albumen (ROC), area (Auc) and the diagnosis effect of the present embodiment detection system is assessed with this under calculated curve.
It is computed, in 34 persons under test of the present embodiment, the AUC using ROC Diagnosis of Breast cancer is 0.9631, thus may be used , check system described in the present embodiment possesses good diagnosis effect.
So far, it has been combined preferred embodiment shown in the drawings and describes technical solution of the present invention, still, this field Technical staff is it is easily understood that protection scope of the present invention is expressly not limited to these specific embodiments.Without departing from this Under the premise of the principle of invention, those skilled in the art can make equivalent change or replacement to the relevant technologies feature, these Technical solution after change or replacement will fall within the scope of protection of the present invention.
The above description is only a preferred embodiment of the present invention, is not intended to restrict the invention；For those skilled in the art For member, the invention may be variously modified and varied.All within the spirits and principles of the present invention, it is made it is any modification, Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (10)

1. a kind of breast cancer detection system based on the extracellular vesica detection of thermophoresis characterized by comprising
To the heating unit heated to the extracellular vesica in person under test's blood；
It is arranged in the heating unit side, to load the sample bin chamber unit of extracellular vesica, the sample bin chamber unit There is highly expressed expression albumen specific binding can occur with aptamer or antibody breast cancer in the outer vesica of inner cell and marks Optical signal generates thermophoretic effect after the heating unit is to sample bin chamber unit heating in the sample bin chamber unit And convection current, extracellular vesica is converged in into the lower side of temperature in the sample bin chamber unit；
The side of the sample bin chamber unit far from the heating unit is set, and the signal to amplify with reflected light signal is put Optical signal can be reflexed to designated position by big unit, the signal amplification unit；
It is arranged in sample bin chamber unit side, to the signal processing list that amplified optical signal is acquired and is calculated Member, the signal processing unit obtain at least one optical signal parameter, and by quantization optical parameter and using no weighting And/or there is weighted model to determine corresponding optical signal parameter, have in the acquisition extracellular vesica of singleton to breast cancer highly expressed Express the expression intensity of albumen.
2. the breast cancer detection system according to claim 1 based on the extracellular vesica detection of thermophoresis, which is characterized in that institute State chemiluminescent process are as follows: the aptamer for having the catalytic materials that shine or antibody are incubated for together with extracellular vesica first, pass through spy Luminous catalysis substance markers on extracellular vesicle surface, and are added to it luminous substrate by anisotropic combination, pass through luminous catalytic materials Catalytic luminescence substrate and reach excited state, and during being converted into ground state discharge luminous energy so that extracellular vesicle surface Mark optical signal.
3. the breast cancer detection system according to claim 1 or 2 based on the extracellular vesica detection of thermophoresis, feature exist In the weighted sum method for calculating extracellular vesica expression albumen intensity in the signal processing unit includes:
Step a: being set as dependent variable Y for the weighting expression intensity of albumen, the extracellular vesica mark that signal processing unit is measured Object light parameter is set as independent variable X, then the optical parameter of unlike signal object is set as according to measuring sequence on extracellular vesica: X1, X2..., Xk；
Step b: it since weighting expression intensity Y and optical parameter X is in a linear relationship, is calculated as follows at this time:
Y=β0+β1X1+β2X2+...+βkXk+ε (1)
Wherein, β0, β1, β2..., βkFor regression parameter, ε is stochastic error；
Step c: in the step b formula (1) and optical parameter X make basic assumption to guarantee be weighted summation to data When parameter Estimation, the validity of statistical check and Estimating Confidence Interval；
Step d: when the formula (1) and optical parameter X meets the hypothesis, expectation is taken to the formula (1) both sides, is obtained:
E(Y|X1,X2...Xk)=β0+β1X1+β2X2+...+βkXk (2)
Wherein, E (Y | X1, X2..., Xk) indicate in given optical parameter XiUnder conditions of albumen weight expression intensity Y condition Mean value；
Step e: after the completion of taking expectation to the formula (1), regression parameter β is provided according to optical parameter X0, β1, β2..., βkAccordingly Estimated valueThe corresponding estimated value of albumen weight expression intensity Y is obtained at this time:
Above-mentioned formula (3) be E (Y | X1, X2..., Xk) point estimate；
Step f: when the formula (1) and optical parameter X meet the hypothesis in the step c, parameter is obtained by least square and is estimated Meter, it is assumed that
It is right respectively in the formula (4)Partial derivative is sought, and the partial derivative is enabled to be equal to 0, is obtained:
Equation group in above-mentioned formula (5) is solved, regression parameter β is obtained0, β1, β2..., βkEstimated valueWith albumen weight expression intensity Y.
4. the breast cancer detection system according to claim 3 based on the extracellular vesica detection of thermophoresis, which is characterized in that institute Stating the optical parameter in step a is one of brightness L, luminous intensity C, absorbance A or light frequency λ or a variety of.
5. the breast cancer detection system according to claim 3 based on the extracellular vesica detection of thermophoresis, which is characterized in that institute Stating the basic assumption made in step c to the formula (1) and optical parameter X includes:
It is assumed that c1: the probability distribution of stochastic error ε has zero-mean, E (ε)=0；
It is assumed that c2: the probability distribution of stochastic error ε has a same variance for different argument list present worths, the variance of ε not with XijVariation and change, D (ε)=σ2；
It is assumed that c3: auto-correlation, cov (ε is not present in stochastic error εi, εj)=0；
It is assumed that c4: εiWith any explanatory variable XiIt is uncorrelated, cov (εi, Xi)=0；
It is assumed that c5: perfect collinearity being not present between independent variable X；
Wherein, above-mentioned hypothesis c1-c4 is identical as the hypothesis of simple regression analysis, and the hypothesis c5 is to be directed to explanatory variable.
6. the breast cancer detection system according to claim 1 or 2 based on the extracellular vesica detection of thermophoresis, feature exist In the weighted sum method for calculating the extracellular vesica expression total abundance of albumen in the signal processing unit includes:
Step a: the extracellular vesica expression total abundance of albumen is set as dependent variable M, the optical parameter of extracellular vesica marker is set as The optical parameter measured is then set as according to the expression protein order of detection by independent variable D respectively:
D1,D2,...,Dk；
Step b:, need to be according to different types of expression since abundance of the variety classes expression albumen between different patients is all different Corresponding weight coefficient α is arranged in albumen1,α2,...αk, then the extracellular vesica expression total abundance M of albumen can be asked by following formula :
M=α1D1+α2D2+...+αkDk (6)
Step c: determining the total quantity N of measurement cancer species, and determines various types of expression albumen in the cancer species quantity With highly expressed number n1,n2,...nk, then respectively expression albumen has highly expressed ratio in cancer are as follows:
Step d: it is averaged to opalescence parameter D is respectively expressed in the step aAnd to optical parameter D Seek variance:
Step f: it is determined according to the data obtained in the step c and step d to weight coefficient α:
After step g: weight coefficient α determines, extracellular vesica is acquired according to formula in step b and expresses the total abundance of albumen:
7. the breast cancer detection system according to claim 1 based on the extracellular vesica detection of thermophoresis, which is characterized in that institute It states sample bin chamber unit to be arranged in the heating unit side, equipped with sample liquids to fill inside the sample bin chamber unit Carry the extracellular vesica and aptamer or antibody, comprising:
Setting is in the heating unit side and is transparent material, to absorb the first thermal conductive surface of the heating unit heat；
It is arranged below first thermal conductive surface and is transparent material, second to absorb the heating unit heat is thermally conductive Face, wherein the thermal conductivity for being more thermally conductive than the first thermal conductive surface of the second thermal conductive surface；
It is arranged between first thermal conductive surface and the second thermal conductive surface and offers through-hole at center, loads the sample liquid The gasket of body.
8. the breast cancer detection system according to claim 1 based on the extracellular vesica detection of thermophoresis, which is characterized in that institute It states signal amplification unit and the side of the sample bin chamber unit far from the heating unit is set, to vesica outside magnocell The optical signal on surface, comprising:
The side of second thermal conductive surface far from the heating unit is set, to observe the object lens of optical signal；
Side of the object lens far from the heating unit and in a certain angle with the object lens is set, to reflect signal Acquisition reflective mirror；
Side of the object lens far from the heating unit and in a certain angle with the object lens is set, to reflection source Amplify reflective mirror；
Setting is in the amplification reflective mirror side, to provide the observation light source of amplification light source for signal.
9. a kind of breast cancer detection method based on the extracellular vesica detection of thermophoresis characterized by comprising
Blood sample of patient is obtained as sample liquids, and by extracellular vesica therein together with aptamer or antibody with signal It is incubated for, there is highly expressed expression albumen to specifically bind breast cancer with extracellular vesicle surface by aptamer or antibody, Extracellular vesicle surface is marked upper optical signal；
Extracellular vesica after incubation is put into sample bin chamber unit, and sample bin chamber unit is heated with generate thermophoretic effect and Extracellular vesica is converged in the low temperature side in the sample bin chamber unit by convection current, with the light letter on vesica outside magnocell Number, corresponding specific single kind numerical value is converted optical signals by being calculated at least one optical signal parameter；
It after the completion of detection, repeats the above steps, uses a variety of pairs of creams in vesica outside different types of aptamer or antibody on cell Gland cancer has highly expressed expression albumen to be marked and detect respectively, obtains the numerical value of a variety of expression albumen in extracellular vesica Group；
Bring the above-mentioned correspondence numerical value group found out to different optical parameters into weighted model and/or without weighted model to corresponding optical parameter Calculated, obtain extracellular vesicle protein weighting expression intensity and/or without weighting expression intensity and expression albumen it is always rich Whether degree by obtaining the SUM expression figure of extracellular vesica in conjunction with three kinds of numerical value, and suffers from cream according to SUM expression figure person under test Gland cancer determines.
10. one kind utilizes chemiluminescent breast cancer detection method based on the extracellular vesica detection of thermophoresis, which is characterized in that packet It includes:
Blood sample of patient is obtained as sample liquids, and by extracellular vesica therein and aptamer or antibody with luminous catalytic materials It is incubated for culture together, has highly expressed expression albumen to carry out breast cancer by aptamer or antibody and extracellular vesicle surface special Property combine, by extracellular vesicle surface label on shine catalytic materials；
Extracellular vesica after incubation is put into sample storehouse chamber unit, and to luminous substrate is added in sample storehouse chamber unit, makes it With extracellular vesicle surface shine catalytic materials catalysis and reach excited state, and during being converted into ground state discharge luminous energy so that Extracellular vesicle surface marks optical signal；
Sample storehouse chamber unit is heated to generate thermophoretic effect and convection current, extracellular vesica is converged in into the sample storehouse chamber unit Interior low temperature side, with the optical signal on vesica outside magnocell；After amplification, optical signal is acquired using signal processing unit And analysis, corresponding specific single kind numerical value is converted optical signals by being calculated different optical parameters；
It after the completion of detection, repeats the above steps, uses a variety of expression in vesica outside different types of aptamer or antibody on cell Albumen is marked and detects respectively, obtains the numerical value group of a variety of expression albumen in extracellular vesica；
Bring the above-mentioned correspondence numerical value group found out to different optical parameters into weighted model and/or without weighted model to corresponding optical parameter Calculated, obtain extracellular vesicle protein weighting expression intensity and/or without weighting expression intensity and expression albumen it is always rich Whether degree by obtaining the SUM expression figure of extracellular vesica in conjunction with three kinds of numerical value, and suffers from cream according to SUM expression figure person under test Gland cancer determines.
CN201811321129.7A 2018-11-07 2018-11-07 Breast cancer detection system and method based on the extracellular vesica detection of thermophoresis Pending CN109374890A (en)

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