CN109387631A - Prostate cancer treatment Scheme Choice evaluation method and system based on thermophoresis detection - Google Patents

Prostate cancer treatment Scheme Choice evaluation method and system based on thermophoresis detection Download PDF

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CN109387631A
CN109387631A CN201811321166.8A CN201811321166A CN109387631A CN 109387631 A CN109387631 A CN 109387631A CN 201811321166 A CN201811321166 A CN 201811321166A CN 109387631 A CN109387631 A CN 109387631A
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expression
extracellular vesica
albumen
extracellular
parameter
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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
    • G01N33/57434—Specifically defined cancers of prostate
    • 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 prostate cancer treatment Scheme Choice evaluation method and system based on thermophoresis detection, 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, corresponding optical parameter is calculated by quantization optical parameter and using weighted model and/or without weighted model, obtains single kind protein expression intensity.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, corresponding optical parameter is calculated by quantization optical parameter and by using weighted model and/or without weighted model, it can fast and accurately show that the expression albumen intensity of extracellular vesica, detection accuracy are high.

Description

Prostate cancer treatment Scheme Choice evaluation method and system based on thermophoresis detection
Technical field
The present invention relates to cancer diagnosis technology field more particularly to a kind of prostate cancer treatment sides based on thermophoresis detection Case selects evaluation method and system.
Background technique
Prostate cancer refers to the epithelial malignancy occurred in prostate.Prostate cancer is asymptomatic in usual earlier, with The development of tumour, symptom caused by prostate cancer can be summarized as: pressure symptom and metastasis symptom two major classes；Wherein pressure symptom Show themselves in that the prostate gland urethra being gradually increased can cause progressive dysuria, show as urine line it is thin, range is short, Bradyuria, urine flow interrupt, after urine the sound of rain pattering, urination not to the utmost, trouble urinating, in addition, there are also frequent micturition, urgent urination, enuresis nocturna to increase, even The urinary incontinence；Oncothlipsis rectum can cause to have difficulty in passing one's motions or intestinal obstruction, can also oppress vas deferens and cause absence of ejaculation, pressuring nerve Cause perineum pain, and can be radiated to sciatic nerve.Metastasis symptom shows themselves in that prostate cancer can invade bladder, seminal vesicle, blood vessel Nerve tract causes blood urine, blood sperm, impotence；Lymph node transfers prognosi can cause double lower limb oedema.Prostate cancer often easily occurs bone and turns It moves, causes ostalgia or pathologic fracture, paraplegia；Prostate cancer can also invade marrow and cause anaemia or whole blood as reducing.
In the prior art for there are mainly three types of carcinoma of prostate detections: prostate puncture and biopsy, digital rectal examination and preceding The test of column gland specific antigen.
Wherein, prostate puncture and biopsy by, using puncturing to obtain prostata tissue, being to patient's prostatic Make a definite diagnosis the important means of prostate cancer；There are two kinds of Perineal approach, per rectum including puncture, can be used for detecting rectal touch (DRE) it touches scleroma, suspects that tumour, the low echo nodules of B ultrasound discovery prostate or MRI note abnormalities signal, suspect tumour, serum Prostate-specific antigen (PSA) > 4ng/ml, PSA is in 4.0~10.0ng/ml, the forefront such as f/tPSA exception or PSAD value are abnormal Gland related symptoms.Wherein, the Standard General for being included in prostate biopsy is blood biochemical detection PSA (prostate specific antigen) concentration Greater than 4 nanograms per milliliters, digital rectal examination is positive, prostate is ultrasonic or MRI is positive, however above-mentioned standard usually specificity is lower, Lead to there are a large amount of receiving to puncture patient's last diagnostic as feminine gender.Since prostate biopsy is intrusive diagnosis and costly, greatly Amount, which punctures negative findings, leads to unnecessary medical burden；It patient suffering and nurses after surgery, step is various；Even Patient can obtain infection, blood urine, hematochezia etc. and puncture side effect after sensing, but also PSA and rectal touch are in prostate cancer screening In value have a greatly reduced quality；And aspiration biopsy can be only applied to the detection to prostate, can not use, be applicable in for other organs Range is low.It is current to be badly in need of while there is high sensitivity to meet traditional prostate biopsy table with specificity and noninvasive technique differentiation Really have patients with prostate cancer in negative patient in traveller on a long journey group.
Digital rectal examination is the alternative program for checking prostate abnormalities, but it is this examine by can not assess entire body of gland and The limitation of tumor size to a certain extent.
Prostate-specific antigen (PSA) test is currently used for prostate cancer screening, however, this measurement is by many Disadvantage, the false positive results including high percentage.PSA also cannot be distinguished from cancer that is aggressive or more slowly growing in diagnosis, So as to cause over-treatment.Recently, it has been suggested that this program is abandoned, especially in elderly men.
China Patent Publication No.: it is anti-that CN1656231 discloses a kind of prostate-specific for analyzing non-composite form in sample Original is provided in the method for improving prostate cancer detection for test method existing for prostate cancer to be detected and determined.Test Prostate cancer can be detected in the significantly high man group of the ratio of Free PSA and t-PSA.Test can also be a small amount of total Prostate cancer is detected in the man group that PSA, i.e. range are 2-4ng/ml.An embodiment according to the present invention, test packet It includes the following steps: (a) determining the amount of t-PSA contained in the biological sample from patient, (b) determine in same sample and dissociate The amount of PSA, and the ratio of Free PSA and t-PSA is calculated, it (c) determines the amount of the pPSA in same sample, (d) determines same The amount of BPSA in sample, (e) determines the amount of inPSA in same sample, and (f) level and Free PSA based on t-PSA, leads to It crosses and is compared the amount of inPSA with the predetermined value established with the control sample of known cancer and benign disease diagnosis, make sample The amount of inPSA contained in product in patient prostate cancer there are related.It can be seen that the detection method is asked there are following Topic:
The first, the described detection method when being detected it needs to be determined that the amount of t-PSA contained in individual biological sample, with And Free PSA total amount and calculate the ratio of Free PSA and t-PSA, need to carry out a large amount of sample collection in the process, examine It is long to survey the period.
The second, the described detection method is also needed after the completion of aforementioned proportion calculates by the amount of inPSA and will use known cancer The predetermined value established with the control sample of benign disease diagnosis is compared, and makes the amount and individual of inPSA contained in sample The presence of middle prostate cancer is related；In the process, it while acquiring great amount of samples, also needs to carry out collected sample more Secondary calculating, and calculating process is cumbersome.
The extracellular vesica that third, the detection method use is only limitted to serum, blood or blood plasma, is not available above-mentioned three Substance other than the extracellular vesica of kind, there is limitation when detecting.
4th, when handling mass data, it may appear that deviation, and the detection method precision is caused to reduce
Due to lacking accurate feasible and easy-operating analysis method, so that expressing egg in the different extracellular vesicas of analysis at present It still faces the challenge on the minute differences of white matter, develops the analysis method of simple and reliable extracellular vesica expression albumen to tumour morning Phase diagnosis is extremely important.
Therefore, there is an urgent need to more specificity and more accurate prostate cancer detection methods, to help early diagnosing and selecting Select most suitable therapy intervention.Early detection can significantly reduce the death rate of prostate cancer, this makes improved diagnosis and more pre- Method becomes important target afterwards.
Summary of the invention
For this purpose, the present invention provide it is a kind of based on thermophoresis detection prostate cancer treatment Scheme Choice evaluation method and be System, to overcome, detection method can not be to the therapeutic effect of carcinoma of prostate progress essence since detection accuracy is low in the prior art Really the problem of detection.
On the one hand, the present invention provides a kind of prostate cancer treatment Scheme Choice based on thermophoresis extracellular vesica detection and comments Valence 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, by having highly expressed expression albumen to carry out prostate cancer in aptamer or antibody and extracellular vesica Specific binding, by the catalytic materials that shine on extracellular vesicle surface label；
Extracellular vesica after incubation is put into sample bin chamber unit, and to luminous substrate is added in sample bin chamber unit, Make to shine catalytic materials catalytic luminescence substrate and reach excited state, and discharges luminous energy so that thin during being converted into ground state Extracellular vesicle surface has optical signal；
Sample bin 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, by least one optical signal parameter calculated with Convert optical signals into corresponding specific single kind numerical value；
It after the completion of detection, repeats the above steps, using different types of aptamer or antibody for more in extracellular vesica Kind expression albumen is marked and detects respectively, obtains the numerical value groups 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 Total abundance, by the SUM expression figure for obtaining extracellular vesica in conjunction with three kinds of numerical value；
Difference patient identical to seriously ill degree is treated respectively using different therapeutic schemes, and repeats above-mentioned step Suddenly, the intensity that albumen is expressed in extracellular vesica SUM expression figure before and after treatment is carried out through different treatment methods by comparison patient Therapeutic scheme is evaluated, expression albumen intensity is lower after treatment, then therapeutic effect is better.
Further, the therapeutic scheme includes: radical prostaectomy, pelvic lymphadenectomy, radiotherapy and hero Hormone castration.
On the other hand, the present invention provides a kind of prostate cancer treatment Scheme Choice based on the extracellular vesica detection of thermophoresis Evaluation system, the extracellular vesica in the sample bin chamber unit mark optical signal and being in contact with aptamer or antibody, After the heating unit that sample bin chamber unit side is arranged in is to sample bin chamber unit heating, the sample warehouse list Thermophoretic effect and convection current are generated in first, and extracellular vesica is converged in into the lower side of temperature in the sample bin chamber unit；
The signal processing unit obtains at least one optical signal parameter, and passes through quantization optical parameter and use Without weighting and/or thering is weighted model to determine corresponding optical signal parameter, to obtain single kind protein expression intensity.
Further, the extracellular vesica and aptamer occur to specifically bind and mark the process of optical signal are as follows: first will Aptamer with signal is incubated for together with extracellular vesica, is connect aptamer with extracellular vesica by specifically binding, with This makes extracellular vesicle surface with 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(YX1,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 E (Y | X1, X2..., XkPoint 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 wavelength lambda 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 is to explanatory variable.
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 beche-de-mer without spike of extracellular vesica marker Amount 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, to load the cell external capsule Bubble 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, it is described thin to load The gasket of extracellular vesica.
It further, further include a signal amplification unit, the signal amplification unit is arranged in the sample bin chamber unit Side far from the heating unit, 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.
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 prostate cancer, and using detection unit to light in signal Physical parameter is detected and is handled, and can fast and accurately show that extracellular vesica has prostate cancer by analyzing optical parameter Highly expressed expression albumen weights expression intensity, and detection accuracy is high.Especially, the present invention is by using the poly- of a variety of physical parameters Product detection, and it is indirect detected by biological respinse, detection process is easier and easy to operate, and sample dosage is small, also, passes through The accumulation of physical parameter detects, and detects compared to biological respinse, and the expression of detection limit is easier to quantify, can be to prostate carninomatosis Change degree is clearly determined.In particular, the present invention has extracellular vesica to prostate cancer the inspection of highly expressed expression albumen Survey and the parametrization of luminosity, the calculation based on weighted model and/or without weighted sum model obtains, then according to standard Protein markers concentration and certain parameter of light canonical function relationship, to determine canceration degree.Such as, by luminous intensity C, The light characteristics such as the concentration of specimens under brightness L, light frequency λ, specific wavelength absorbance A detection physical quantity is determined.Also, It in detection process based on same antibody or aptamer, by the detection and calculating to a variety of physical parameters, is compared to each other, chooses most The expression of excellent 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, and optical signal is marked in extracellular vesicle surface with this, when detecting, the luminous catalytic materials are able to maintain the long period It shines.
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.
In particular, the present invention is by treating patient using different treatment methods, it is thin before and after treatment by detecting respectively The gene expression abundance figure that albumen is expressed in extracellular vesica, can be accurately to different treatment methods to preceding by comparison gene expression abundance figure The therapeutic effect of column gland cancer is evaluated.
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 extracellular vesica of its inside and to mobile at low temperature, meanwhile, temperature increases Extracellular vesica can generate thermal convection and extracellular vesica is made to accumulate in designated position afterwards, with the light letter of vesica outside this magnocell Number, in this way, can more accurately observe the specific number of extracellular vesica optical parameter when detecting to optical parameter Value, further improves the detection accuracy of the detection system.In particular, a variety of smooth objects can be acquired simultaneously by this kind of mode Manage parameter, 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.Cell in the sample bin chamber unit Outer vesica 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 System is not specifically limited in the selection of extracellular vesica, as long as the extracellular vesica can drive carefully under thermal convection effect Extracellular vesica is mobile and builds up.Further, the side that the extracellular vesica and aptamer or antibody pass through specific binding Formula links together, in this way, being linked on extracellular vesica of can consolidating of signal also can when extracellular vesica is built up It is enough more accurately to observe the optical parameter of extracellular vesica to come, further improve the detection accuracy of the detection system. The power that extracellular vesica is subject under thermophoretic effect is directly proportional to its diameter square, and unrelated with extracellular vesica quantity, therefore, It only needs a small amount of blood sample can be completed when detecting, 0.1 microlitre of sample dosage is only needed for extracellular vesica, and be not necessarily to Preposition processing is carried out to sample.
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 pair to expression albumen high in various cancers It is marked, and measures the abundance map of corresponding expression albumen with this and calculates weighting expression intensity, in this way, the detection system System can not only detect prostate cancer, while can also quickly and accurately be detected to other cancers, such as: lung cancer, Cancer of pancreas, colorectal cancer, gastric cancer, prostate cancer, head-neck carcinoma, cutaneum carcinoma, kidney, carcinoma of testis, thyroid cancer, bladder cancer, uterus Cancer, carcinoma of vagina, carcinoma of endometrium, oophoroma, cancer of the esophagus, carcinoma of mouth, salivary-gland carcinoma, laryngocarcinoma, peritoneal cancer, rhinocarcinoma, laryngocarcinoma, defeated ovum Pipe cancer, kidney mother cell cancer, lymph cancer, cholangiocarcinoma and swing sarcoma, synovial sarcoma, medulloblastoma, trophocyte's tumor, Glioma, spongioblastoma, cholesteatoma, chondrosarcoma, ependymoma, neurinoma, neuroma, rhabdomyosarcoma.
Detailed description of the invention
Fig. 1 is that the present invention is based on the knots of the prostate cancer treatment Scheme Choice evaluation system of the extracellular vesica detection of thermophoresis Composition；
Fig. 2 is that the present invention is based on the extracellular vesica detections of thermophoresis to utilize chemiluminescent prostate cancer treatment Scheme Choice The structure chart of evaluation system；
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 prostate cancer treatment Scheme Choice evaluation system detections of the extracellular vesica detection of thermophoresis The excretion body surface of patients with prostate cancer reaches protein abundance map；
Fig. 5 is that of the invention use utilizes chemiluminescent prostate cancer treatment scheme based on the extracellular vesica detection of thermophoresis The excretion body surface of evaluation system detection patients with prostate cancer is selected to reach protein abundance map；
Fig. 6 is that the present invention is based on the prostate cancer treatment Scheme Choice evaluation system detections of the extracellular vesica detection of thermophoresis Protein abundance comparison diagram is reached using the radical prostaectomy excretion body surface pretherapy and post-treatment to patients with prostate cancer.
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 prostate cancer treatment Scheme Choice evaluation system based on the extracellular vesica detection of thermophoresis, Refering to Figure 1, it is the structural schematic diagram of the prostate cancer detection system detected the present invention is based on the extracellular vesica of thermophoresis, The system of the present embodiment includes heating unit 1, sample bin chamber unit 2, signal amplification unit 3 and signal processing unit 4, wherein The top of the sample bin chamber unit 2 is arranged in the heating unit 1, to add to the sample in the sample bin chamber unit 2 Heat；The sample bin chamber unit 2 is provided with extracellular vesica, to load extracellular vesica and with the aptamer of fluorescent marker； The signal amplification unit 3 is arranged below the sample bin chamber unit 2, to amplify the fluorescence signal of the fluorescent marker, The setting of signal processing unit 4 is in 3 side of signal amplification unit, to acquire and record the amplified fluorescence letter Number, and one or more of the brightness to fluorescence signal, luminous intensity and optical wavelength parameter are carried out or are obtained, while being used and being added Power and the detection for carrying out cancerous lesion degree to the extracellular vesica of sample to be tested without weighted sum.
Specifically, in the prostate cancer treatment Scheme Choice evaluation system work based on the extracellular vesica detection of thermophoresis Before, extracellular vesica and aptamer with fluorescent marker are first put into sample bin chamber unit 2, pass through aptamer and extracellular vesica table A kind of pair of prostate cancer in face has highly expressed expression albumen to specifically bind, by fluorescent marker in extracellular vesica On.After the completion of label, the heating unit 1 starts to heat sample bin chamber unit 2, after sample bin chamber unit 2 is heated, inside Extracellular vesica start to generate thermophoretic effect and occur convection current, and by labeled extracellular vesicular aggregates in designated position； After the completion of aggregation, signal amplification unit 3 can emit comparison light source, 4 meeting of signal processing unit to the position of extracellular vesicular aggregates The relevant information for acquiring the extracellular vesica of aggregation, and analyzes it accordingly, by the brightness L of light, luminous intensity C, One or more of wavelength lambda parameter obtains, and takes weighted sum to carry out cancerous lesion degree without the mode of weighted sum and is sentenced It is disconnected.It will be appreciated by those skilled in the art that the present embodiment is controlled based on the carcinoma of prostate of the extracellular vesica detection of thermophoresis Treat Scheme Choice evaluation system cannot be only used for that extracellular vesica is assembled and detected, can also to extracellular vesica or other The micro-nano biomone of type is detected, as long as meeting the prostate cancer detection system based on the extracellular vesica detection of thermophoresis System can reach its specified working condition.
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 prostate cancer detects to person under test by first by fluorescence Label is connected with aptamer, then aptamer is incubated for together with the extracellular vesica of sample to be tested extracellular vesica is put on fluorescence mark Note, 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, it will be able to which the state of an illness of patient is quickly diagnosed.
System embodiment two
The embodiment of the present invention is to utilize chemiluminescent prostate cancer treatment scheme based on the extracellular vesica detection of thermophoresis Selection evaluation system please refers to shown in Fig. 2 as the preferred embodiment of the present invention, and it is thin to be based on thermophoresis for the embodiment of the present invention Structural schematic diagram of the extracellular vesica detection using chemiluminescent prostate cancer treatment Scheme Choice evaluation system, the present embodiment System include heating unit 1, sample bin chamber unit 2 and signal processing unit 4, the said units and one phase of above-described embodiment Together.
Unlike above-described embodiment one, signal of the invention uses chemiluminescent labeling method, is based in use Before the extracellular vesica detection of thermophoresis utilizes chemiluminescent prostate cancer treatment Scheme Choice evaluation system, first by cell external capsule Bubble sample to be tested is incubated for together with the antibody for being marked with luminous catalytic materials, and luminous catalytic materials can be enzyme, passes through antibody and cell Outer vesica expression protein-specific is combined extracellular vesica marker enzyme, after the completion of label, will be incubated for the sample completed and is put into institute State sample bin chamber unit 2, and to luminous substrate is added inside the sample bin chamber unit, enzymatic luminous substrate is simultaneously reached Excited state, and optical signal is issued when it is converted into ground state.
It is evaluated based on the extracellular vesica detection of thermophoresis using chemiluminescent prostate cancer treatment Scheme Choice when described When system starts, the heating unit 1 heats the sample bin chamber unit 2, the extracellular vesica for keeping it internal Generate thermophoretic effect and start it is mobile to the low one side of temperature, while the two sides temperature difference of the sample bin chamber unit make it is described Start to generate convection current inside sample bin chamber unit 2, and extracellular vesica is accumulated in into designated position, after the completion of accumulation, the letter Number acquisition unit 3 starts to be acquired the optical signal of extracellular vesica, and obtains the extracellular vesicle surface expression albumen Abundance map.Please continue to refer to shown in Fig. 2, signal processing unit 4 described in the embodiment of the present invention is arranged in the sample warehouse 2 lower section of unit, the extracellular vesica of the aggregation is observed and be acquired.
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 Prostate cancer, which has highly expressed expression albumen that rear extracellular vesicle surface has been marked, can maintain prolonged high light letter Number, therefore amplify without using the amplification reflective mirror 33 and observation light source 34 to optical signal can also be to extracellular for the present embodiment The optical signal of vesica 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 extracellular In the sample bin chamber unit 2 of vesica, so, just eliminate luminous catalytic materials in Excess antibody and luminous substrate catalysis and The phenomenon for reacting and shining together, so that the cancer detection system for utilizing chemical detection based on the extracellular vesica of thermophoresis When detecting to extracellular vesica, there is the cancer detection system advantage based on the extracellular vesica detection of thermophoresis On the basis of, 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 patients with prostate cancer are chosen below, are first passed through common detection methods and patient are detected to obtain each patient The practical weighting expression intensity Y of each expression albumen in vivo0, and respectively using above-mentioned four kinds of measurement methods respectively in each patient The weighting expression intensity of expression albumen measures, and obtains expression protein measurement intensity YL,YC,Yν,YH, asked by following formula Each expression protein measurement intensity Y outL,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 be detected as detection sample, comprising the following steps:
Step 1: extracting blood sample out of person under test body, excretion body therein is incubated for together with the aptamer with fluorescent marker Culture specifically binds aptamer and the expression albumen in excretion body surface face with fluorescent marker, with this by excretion body surface Face marks 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 extracellular vesica inside sample bin chamber unit 2, after heating, in extracellular vesica Excretion, which is known from experience, generates thermophoretic effect, and mobile to low-temperature region；Meanwhile the extracellular vesica expanded by heating generates buoyancy, from And 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, The excretion body of surrounding is converged 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 cell external capsule in protein graphical spectrum and the step 7 is reached according to the excretion body surface obtained in the step 6 Bubble SUM expression figure makes canceration judgement.
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 extracellular vesica 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 (on excretion body carry on albumen): CD9, CD63 and Tri- kinds of excretion bodies of CD81 generally have more highly expressed albumen；And cancer is related and protein markers in the expression of excretion body surface face Object: EpCAM, PTK7, HepG2, HER2, PSA, PSMA, CA125, EGFR, MUC-1, CD133, CD24, CEA and CD30.To upper The excretion physical examination for stating marker is surveyed, its expression and distribution is analyzed.
The present embodiment collects the patient for receiving prostate biopsy in March, 2014 in certain hospital's Urology Surgery in March, 2018. The standard of being included in includes:
(1) having prostate biopsy indication, (nanograms per milliliter of PSA > 4, digital rectal examination are positive, prostate is ultrasonic or MRI sun Property)；
(2) nanograms per milliliter of PSA < 20.
Exclusion criteria includes:
(1) bacillary acute prostatitis is diagnosed as before biopsy in 3 months；
(2) 5 instrument reductase inhibitors, anabolic steroids or antiandrogen drug are taken before biopsy in 12 months；
(3) being inscribed within 6 weeks before biopsy and having received digital rectal examination, prostate biopsy inspection or pathological diagnosis is prostate specific type Tumour (such as sarcoma, small cell carcinoma).
Totally 50 patients, which meet, is included in and exclusion criteria, including 15 patients with prostate cancer, 10 precancerous lesion patients with Prostatitis patient and 25 patients with normal prostate tissue.
Patients serum will be punctured to be incubated for jointly with aptamer, using 16 kinds of different aptamers to excretion in blood serum sample 16 kinds of expression albumen of body (CD9, CD63, CD81, PTK7, EpCAM, HepG2, HER2, PSA, CA125, PSMA, EGFR, MUC-1, CD133, CD24, CEA, CD30) abundance detected.The excretion body operating method and laser of use, sample warehouse list Member and microscope and CCD camera are all the same.Testing result as shown in figure 4, wherein No. 1-15 be patients with prostate cancer, No. 16-25 For prostate precancerous lesion and prostatitis patient, No. 26-50 is the patient with normal prostate tissue.
As can be seen from FIG. 4, all puncture patients serum's excretion bodies contain CD9, CD63, CD81 protein expression, and forefront The expression of adenocarcinoma patients is higher than the expression of precancerous lesion and prostatitis patient, the table of precancerous lesion patient and prostatitis patient Up to the expression for being higher than normal tissue patient, the conspicuousness of differential expression has statistical significance (P < 0.05).And cancer correlation mark Will object EpCAM, PTK7, HepG2, HER2, PSA, PSMA, CA125, EGFR, MUC-1, CD133, CD24, CEA, CD30 and excretion Expression of body marker CD9, CD63, CD81 albumen between different type patient also has difference, the expression of patients with prostate cancer Higher than the expression of precancerous lesion and prostatitis patient, the expression of precancerous lesion and prostatitis patient is higher than normal tissue patient Expression, the conspicuousness of differential expression has statistical significance (P < 0.05).
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 patients with prostate 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 for calculating a certain expression albumen intensity of extracellular vesicle surface, as shown in formula (10), at this point, After assuming to above-mentioned formula (10), 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 obtain formula (13) in the formula (13) respectively It is rightPartial derivative is sought, and the partial derivative is enabled to be equal to 0, obtains formula (14), after solving to formula (14), i.e., Regression parameter β can be obtained0, 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 into the weighted sum model for calculating extracellular vesica expression protein abundance, as shown in formula (6), meanwhile, make With formula (7) to the fluorescent brightness L1,L2,...LkSeek variances sigma2, and will test cancer sum N and a certain type expression albumen exist There is highly expressed number n in the cancer species quantity1,n2,...nkWith the variances sigma2It is brought into formula (8) together with determination Each weighting 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 50 puncture patients of the present embodiment, the AUC using ROC diagnosis of prostate cancer is 0.9521, by This can be obtained, and check system described in the present embodiment possesses good diagnosis effect.
Embodiment 2
The present embodiment selects excretion body to be detected as detection sample, comprising the following steps:
Step 1: extracting blood sample out of person under test body, excretion body therein is incubated together with the antibody with the catalytic materials that shine Culture is educated, 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 luminous energy is discharged during being converted into ground state, so that Excretion body has 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 extracellular vesica inside product warehouse unit 2, after heating, the excretion in extracellular vesica, which is known from experience, generates thermophoretic effect, and to Low-temperature region is mobile；Meanwhile the extracellular vesica expanded by heating generates buoyancy, to be produced in the sample bin chamber unit 2 Raw convection current, the direction of convection current are directed toward the heating region of sample bin chamber unit 2 from surrounding, the excretion body of surrounding are converged in sample bin The low temperature side of chamber unit 2；
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 Whether expression figure person under test decisions making with prostate cancer.
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 excretion body marker (on excretion body carry on albumen): CD9, CD63 and Tri- kinds of excretion bodies of CD81 generally have more highly expressed albumen；And cancer is related and protein markers in the expression of excretion body surface face Object: EpCAM, PTK7, HepG2, HER2, PSA, PSMA, CA125, EGFR, MUC-1, CD133, CD24, CEA and CD30.To upper The excretion physical examination for stating marker is surveyed, its expression and distribution is analyzed.
50 patients with prostate cancer are selected to be detected, using 16 kinds of different aptamers to 16 kinds of excretion body in blood serum sample The abundance of expression albumen is detected.Testing result is as shown in Figure 5.
As can be seen from FIG. 5, all puncture patients serum's excretion bodies contain CD9, CD63, CD81 protein expression, and cancer Research of predicting markers EpCAM, PTK7, HepG2, HER2, PSA, PSMA, CA125, EGFR, MUC-1, CD133, CD24, CEA, CD30 Also there is difference from expression of excretion body marker CD9, CD63, CD81 albumen between different patients.
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 patients with prostate 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 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 (ROC) is drawn to SUM and the total abundance M of a certain type expression albumen of excretion body, meter It calculates area under the curve (Auc) and assesses the diagnosis effect of the present embodiment detection system with this.
It is computed, in 50 puncture patients of the present embodiment, the AUC using ROC diagnosis of prostate cancer is 0.9631, by This can be obtained, and check system described in the present embodiment possesses good diagnosis effect.
Embodiment 3
The present embodiment will use the prostate cancer treatment scheme of the present invention based on the extracellular vesica detection of thermophoresis to select It selects and evaluation method is respectively evaluated different patients using the therapeutic effect after different therapeutic schemes, comprising the following steps:
Step 1: extracting blood sample out of person under test body, excretion body therein is incubated together with the antibody with the catalytic materials that shine Culture is educated, 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 luminous energy is discharged during being converted into ground state, so that Excretion body has 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 extracellular vesica inside product warehouse unit 2, after heating, the excretion in extracellular vesica, which is known from experience, generates thermophoretic effect, and to Low-temperature region is mobile；Meanwhile the extracellular vesica expanded by heating generates buoyancy, to be produced in the sample bin chamber unit 2 Raw convection current, the direction of convection current are directed toward the heating region of sample bin chamber unit 2 from surrounding, the excretion body of surrounding are converged in sample bin The low temperature side of chamber unit 2；
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 Whether expression figure person under test decisions making with prostate cancer.
Step 8: patient to be detected being treated, is accomplished 6 weeks after treatment, it is right that step 1- step 7 is repeated after the completion of mastery Person under test carries out secondary sample and detection, and compares abundance map and excretion body SUM table that pretherapy and post-treatment excretion body surface reaches albumen Up to figure, to observe therapeutic effect.
For current thermophoresis system, the present embodiment selects on excretion body marker to be detected as antigen, the effect of antibody It is realized by aptamer, enzyme is then linked on aptamer, and luminous substrate is added before detection to sample.
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.
Wherein, the marker of the present embodiment detection includes excretion body marker (albumen on carrying on excretion body): CD9, Tri- kinds of excretion bodies of CD63 and CD81 generally have more highly expressed albumen；And cancer is related and egg in the expression of excretion body surface face White marker: EpCAM, PTK7, HepG2, HER2, PSA, PSMA, CA125, EGFR, MUC-1, CD133, CD24, CEA and CD30.Excretion physical examination for above-mentioned marker is surveyed, and is analyzed its expression and distribution, is analyzed by data, and the above mark is covered in proposition One multiple-factor mathematical synthesis index of object obtains best accuracy and sensitivity in prostate cancer detection.
Specifically, the therapeutic scheme that the present embodiment is selected includes:
Radical prostaectomy: radical prostatectomy refers to excision prostate and surrounding seminal vesicle, ejaculatory duct, defeated A part of spermaduct, while watching pelvic lymph node and cleaning whether there is or not transfer is parallel.Operation is uniquely to can be radically cured prostate cancer Method, radical Retropubic Prostatectomy are more commonly used.
Pelvic lymphadenectomy (PLND): enlargement of lymph nodes, matter are hard, movable, clinical diagnosis is Cervical Lymph Node Metastasis (or suspicious transfer), original site cancerous swelling are controlled or estimate can control.Feasible radical-ability excision of cervical lymph nodes.Lymph node The value performed an operation is that can make a definite diagnosis by postoperative pathological section biopsy is any disease.But it in the treatment, drenches It is useless for fawning on disease surgery.
Radiotherapy (RT): by the radiation exposure tumour with various different-energies, to inhibit and kill cancer cell.
Androgen ablation therapies (ADT): 1. operation castrations include cutting off bilateral testes to patients with prostate cancer.2. radiotherapy is gone Gesture: it is noninvasive because exposure dose is low, therefore substantially, but treatment cycle is longer, and curative effect is thorough not as good as operation castration, at present clinically No longer conventional application.3. medical castration: gonadotropin releasing hormone analogues are by negative feedback under hypothalamus, inhibition Thalamus generates gonadotropin-releasing hormone (GRH).Medical castration is to reduce blood by drug under the premise of retaining testis or ovary Clear testosterone or estrogen level, achieve the purpose that castration.Medical castration has curative effect similar with operation castration, overcomes operation Castration and the shortcomings that radiotherapy castration, therefore safer, and reversible after being discontinued, the quality of life of patient is more preferable, be it is current it is clinical most Frequently with castration method.
Present case collects the patient for receiving radical prostaectomy in March, 2013 in certain hospital's Urology Surgery in March, 2017. The standard of being included in includes: not receive before radical correction to assist in the treatment of (radiotherapy or Androgen deprivation therapy), and the preoperative distal end that do not occur turns It moves.This exclusion criteria purpose is the influence for excluding radiotherapy chemotherapy to excretion body marker expression amount.Totally 40 patients, which meet, is included in Standard.Each patient is taking blood primary and is taking blood again after 6 weeks after surgery before puncture is made a definite diagnosis.
The preoperative serum with postoperative serum of patient is incubated for using aptamer in the present embodiment, and incubation method is identical. Using 16 kinds of different aptamers to 16 kinds of expression albumen of excretion body in blood serum sample (CD9, CD63, CD81, PTK7, EpCAM, HepG2, HER2, PSA, CA125, PSMA, EGFR, MUC-1, CD133, CD24, CEA, CD30) abundance detected.Using Excretion body operating method and laser, sample bin chamber unit and microscope and CCD camera it is all the same,
After testing with after comparison it can be concluded that, radical prostaectomy can reach best treatment to prostate patient and imitate The protein abundance map of fruit, preoperative blood sample and postoperative blood sample is as shown in Figure 6.
Person under test's performance curve (ROC) is drawn to SUM and the total abundance M of excretion body single kind expression albumen, meter It calculates area under the curve (Auc) and assesses the diagnosis effect of the present embodiment detection system with this.
It is computed, in 40 puncture patients of the present embodiment, the AUC using ROC diagnosis of prostate cancer is 0.9631, by This can be obtained, and 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 prostate cancer treatment Scheme Choice evaluation method based on the extracellular vesica detection of thermophoresis, which is characterized in that Include:
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, it is special by thering is highly expressed expression albumen to carry out prostate cancer in aptamer or antibody and extracellular vesica Property combine, by extracellular vesicle surface label on shine catalytic materials；
Extracellular vesica after incubation is put into sample bin chamber unit, and to luminous substrate is added in sample bin chamber unit, makes to send out Photocatalysis object catalytic luminescence substrate and reach excited state, and discharge luminous energy so that extracellular during being converted into ground state Vesicle surface has optical signal；
Sample bin chamber unit is heated to generate thermophoretic effect and convection current, extracellular vesica is converged in into the sample bin chamber unit Interior low temperature side, with the optical signal on vesica outside magnocell, by being calculated at least one optical signal parameter with by light Signal is converted into corresponding specific single kind numerical value；
It after the completion of detection, repeats the above steps, using different types of aptamer or antibody for a variety of tables in extracellular vesica It is marked and detects respectively up to albumen, obtain 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 Degree, by the SUM expression figure for obtaining extracellular vesica in conjunction with three kinds of numerical value；
Difference patient identical to seriously ill degree is treated respectively using different therapeutic schemes, and is repeated the above steps, and is led to Comparison patient is crossed to carry out expressing the intensity of albumen to treatment in extracellular vesica SUM expression figure before and after treatment through different treatment methods Scheme is evaluated, and expression albumen intensity is lower after treatment, then therapeutic effect is better.
2. the prostate cancer treatment Scheme Choice evaluation side according to claim 1 based on the extracellular vesica detection of thermophoresis Method, which is characterized in that the therapeutic scheme includes: radical prostaectomy, pelvic lymphadenectomy, radiotherapy and androgen Castration.
3. the prostate cancer treatment Scheme Choice according to claim 1 based on the extracellular vesica detection of thermophoresis evaluates system System, which is characterized in that the extracellular vesica in the sample bin chamber unit marks light and being in contact with aptamer or antibody Signal, after the heating unit that sample bin chamber unit side is arranged in is to sample bin chamber unit heating, the sample Thermophoretic effect and convection current are generated in warehouse unit, and extracellular vesica is converged in into temperature lower one in the sample bin chamber unit Side；
The signal processing unit obtains at least one optical signal parameter, and by quantization optical parameter using no weighting And/or there is weighted model to determine corresponding optical signal parameter, to obtain single kind protein expression intensity.
4. the prostate cancer treatment Scheme Choice according to claim 3 based on the extracellular vesica detection of thermophoresis evaluates system System, which is characterized in that the extracellular vesica and aptamer or antibody occur to specifically bind and mark the process of optical signal are as follows: first The aptamer for having signal or antibody are incubated for together with extracellular vesica, by specifically binding aptamer or antibody and cell Outer vesica connection, makes extracellular vesicle surface with signal with this.
5. the prostate cancer treatment Scheme Choice according to claim 3 or 4 based on the extracellular vesica detection of thermophoresis is commented Valence system, which is characterized in that the weighted sum method of extracellular vesica expression albumen intensity is calculated in the signal processing unit Include:
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.
6. the prostate cancer treatment Scheme Choice according to claim 5 based on the extracellular vesica detection of thermophoresis evaluates system System, which is characterized in that optical parameter in the step a be one of brightness L, luminous intensity C, absorbance A or light wavelength lambda or It is a variety of.
7. the prostate cancer treatment Scheme Choice according to claim 5 based on the extracellular vesica detection of thermophoresis evaluates system System, which is characterized in that 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 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 explanatory variable.
8. the prostate cancer treatment Scheme Choice according to claim 3 or 4 based on the extracellular vesica detection of thermophoresis is commented Valence system, which is characterized in that the weighted sum side of the extracellular vesica expression total abundance of albumen is calculated in the signal processing unit Method 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 For independent variable D, then the optical parameter measured is set as respectively according to the expression protein order of detection:
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:
。
9. the prostate cancer treatment Scheme Choice according to claim 3 based on the extracellular vesica detection of thermophoresis evaluates system System, which is characterized in that sample bin chamber unit setting in the heating unit side, to load 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, it is described extracellular to load The gasket of vesica.
10. the prostate cancer treatment Scheme Choice evaluation according to claim 3 based on the extracellular vesica detection of thermophoresis System, which is characterized in that further include a signal amplification unit, the signal amplification unit setting is remote in the sample bin chamber unit Side from the heating unit, the optical signal to vesicle surface outside magnocell, comprising:
It is arranged in second thermal conductive surface side, 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.
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