CN208156013U - Cancer detection system based on excretion body - Google Patents
Cancer detection system based on excretion body Download PDFInfo
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- CN208156013U CN208156013U CN201820486575.2U CN201820486575U CN208156013U CN 208156013 U CN208156013 U CN 208156013U CN 201820486575 U CN201820486575 U CN 201820486575U CN 208156013 U CN208156013 U CN 208156013U
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Abstract
The utility model relates to a kind of cancer detection systems based on excretion body, including heating unit, sample bin chamber unit, signal acquisition unit, the excretion body being mounted in the sample bin chamber unit in conjunction with aptamer corresponding to fluorescent marker, the aptamer and one of target protein in excretion body surface face are specifically bound, after the heating unit is to sample bin chamber unit heating, thermophoretic effect is generated in the sample bin chamber unit, the excretion body is converged in into the lower side of temperature in the sample bin chamber unit, with the fluorescence signal amplification that will be marked on the indoor excretion body of sample；The signal acquisition unit obtains amplified fluorescence signal, obtains the abundance of excretion body surface face target protein, by conjunction with the different target proteins in excretion body surface face and detecting various aptamers respectively, obtains excretion body surface face protein graphical spectrum, makes canceration detection.The utility model only needs micro-example that the detection of cancer can be completed, and has a wide range of application.
Description
Technical field
The utility model relates to cancer detection field more particularly to a kind of cancer inspections using thermophoretic effect based on excretion body
Examining system.
Background technique
Micro-nano particle is detected in the prior art, to measure particle size, shape, concentration, activity etc., in blood
The subjects such as, immunology, molecular biology have relatively broad application.In the prior art frequently with streaming particle detection method pair
Micro-nano particle is detected, and is the technology that the fine-grained particles in liquid are carried out with quantitative analysis and sorting one by one, is being examined
Coulter principle employed in survey refers to：When the particle to suspend in the electrolytic solution passes through aperture with electrolyte, replace same volume
Long-pending electrolyte causes two resistance between electrode inside and outside aperture that instantaneous variation occurs in the circuit of constant current design, generates current potential
Pulse, the size and number of pulse signal are directly proportional to the size and number of particle.Sample focusing is the pass of streaming detection of particulates
Key technology is all to realize to focus to sample liquid by external force in current detection.Focus again be divided by sheath fluid focus and
Focusing without sheath fluid.
Wherein, sheath fluid is focused as disclosed in Chinese patent 201210482142.7《Microfluid particle instrument and production method》
In, sample liquid is injected from sample liquid entrance respectively using the pressure of extraneous syringe pump, injects sheath fluid from sheath fluid entrance, then sample
Liquid and two-way sheath fluid flow to sheath stream assembling area simultaneously, and the fine-grained particles in sample liquid are sandwiched linear row by the aggtegation of sheath fluid
Column flow into detection zone and are detected.Two sheath streams and sample liquid require driving source in this method, using a motor control
The mode of three pipelines, not only equipment becomes very huge in this way, and cost also improves, more importantly due to being detected every time
When need replacing chip, then every time detection require again to be attached in three channels with motor, this junction
Sealing problem just influences whether the size of the pressure to three channels, causes focusing effect bad, and test result is just not smart enough
Really.
Wherein, without the focusing of sheath fluid, as disclosed in Chinese patent 201310283051.5《One kind being used for streaming particle instrument
Microfluidic chip structure and preparation method thereof》, use taper focusing structure, it is believed that it is with similar with traditional sheath liquid stream
The focusing effect of system, so that fine-grained particles individually flow into microchannel, microchannel, which fetters particle by channel, passes through it individually
Detection zone causes the inaccurate of testing result under the testing conditions of high concentration sample.
In the technical solution of above two detection micro-nano particle, on the one hand, potential pulse is generated by electrolysis, using electricity
Chemical method is separated and is detected to nano particle, and a fluid stream of the particle containing micro-nano is formed, and the amount of required sample is very big；Another party
Face, by the driving source of such as motor, while using the single channel of fixed structure, limit micro-nano particle flow direction and
Direction is built up, during applying outer active force and channel limits, external force is often directed to micro-nano particle in fluid
Force be uncontrollable.
Especially for micro-nano biomone, such as detection of excretion body, excretion body is film bubble secreted by cell, for thin
Intercellular exchange is increasingly becoming a kind of emerging non-intruding because it contains albumen relevant to mother cell and inhereditary material in recent years
The biomarker of formula diagnosing tumor.
It generallys use in the prior art：First, enzyme linked immunosorbent assay (ELISA), i.e. ELISA, refer to soluble antigen or resist
Body is integrated on the solid phase carriers such as polystyrene, the qualitative and quantitative inspection being immunoreacted using antigen-antibody binding specificity
Survey method, in measurement, by examining sample (measuring antibody or antigen therein) and enzyme-labelled antigen or antibody by different steps
It reacts with the antigen or antibody of surface of solid phase carriers；Make the antigen antibody complex formed on solid phase carrier with the method for washing
It is separated with other substances, finally combines the amount of tested substance in enzyme amount and the sample on solid phase carrier at certain ratio.Add
After the substrate for entering enzyme reaction, substrate becomes color products, the direct phase of amount of the amount and tested substance in sample of product by enzymatic
It closes, therefore can be according to the depth publication qualitative or quantitative analysis of color reaction.
Second, immunoblotting, i.e. Western Blot, the basic principle is that by specific antibody to gel electrophoresis
Processed cell or biological tissue samples are coloured；Specific protein is obtained by the position and color depth of analysis coloring
The information of expression in the cell or tissue analyzed.
On the one hand above two technical solution is separated and is built up to sample, special device and method need to be used；Separately
On the one hand, detection method is needed using a large amount of sample, and the process for often carrying out canceration detection for serum does not have adaptability.
This system has huge advantage, including sample dosage pole compared with traditional ELISA and Western blotting
Few (0.1 microlitre), it is easy to operate, it is not necessarily to specific apparatus, is purified without sample pre-treatments and excretion body, is common to aptamer and antibody
(the generally multi-purpose antibody of conventional method).This method is not limited to excretion body, and the micro-nanos biomone such as other extracellular vesica, cells is equal
It can.
The lower surface design of sample room is the fabulous transparent material of thermal conductivity, causes excretion body to the lower sample room of temperature
Lower surface migration.Simultaneously because sample liquids expanded by heating generates buoyancy to generate convection current in sample room, convection current can add
Speed and the convergence for reinforcing excretion body, to improve signal enlargement ratio.
But due to lacking accurate feasible and easy-operating analysis method, so that analyzing different excretion body surface proteins at present
It still faces the challenge on the minute differences of matter, develops the analysis method of simple and reliable excretion body surface protein to early diagnosis of tumor
It is extremely important.
Utility model content
The purpose of this utility model is to provide a kind of cancer detection systems based on excretion body, to overcome above-mentioned technology
Defect.
To achieve the above object, the utility model provides a kind of cancer detection system based on excretion body, including heating list
Member, sample bin chamber unit, signal acquisition unit, wherein the outside of the sample bin chamber unit is arranged in the heating unit, uses
With the sample heating into the sample bin chamber unit；
The excretion body being mounted in the sample bin chamber unit in conjunction with aptamer corresponding to fluorescent marker, the aptamer and outer
One of target protein specific binding for secreting body surface face, after the heating unit is to sample bin chamber unit heating, institute
It states and generates thermophoretic effect in sample bin chamber unit, the excretion body is converged in into temperature lower one in the sample bin chamber unit
The fluorescence signal marked on the indoor excretion body of sample is amplified in side；
The signal acquisition unit obtains amplified fluorescence signal, obtains the abundance of excretion body surface face target protein, passes through
Various aptamers in conjunction with the different target proteins in excretion body surface face and are detected respectively, excretion body surface face protein graphical spectrum is obtained, makes
Detection is sentenced in canceration.
Further, the sample bin chamber unit includes loading the excretion body sample and to provide generation thermophoretic effect
The closed sample room in space, the sample room include：It is led to close the sample room and build up the second of the micro-nano particle
Hot face.
Further, nearby temperature is lower than the temperature of the micro-nano particle fluid to second thermal conductive surface, described the
The temperature difference is generated between two thermal conductive surfaces and micro-nano particle fluid, to generate thermophoretic effect, drives micro-nano particle fixed to the second thermal conductive surface
To movement.
Further, the heating unit is laser, is irradiated to the sample bin chamber unit, and light beam passes sequentially through institute
Micro-nano particle fluid and the second thermal conductive surface are stated, to generate thermophoretic effect to the micro-nano particle solution.
Further, the sample room further includes：To close the first thermal conductive surface of the sample room, described second is thermally conductive
Face and the first thermal conductive surface can be such that light beam passes through.
Further, second thermal conductive surface is transparent material, is sapphire material；
First thermal conductive surface is any one of glass, polymethyl methacrylate, dimethyl silicone polymer, sapphire
Or appoint several combinations.
Further, first thermal conductive surface and the second thermal conductive surface are oppositely arranged, low near second thermal conductive surface
Warm direction forms the convection current that illuminated laser spot is directed toward from surrounding.
Further, the system also includes signal acquisition units, the micro-nano particle in the sample bin chamber unit
After accumulation, the indication information of amplified micro-nano particle is acquired.
Further, the excretion body can be replaced using extracellular vesica, cell.
The beneficial effects of the utility model are compared with prior art, and the utility model micro-nano detection system passes through to micro-
It receives the wherein direction of the sample bin chamber unit where particle and is heated, introduce thermophoretic effect and convection current, make sample warehouse list
Member generates the temperature difference, generates low temperature in the side far from heating unit, thermophoretic effect makes micro-nano particle migration in sample and is accumulated to
Sample bin chamber unit, to complete the accumulation of micro-nano particle；Simultaneously as sample liquids expanded by heating generates buoyancy in sample
Convection current is generated in warehouse unit, is directed toward sample bin chamber unit from surrounding in the direction of the low-temperature region of sample bin chamber unit, convection current
Heating region, further promote micro-nano particle accumulation.
Further, this system passes through the aptamer or antibody incubation of the sample to be tested containing excretion body and fluorescent marker
Excretion body is marked upper fluorescence in conjunction with the protein-specific of excretion body surface face by aptamer or antibody；The sample being incubated for is put into up and down
Transparent sample room, and be placed on fluorescence microscope objective table and be observed, infrared laser shines through sample room effect
It is by thermophoresis effect that excretion body in sample is highly enriched at the laser spot of sample room bottom with sample, so that excretion body
Fluorescence is highly enlarged, and the abundance of certain excretion body surface protein is detected by fluorescence power.
Further, this system heats sample room irradiation using laser, by being arranged in sample room opposite flank
The different transparent thermal conductive surface of heating conduction, generates the temperature difference between two thermal conductive surfaces, to generate thermophoretic effect, drives micro-nano particle
From the lower second thermal conductive surface displacement of the first thermal conductive surface low temperature.It heats, is not needed using other auxiliary especially with light beam
Equipment only needs sample room transparent thermal conductive surface setting up and down.Also, micro-nano particle is straight in thermophoretic effect lower stress and particle
Diameter square is directly proportional, and how much unrelated with micro-nano number of particles, therefore, only needs micro micro-nano particle that convergence and inspection can be completed
Survey, 0.1 microlitre of sample dosage only needed for excretion body, it is easy to operate, be not necessarily to specific apparatus, and without sample pre-treatments and
The purification of excretion body, is common to aptamer and antibody；It is not limited to excretion body, the micro-nanos biomone such as other extracellular vesica, cells is equal
It can.
Especially, Tthe utility model system can choose and complete measurement under specific temperature, do not limited by actual temp,
It only needs that the temperature difference can be generated to build up particle；Measurement, including research film can be also completed in a variety of different solution environmentals
The detergent environment of complexity needed for albumen；It can also be to various different size of molecules：Such as ion, nucleic acid fragment, nucleosome, rouge
Plastid is detected, and when specifically being detected, system can adjust temperature according to the physical property of particle itself and the size of particle
The parameters such as the frequency of height, the type of fluid and laser irradiation between poor, upper and lower thermal conductive surface are adjusted, above-mentioned each parameter
Adjustment, can be realized quantitative adjustment, control is precisely, easy to adjust.
The large biological molecules such as the utility model floating preteins, nucleic acid or excretion body are not exposed to the albumen on surface, nucleic acid
Equal large biological molecules, micron-scale spherome surface modification can with the antibody or aptamer in conjunction with target protein, nucleic acid specificity,
Immune microsphere is obtained, and it with the sample incubation containing target biomacromolecule and in conjunction with target biomacromolecule and is marked
Fluorescence is converged with the particle to free state, and is detected after convergence.
The utility model is based on thermophoretic effect and builds up particle, does not limit the loading container of micro-nano particle, especially in body
In the biggish container of product, it is easier to which particle is built up under thermophoretic effect, is carried out without being considered as the carrier containers such as capillary
Guidance.Albumen, the nucleic acid on surface etc. are not exposed in the large biological molecules such as the utility model floating preteins, nucleic acid or excretion body
Large biological molecule can obtain in the spherome surface modification of micron-scale with the antibody or aptamer in conjunction with target protein, nucleic acid specificity
It with the sample incubation containing target biomacromolecule and in conjunction with target biomacromolecule and is marked glimmering to immune microsphere, and by it
Light is converged with the particle to free state or the target biomacromolecule for being not exposed to surface, and is detected after convergence.
Detailed description of the invention
Fig. 1 is the structural block diagram of the micro-nano particle detection systems of the utility model；
Fig. 2 is the signal detection flow diagram based on excretion body of the utility model；
Fig. 3 is the comparison map of the excretion body of the utility model embodiment 1 before and after the test；
Fig. 4 be the utility model embodiment 2 excretion body each surface protein map after sensing and corresponding each surface
Expressing quantity schematic diagram；
Fig. 5 is all kinds of cancer patients of the utility model embodiment 2 in all kinds of protein expressions of the serum excretion body of Healthy People
The schematic diagram of amount；
Fig. 6 is that the fluorescence measurement gray value of the fluorescence polystyrene microsphere of the different-diameter of the utility model embodiment 3 shows
It is intended to；
Fig. 7 is 11 kinds of albumen marker expression amounts in the ovarian cancer patients and Healthy Human Serum of the utility model embodiment 4
Schematic diagram；
11 unlike signal objects that Fig. 8 is the utility model embodiment 4 and its summation are as distinguishing cancer and health standards
Accuracy schematic diagram.
Specific embodiment
Below in conjunction with attached drawing, the above and other technical features and advantages of the present invention are described in more detail.
Preferred embodiments of the present invention are described with reference to the accompanying drawings.It should be understood by those skilled in the art that
It is that these embodiments are used only for explaining the technical principle of the utility model, it is not intended that limit the protection of the utility model
Range.
It should be noted that term " on ", "lower", "left", "right", "inner", "outside" etc. in the description of the present invention,
The direction of instruction or the term of positional relationship are direction based on the figure or positional relationship, this, which is intended merely to facilitate, retouches
It states, rather than indication or suggestion described device or element must have a particular orientation, be constructed and operated in a specific orientation, because
This should not be understood as limiting the present invention.In addition, term " first ", " second " are used for description purposes only, and cannot manage
Solution is indication or suggestion relative importance.
In addition it is also necessary to explanation, in the description of the present invention, unless otherwise clearly defined and limited, art
Language " installation ", " connected ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or
It is integrally connected；It can be mechanical connection, be also possible to be electrically connected；It can be directly connected, it can also be by between intermediary
It connects connected, can be the connection inside two elements.To those skilled in the art, can understand as the case may be above-mentioned
The concrete meaning of term in the present invention.
Refering to Figure 1, its structural block diagram for the micro-nano particle detection systems of the utility model, the present embodiment are
System includes heating unit 1, sample bin chamber unit 2, signal acquisition unit 4, wherein the heating unit 1 is arranged in the sample
The outside of warehouse unit 2, to the sample heating into the sample bin chamber unit 2；It is mounted in the sample bin chamber unit 2
Micro-nano particle generates thermophoresis in the sample bin chamber unit 2 after the heating unit 1 is to the sample bin chamber unit 2 heating
Micro-nano particle is converged in the side in the sample bin chamber unit 2 far from the heating unit 1 by effect；The signal is adopted
Collect unit 4, after the micro-nano particle in the sample bin chamber unit 2 is built up, acquires the coherent signal of the micro-nano particle
Information, and corresponding analysis is carried out to corresponding micro-nano particle.This system utilizes thermophoretic effect, i.e., object is under temperature gradient effect
Directional migration, by infrared laser be radiated at sample locally generate temperature gradient field, so that the excretion body in sample is moved to temperature
Spend lower.Heated by the wherein direction to the sample bin chamber unit 2 where micro-nano particle, introduce thermophoretic effect and
Convection current, the side for making sample bin chamber unit 2 generate micro-nano particle fluid and sample bin chamber unit 2 in the temperature difference generate low temperature temperature
Difference, and the temperature of the side of sample bin chamber unit 2 is made to be lower than the temperature of micro-nano particle fluid, thermophoretic effect makes micro-nano grain in sample
Son migrates and is accumulated to the low temperature side of sample bin chamber unit 2；Simultaneously as sample liquids fluid expanded by heating generate buoyancy from
And convection current is generated in sample bin chamber unit 2.Sample is directed toward from surrounding in the direction of the low-temperature region of sample bin chamber unit 2, convection current
The heating region of product warehouse unit 2, arrow direction, plays the role of conveyer belt and converges in micro-nano particle around as shown in figure 1
The 2 low temperature side of sample bin chamber unit of sample bin chamber unit 2, to play the role of building up micro-nano particle.
Specifically, the heating unit 1 of the present embodiment is laser, the outside of sample bin chamber unit 2 is set, to sample
Be irradiated in product warehouse unit 2, to generate circular heating region inside it, certain heating region may be it is linear or
Person's other way.It will be appreciated by persons skilled in the art that heating method is not limited in laser irradiation, laser irradiation direction
It only needs to ensure to generate heat source, the selection of power depends on direction of illumination, and the factors such as spot diameter, wavelength can root
Change according to actual micro-nano particle and use environment.
Specifically, sample bin chamber unit 2 includes loading micro-nano particle samples and generating thermophoretic effect space to provide
Closed sample room 24, sample room 24 includes the first thermal conductive surface 21 to close sample room 24, and to close sample room
24 the second thermal conductive surface 22, in the present embodiment, the temperature for being mounted with micro-nano particle fluid and the second thermal conductive surface of sample room 24
The temperature difference is generated between 22, to generate thermophoretic effect, is driven micro-nano particle to orient from micro-nano particle fluid to the second thermal conductive surface 22 and is moved
It is dynamic.Therefore, the temperature near second thermal conductive surface 22 is lower than the temperature of micro-nano particle fluid by the present embodiment.
In the present embodiment, sample room 24 is heated using laser, first thermal conductive surface 21, the second thermal conductive surface
22 are oppositely arranged, and the thermal conductivity of second thermal conductive surface 22 is greater than the first thermal conductive surface 21, and two thermal conductive surfaces are transparent material,
It is greater than the first thermal conductive surface 21, therefore, the second thermal conductive surface convenient for being observed the heat dissipation performance of the second thermal conductive surface 22 to micro-nano particle
22 temperature is lower than the temperature of the first thermal conductive surface 21.The sample room 24 further includes the gasket 23 to sealed sample room 24, this
Field technical staff is it is understood that two thermal conductive surfaces 21 can be oppositely arranged or be disposed adjacent, or between each other according to default
Angle arrangement, need to only drive micro-nano particle to move, build up along a direction of setting.It will be appreciated by those skilled in the art that
, the fluid of the present embodiment can be liquid, the mixed liquor of such as water or water, or and gas, such as heat gas or nature
State gas only needs that micro-nano particle can be loaded, and micro-nano particle can allow for move freely in a fluid.Meanwhile first
Thermal conductive surface 21 and the second thermal conductive surface 22 be it is transparent, the first thermal conductive surface and the second thermal conductive surface can be sequentially passed through by infrared ray,
And heat is brought into the fluid.
As preferably embodiment, the first thermal conductive surface 21 is glass, polymethyl methacrylate (PMMA), poly dimethyl silicon
Oxygen alkane (PDMS), sapphire etc., the second thermal conductive surface 22 are the good sapphire sapphire of thermal conductivity or diamond.Laser is by sequentially
The first thermal conductive surface 21, the sample room 24 for loading micro-nano particle, the second thermal conductive surface 22 are irradiated, generates low temperature in the second thermal conductive surface 22
Area.Laser spot is adjusted in sample room 24, laser absorbs laser and temperature by the sample liquids in region in sample room 24
Increase, thermophoretic effect makes in sample micro-nano particle migration to lower second thermal conductive surface 22 of temperature, simultaneously because sample liquids by
Thermal expansion generates buoyancy to generate convection current in sample room；Low temperature direction near the second thermal conductive surface 22, the direction of convection current
It is directed toward illuminated laser spot from surrounding, plays the role of conveyer belt for micro-nano particle around and converges in the sample room below illuminated laser spot
The region of second thermal conductive surface 22, to enhance the accumulation of micro-nano particle.
In the present embodiment, micro-nano particle selection is excretion body, and excretion body is film bubble secreted by cell, is handed over for iuntercellular
Stream is increasingly becoming a kind of emerging non-intrusion type tumour because it contains albumen relevant to mother cell and inhereditary material in recent years
The biomarker of diagnosis.
Concrete principle of the present embodiment based on excretion body is as described below.
Excretion body heat swimming motion model：
Wherein vTFor thermophoresis speed, STFor Soret coefficient, D is diffusion coefficient,For temperature gradient, model formation right end
Negative sign indicates that thermophoresis direction is low temperature direction.
Soret coefficient formulas in above-mentioned formula (1)：
Wherein A is excretion body surface area, and k is Boltzmann constant, and T is temperature, shydFor aquation entropy, β is coefficient,
σeffFor the equivalent charge density in excretion body surface face, λDHFor Debye length, ε0For permittivity of vacuum, ε relative dielectric constant.It is comprehensive
Above-mentioned formula (1)-(2), it is known that excretion body heat swimming stress is directly proportional to diameter square.
Excretion body migration models in thermal convection：
Res=ρ a | u-Vp|/η (5)
Wherein, VpFor movement velocity of the excretion body under thermal convection effect, a is excretion body diameter, and u is thermal convection speed, CD
For viscosity coefficient, can be calculated according to formula (4), wherein a1、a2、a3For constant, ResIt, can be according to formula for relative motion Reynolds number
(5) it calculates, g is acceleration of gravity, ρpFor excretion body averag density, ρ is sample liquids density.Aggregative formula (3)-(5), it is known that
The viscous resistance of excretion body thermal convection is directly proportional to diameter.
Compare thermophoretic forces and thermal convection viscous resistance, it is known that object is bigger, and the thermophoretic forces being subject to more account for leading, more tends to
It is gathered in sample room bottom surface；Object is smaller, is more accounted for by thermal convection viscous resistance leading, more tends to follow thermal convection without poly-
Collection.
With continued reference to shown in Fig. 1, signal amplification unit 3 is poly- including the micro-nano particle that the sample bin chamber unit 2 is arranged in
The microscope in product region comprising be directed at the object lens 31, reflective mirror 32 and observation light source 33 of the micro-nano particle of accumulation, pass through
Microscope more can clearly observe micro-nano particle.The signal acquisition unit 4 be CCD camera, it is of course also possible to be it is any can
The instrument for detecting optical signal, takes pictures to micro-nano particle by microscope, obtains information.
In the present embodiment, for excretion body signal detection, the aptamer of excretion body sample and fluorescent marker is incubated first
It educates, the target protein of the aptamer and excretion body surface face that make fluorescent marker is specifically bound, so that excretion body is marked upper fluorescence；It will incubate
Excretion body sample after educating is put into sample room 24, and is heated by laser and introduce thermophoretic effect and convection current, and sample is indoor outer
Secrete the fluorescence signal amplification marked on body；Through the fluorescence signal of CCD recording laser irradiation front and back, before analysis laser irradiation
Fluorescence signal afterwards obtains the abundance of excretion body surface face target protein；It, can using a series of aptamers that can combine different target proteins
It obtains excretion body surface face protein graphical spectrum, and finally determines the corresponding index parameter of excretion body by the analysis.
In the present embodiment, the detection method of micro-nano particle includes：
Step a heats the micro-nano particle samples in sample bin chamber unit 2 from side, produces in sample bin chamber unit 2
Raw thermophoretic effect, converges in the low temperature side in the sample bin chamber unit 2 for micro-nano particle；
In above-mentioned steps a, sample fluid expanded by heating generates buoyancy to generating convection current in sample bin chamber unit 2,
In the low-temperature region of sample bin chamber unit 2, the heating region of sample bin chamber unit 2 is directed toward in the direction of convection current from surrounding, by surrounding
Micro-nano particle converges in the low temperature side of sample bin chamber unit 2.
Step b acquires micro-nano particle by the micro-nano particle built up to the low temperature side in the sample bin chamber unit 2
Corresponding index information and corresponding index is analyzed.
Specifically, the present embodiment carries out signal detection to excretion body, as shown in connection with fig. 2, which is：
The aptamer of excretion body sample and fluorescent marker is incubated for by step a1, makes aptamer and the excretion body surface face of fluorescent marker
Target protein specific binding, so that excretion body is marked upper fluorescence；
Excretion body sample after incubation is put into sample room by step a2, and is heated by laser and to be introduced thermophoretic effect and right
Stream, converges in the indoor low temperature side of the sample, the fluorescence signal that will be marked on the indoor excretion body of sample for excretion body
Amplification；
Step a3 obtains the fluorescence signal of light irradiation front and back, by the fluorescence signal before and after analysis laser irradiation, obtains out
Secrete the abundance of body surface face target protein；
Step a4 obtains excretion body surface face protein graphical spectrum using a series of aptamers that can combine different target proteins.
Above-mentioned micro-nano particle detection systems and method are illustrated below by specific embodiment.
Embodiment 1
The aptamer of excretion body sample and fluorescent marker is incubated for, the aptamer of selection is through in-vitro screening technology SELEX (index
Enrichment Fas lignand system is evolved) oligonucleotide fragment of energy specific binding protein or other small-molecule substances that filters out, tool
For body, the aptamer of fluorescent marker is the single stranded DNA of 40 bases, and the ball of string diameter in sample liquids is less than 5 nanometers, and excretion
Body diameter is 30-150 nanometers；The aptamer of specific recognition CD63 albumen is trained applied to A375 cell (human melanoma cell)
Support the excretion body in base supernatant.Fluorophor can be modified in aptamer end by standard approach, when aptamer and excretion body surface face
When specificity between target protein interacts, the fluorescence of aptamer carrying is marked in excretion body.The excretion body sample of the present embodiment
Incubation conditions for cell culture medium supernatant, sample are：2 hours incubation times, are incubated for 0.1 every liter of micromole of aptamer concentrations
Temperature room temperature.
Wherein, laser is heated using the infrared laser of 1480nm wavelength for sample, and power is 200 milliwatts, and focus goes out
About 200 microns of laser spot diameter.Because the general main component of sample liquids is water, water nearby has an absorption to 1480nm wave band
Peak, it will be appreciated by persons skilled in the art that heating method is not limited in laser irradiation, wavelength is also not limited to
1480nm, laser irradiation direction are not limited to irradiate from the top down, and the selection of power depends on direction of illumination, spot diameter, wavelength
Etc. factors, be not limited to 200 milliwatts.In the present embodiment, laser irradiates from top to bottom, and the upper thermal conductive surface of sample room uses bright material
Matter, such as glass, PMMA, PDMS, lower thermal conductive surface uses the better sapphire of thermal conductivity, and forming low-temperature space in bottom surface makes excretion body heat
Swimming converges 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 room
It is 240mm.
Operated according to the above-mentioned signal detecting method based on excretion body, when aptamer identify excretion body surface protein and with
Combination when, the sample room bottom section that the fluorescent marker on aptamer follows excretion body to be accumulated below laser spot, and producing
Raw enhancing fluorescence signal；When the unidentified excretion body surface protein of aptamer, free aptamer cannot be converged since size is small, signal
Do not enhance.As shown in connection with fig. 3, in the present embodiment, CD63 albumen is widely present in the excretion body surface face of various types of cells, passes through laser
After irradiation, there is apparent fluorescence signal, shows that the excretion body surface face of A375 cell has CD63 albumen.
The fluorescence signal marked on aptamer after being excited and received in conjunction with excretion body using fluorescence microscope, excites and connects
The wavelength for receiving fluorescence is related to the fluorescence radiation group properties of label, in the present embodiment, luminophore Cy5 excitation/emission wavelength
For 649/666nm, the CCD record that fluorescence signal is connect with fluorescence microscope.Pass through the fluorescence of CCD recording laser irradiation front and back
Signal obtains the abundance of excretion body surface face target protein by the fluorescence signal before and after analysis laser irradiation.
Embodiment 2
The present embodiment, using cervical cancer patient blood serum sample, using 7 kinds of different aptamers to excretion body 7 in blood serum sample
The abundance of kind surface protein (CD63, PTK7, EpCAM, HepG2, HER2, PSA, CA125) is detected, and and Healthy Human Serum
Sample compares.
The excretion body operating method and laser of use, sample room and microscope and CCD camera are all the same.
As shown in connection with fig. 4, it is known that this cervical cancer patient serum excretion body height expresses CD63 albumen and cancer Research of predicting markers
PTK7, EpCAM, HepG2, HER2, PSA and CA125, wherein CA125 can be used as the marker of traditional cervical carcinoma, also there is portion
Dividing cervical cancer patient, there are HER2 high expression.It is generally acknowledged that tumor markers PTK7, EpCAM are related to kinds cancer, HepG2 master
There is specificity for liver cancer, PSA has specificity mainly for prostate cancer.So these tumor markers be not and certain
Cancer has stringent correspondence correlativity.But since tumour is during growth or cancer are to other organ metastasis, pass through
Multiple division growth, cell constantly generate gene mutation, show the change in terms of molecular biology or gene but, so these
Tumor markers are not to have stringent corresponding correlativity with certain cancer.In the present embodiment in this cervical cancer patient serum
It detects PTK7, EpCAM, HepG2, embodies the potentiality of gene mutation or transfer of the method in capture tumour.Furthermore CD63
As the albumen that excretion body is generally expressed, cancer patient's excretion body expression also above Healthy People, with existing traditional detection
The result that method obtains is consistent.
This method is further applied into a large amount of true clinical blood serum samples, includes 3 cervical carcinomas, 2 oophoromas, 2
Lymph cancer, 2 breast cancer and 2 Healthy Peoples.As shown in connection with fig. 5, this method is capable of detecting when all kinds of cancer patients and health
The difference of all kinds of expressing quantities of the serum excretion body of people.To between variety classes cancer, serum excretion body protein expression quantity
Difference, be mainly manifested in HER2 expressed in breast cancer and cervical carcinoma it is higher, CA125 expressed in oophoroma and cervical carcinoma compared with
Height, PSA are not expressed in type in cancer detected, and EpCAM and PTK7 and CD63 have higher table in kinds cancer
It reaches.These results are consistent with existing method testing result.
Illustrate that this method can delicately detect the excretion body surface protein in cancer patient's serum and Healthy Human Serum,
The difference of expression quantity including cancer markers.And show detection method using excretion body as cancer marker more
It is convenient, sensitive, effective：The tumor marker species of conventional cancer screening or physical examination detection are limited (to be limited to available expensive anti-
Body and reagent) and sensitivity it is not high and lead to false negative, i.e., marker is not detected in patient, for example, cervical carcinoma in the present embodiment
CA125 expression of results is in normal range value in Venous Blood examining report.And this method is not necessarily to expensive antibody, according to inspection
Survey needs can be used can be with the aptamer in conjunction with the protein-specific of corresponding tumor markers.
Embodiment 3
The present embodiment, the micro-nano particle used are for abiotic micro-nano particle, specially fluorescence polystyrene microsphere, brand
Thermofisher, diameter are 50 to 200 nanometers, and mass fraction 0.001% is dissolved in the water-soluble of the Tween20 containing 0.02%
In liquid.Laser, sample room and microscope and CCD camera are identical as above-described embodiment 1,2.
In conjunction with shown in lower Fig. 5, the fluorescent microsphere of all different-diameters is highly converged at laser spot, and according to fluorescence
Measurement gray value convergence degree visible with fluorescence picture and fluorescence intensity enhance as particle diameter increases, with the present embodiment
Working principle is consistent, i.e., bulky grain is more likely to converge.This example demonstrates that either biological or abiotic micro-nano particle,
It is suitable for the design of the technical program.
Embodiment 4
The present embodiment micro-nano particle is the egg that the large biological molecules such as floating preteins, nucleic acid or excretion body are not exposed to surface
The large biological molecules such as white, nucleic acid cannot directly build up the large biological molecule of free state using the thermophoretic effect of the various embodiments described above,
Therefore, the mechanism of the present embodiment is, modifying in the spherome surface of micron-scale can be in conjunction with target protein, nucleic acid specificity
Antibody or aptamer obtain immune microsphere, and divide greatly by it with the sample incubation containing target biomacromolecule and with target organism
Son combines and mark fluorescent.It is acted on by above-mentioned thermophoresis and converges microballoon height, so that target biomacromolecule fluorescence signal is high
Degree amplification, and its abundance is detected by fluorescence power.
The present embodiment includes based on microsphere supported particle detecting method：
Step a11, prepares immune microsphere, and microballoon and antibody or aptamer are incubated for jointly, is fixed on antibody or aptamer micro-
Ball surface obtains immune microsphere；In this process, the antibody by extra not in conjunction with microballoon or aptamer wash away；In this implementation
In example, microballoon uses polystyrene microsphere.
Step b11, immune microsphere and sample to be tested are incubated for, the target protein or nucleic acid in sample to be tested with
Antibody or aptamer on immune microsphere are specifically bound, to be fixed on immune microsphere；
Step c11 by the immune microsphere for being combined with target biological molecules made from above-mentioned steps b11 and carries fluorophor
Antibody or aptamer combine, by specific recognition, by the target biological molecules mark fluorescent on immune microsphere；
Step d11 carries out the immune microsphere sample for being combined with target biological molecules in sample bin chamber unit 2 from side
Heating generates thermophoretic effect in sample bin chamber unit 2, the immune microsphere for being combined with target biological molecules is converged in the sample
Low temperature side in product warehouse unit 2, and amplify signal since fluorescent marker is enriched with；In this process, by generating heat
Swimming is amplified since target biological molecules are captured by immune microsphere to which equivalent dimension becomes larger by highly enriched and signal, rather than mesh
Mark biomolecule, which is in free state equivalent dimension very little signal, to be amplified.
Step e11 is combined with target biological molecules by what is built up to the low temperature side in the sample bin chamber unit 2
Immune microsphere, acquisition are combined with the corresponding index information of the immune microsphere of target biological molecules and analyze corresponding index.
In this process, the fluorescence signal for obtaining light irradiation front and back obtains excretion body by the fluorescence signal before and after analysis laser irradiation
The abundance of surface target protein；Using a series of aptamers that can combine different target proteins, excretion body surface face protein graphical spectrum is obtained.
This example captures the protein marker that dissociates in ovarian cancer patient whole blood using the immune microsphere of pan coating antibody,
Thermophoresis is generated using infrared laser, detection is amplified to protein marker fluorescence signal, and determines the rich of testing protein marker
Degree, result meet with traditional detection method result, provide molecular information for cancer detection.In the present embodiment, for ovary
Cancer chooses EpCAM, CA-125, CA19-9, CD24, HER2, MUC18, EGFR, CLDN3, CD45, CD41, D2-40 as albumen
Immune microsphere is respectively prepared in the corresponding specific antibody of these protein markers (buying from abcam company) by marker, and every kind
Microballoon is prepared separately in antibody, specifically for a kind of detection of marker.Normal stream is prepared with for coated antibody immune microsphere
Journey can refer to, herein briefly narration：The antibody of 1 micron polystyrene microspheres of diameter and 5 μ g/ml concentration is incubated at room temperature 1
Hour, extra unreacted antibody is removed with ultrafiltration after incubation.It is not limited to 1 micron in the diameter of this microballoon, as long as size reaches
It can be converged to thermophoresis；Material is not limited to polystyrene, as long as antibody and egg to be measured successfully can be had no effect on for antibody attachment
The white active material of marker can be used, and antibody concentration and incubation temperature time are not limited to specific value described in this example,
Change with reference to actual use antibody brand batch and specific experiment condition.
The present embodiment is prepared for 11 kinds of immune microspheres using above step, to detect above-mentioned 11 kinds of markers respectively, will take
1.1 μ L of patients serum divides equally 11 parts after diluting 100 times, mixes be incubated at room temperature 1 hour with 11 kinds of immune microspheres respectively, will have
There is the antibody of fluorescent marker to be incubated for the microballoon for capturing testing protein marker, fluorescent marker is carried out to protein marker.
And it is detected using the detection system of the various embodiments described above.Above-mentioned step is repeated to 10 ovarian cancer patients and 10 Healthy Peoples
Suddenly, 11 kinds of albumen marker expression amounts in 20 blood serum samples are measured, refering to shown in Fig. 7 and Fig. 8.Due to the heterogeneity of cancer,
The marker expression of specific each patient's serum is not exactly the same, but generally expression quantity is apparently higher than healthy sample.Important
It is every kind of protein marker as single cancer detection standard, accuracy rate is not high.Using the sum of expressing quantity in 11 as inspection
Mark is quasi-, and survey can accurately distinguish oophoroma and healthy sample.It will be greatly improved using the marker for more having diagnostic significance
Accuracy rate of diagnosis, but cost increases with marker quantity and is risen, especially for the more rare valuableness of antibody of certain marker.
The detection method of the present embodiment, everyone only needs 1ng antibody for every kind of marker, and cost is not necessarily to other expensive reagents less than 1 yuan.
So far, it has been combined preferred embodiment shown in the drawings and describes the technical solution of the utility model, still, this
Field technical staff is it is easily understood that the protection scope of the utility model is expressly not limited to these specific embodiments.?
Under the premise of the principles of the present invention, those skilled in the art can make equivalent change to the relevant technologies feature
Or replacement, the technical solution after these changes or replacement are fallen within the protection scope of the utility model.
Claims (10)
1. a kind of cancer detection system based on excretion body, which is characterized in that including heating unit, sample bin chamber unit, wherein
The side of the sample bin chamber unit is arranged in the heating unit, to the excretion body into the sample bin chamber unit
Sample heating；
The excretion body being mounted in the sample bin chamber unit in conjunction with aptamer corresponding to fluorescent marker, in the heating unit pair
After the sample bin chamber unit heating, thermophoretic effect is generated in the sample bin chamber unit, the excretion body is converged in described
The lower side of temperature in sample bin chamber unit amplifies the fluorescence signal marked on the indoor excretion body of sample, to examine
It surveys.
2. the cancer detection system according to claim 1 based on excretion body, which is characterized in that the system also includes letters
Number acquisition unit, the signal acquisition unit obtain amplified fluorescence signal, obtain the abundance of excretion body surface face target protein, lead to
It crosses various aptamers and in conjunction with the different target proteins in excretion body surface face and to detect respectively, obtain excretion body surface face protein graphical spectrum.
3. the cancer detection system according to claim 2 based on excretion body, which is characterized in that the sample bin chamber unit
Including loading the excretion body sample and to provide the closed sample room for generating thermophoretic effect space.
4. the cancer detection system according to claim 3 based on excretion body, which is characterized in that the sample room includes：
To close the sample room and build up the second thermal conductive surface of the excretion body.
5. the cancer detection system according to claim 4 based on excretion body, which is characterized in that second thermal conductive surface is attached
Nearly temperature is lower than the temperature of the excretion body fluid, to generate the temperature difference between second thermal conductive surface and excretion body fluid, with
Thermophoretic effect is generated, drives excretion body to the second thermal conductive surface displacement.
6. the cancer detection system according to claim 4 based on excretion body, which is characterized in that the heating unit is sharp
Light device is irradiated to the sample bin chamber unit, and light beam passes sequentially through the excretion body fluid and the second thermal conductive surface, to described
Excretion liquid solution generates thermophoretic effect.
7. the cancer detection system according to claim 6 based on excretion body, which is characterized in that second thermal conductive surface is
Transparent material is sapphire material or diamond material.
8. the cancer detection system according to claim 6 based on excretion body, which is characterized in that also wrap the sample room
It includes：To close the first thermal conductive surface of the sample room, second thermal conductive surface and the first thermal conductive surface can be such that light beam passes through, institute
The first thermal conductive surface is stated to be any one of glass, polymethyl methacrylate, dimethyl silicone polymer, sapphire or appoint several
Combination.
9. the cancer detection system according to claim 8 based on excretion body, which is characterized in that first thermal conductive surface and
Second thermal conductive surface is oppositely arranged, the low temperature direction near second thermal conductive surface, is formed from surrounding and is directed toward illuminated laser spot
Convection current.
10. the cancer detection system according to claim 1-8 based on excretion body, which is characterized in that the system
System further includes signal acquisition unit, after the excretion body in the sample bin chamber unit is built up, to amplified excretion body
Indication information is acquired.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593916A (en) * | 2018-04-08 | 2018-09-28 | 国家纳米科学中心 | Cancer detection system and method based on excretion body |
| CN110607270A (en) * | 2019-10-23 | 2019-12-24 | 中国科学院化学研究所 | Method for jointly characterizing exosome membrane marker and RNA (ribonucleic acid) based on aptamer immune PCR (polymerase chain reaction) |
| CN112557475A (en) * | 2020-02-19 | 2021-03-26 | 南京启医科技有限公司 | Exosome separation detection system based on micro-fluidic and ELISA analysis |
-
2018
- 2018-04-08 CN CN201820486575.2U patent/CN208156013U/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593916A (en) * | 2018-04-08 | 2018-09-28 | 国家纳米科学中心 | Cancer detection system and method based on excretion body |
| CN110607270A (en) * | 2019-10-23 | 2019-12-24 | 中国科学院化学研究所 | Method for jointly characterizing exosome membrane marker and RNA (ribonucleic acid) based on aptamer immune PCR (polymerase chain reaction) |
| CN112557475A (en) * | 2020-02-19 | 2021-03-26 | 南京启医科技有限公司 | Exosome separation detection system based on micro-fluidic and ELISA analysis |
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