CN104714013B

CN104714013B - Graphene chip for capturing cancer cells in whole blood and preparation method thereof

Info

Publication number: CN104714013B
Application number: CN201510170415.8A
Authority: CN
Inventors: 王树涛; 李瑛颖; 江雷
Original assignee: Beijing Tianheng Shengtong Technology Development Co Ltd
Current assignee: Beijing Tianheng Shengtong Technology Development Co Ltd
Priority date: 2015-04-12
Filing date: 2015-04-12
Publication date: 2017-04-26
Anticipated expiration: 2035-04-12
Also published as: CN104714013A

Abstract

The invention provides a graphene chip for capturing cancer cells in whole blood and a preparation method thereof, and relates to the field of biotechnology and clinic, in particular to a graphene biochip used for specifically identifying and capturing circulating cancer cells by using the surface with a multi-matching structure of a graphene material and a preparation method of the graphene biochip. A special structure is prepared on the surface of graphene and is entitled with a variety of chemical and physical properties matched with cells, then a specific identification antibody on the surface of a circulating cancer cell is fixed on the graphene surface with the special structure, the graphene is placed in a cell culture box, a sample (blood) to be measured is dropwise added onto the surface of the specific antibody on the surface of the cancer cell, and then the graphene is placed in a cell culture cabinet to achieve a multi-matching effect by means of the action of the specific antibody on the surface of the cancer cell and the surface of the special structure of the graphene, so that the circulating cancer cells in the sample to be measured can be effectively and accurately identified and captured. The method provided by the invention can be used for effectively capturing the circulating cancer cells, is low in cost, simple to operate and high in sensitivity, and can be applied to clinical detection.

Description

A kind of Graphene chip of whole blood capture cancerous cell and preparation method thereof

Technical field

The invention belongs to technical field of function materials, biomedical material, are more particularly to enriched with for circulating tumor cell With the biochip and preparation method thereof of detection.

Background technology

Circulating tumor cell（CTCs）Detection, identification and separate, the identification and understanding to stem cell cancer marker thing has Very great meaning.Although CTCs quantity in blood is considerably less（It is several to per milliliters up to a hundred）It is entrained in a large amount of blood thin Born of the same parents（10⁹It is individual per milliliter）In, but cancer metastasis is being checked, the diagnosis of patient is predicted, in the result of monitoring treatment, is but played non- Often important effect.Illustrate, CTCs separates and then be transferred to by blood vessel the difference of body from primary tumor Organ, the derivative epithelial cell of the tumor that these live is likely to be the root of cancer metastasiss, and this phenomenon exists Confirmed in cancer patient epithelium blood.That is, CTCs can not only replace the biopsy of intrusive mood to send out Now, inspection cancer, and his number is of great importance for clinical treatment.In the last few years, there are many platforms, such as miniflow Body, immunomagnetic beadses etc. have grown up, and these inspections and diagnosis to early-stage cancer are all requisite.However, now The method of separation and counting to CTCs is all extremely complex, and efficiency is very low.

The content of the invention

Present invention aim at providing a kind of Graphene chip that cancerous cell is captured for whole blood.

Another object of the present invention be provide a kind of preparation cost it is cheap, simple and convenient and have it is extremely highly sensitive for The preparation method of the Graphene chip of whole blood capture cancerous cell.

The present invention is by from novel grapheme material, making the surface of different base pattern, by biotin/life Thing Avidin（biotin/streptavidin）The antibody anti-EpCAM. of the upper specificity of the connection that interacts is in substrate topology Effect, realizes the efficient specificity capture to cancerous cell under hardness effect and the collaboration of antibody specificity recognition reaction.And in disease The high capture rate of comparison is reached in human blood, simple and feasible method is provided for later Clinical detection.

The present invention for whole blood capture cancerous cell Graphene chip, be using grapheme material formed with flower The substrate of valve structure, the graphenic surface are modified with the antibody of specific recognition tumor cell（Anti-EpCAM）.

The material of the Graphene is improved redox graphene material, in the method used by the process for being aoxidized It is oxidation stripping method.

The oxidation graphene film is that by vacuum-thermal reduction, reduction temperature is more than 100 DEG C of vacuum.

The surface modification of the redox graphene has the antibody of specially recognizing tumor cells（Anti-EpCAM）.

The concentration of redox graphene substrate graphene oxide solution used in preparation process is 0 ~ 7mg/ m。

The Graphene chip that cancerous cell is captured for whole blood of the present invention has petal-like micro nano structure, by micro-nano The topological structure of rice structure and circulating tumor cell is produced and strengthens adhesion effect, in addition, which is distinctive close to living things system Hardness provides the condition for more matching for the adhesion of cell, at the same the thin film of prepared redox graphene have it is super hydrophilic Property, there is ultralow background to adsorb the condition of the indispensability for providing for which.

Test result indicate that, the present invention's captures circulating tumor of the Graphene chip of cancerous cell to specificity for whole blood Cell（Such as breast cancer cell MCF7, prostate gland cancer cell PC3）Bioaccumulation efficiency up to more than 90%, and nonspecific cell System（Such as JurkatT lymphocytes, Daudi lymphocytes, Hela uterine cancer cells）But very little adhesion is on biochip, so as to Can realize in biological sample（Blood）In efficient circulation tumor cell capture.For example, by patient blood（1 milliliter is needed only） Instill on the biochip of the present invention, it is possible to achieve the efficient and highly sensitive enrichment to circulating tumor cell in blood sample, and Normal cell but very little adhesion on biochip.The biochip of the present invention, in clinical experiment, can reach extraordinary catching Obtain efficiency and extremely low background absorption.

The preparation method of the Graphene chip for capturing cancerous cell for whole blood of the present invention is comprised the following steps：

（1）Graphite powder is added in the flask containing fuming nitric aicd and concentrated sulphuric acid, is added in the case of zero degrees celsius Potassium chlorate, all of process is all in the case of logical nitrogen.Then the object in flask is stirred at room temperature.

（2）By step（1）The product for obtaining adds hydrochloric acid, washes sulfate radical elimination.

（3）By step（2）It is neutral that the product high purity water for obtaining cleans to pH value.

（4）By step（3）The method of the product high temperature drying for obtaining, obtains powder.

（5）By step（4）The powder for obtaining is weighed, and is made into the GO solution of the concentration of different ratio.

（6）By step（5）The solution for obtaining carries out vacuum filtration film forming, makes the thin film of graphene oxide.

（7）By step（6）The thin film for obtaining is put into vacuum and carries out heating reduction, and the temperature of heating is more than 100 DEG C.

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, obtain PCA-rGO substrates.

（9）By step（8）The PCA-rGO thin film for obtaining is fixed on the glass sheet, obtains rGO substrates.

（10）By step（9）The rGO substrates for obtaining are placed in six orifice plates, add N- hydroxy thiosuccinimides （NHS）With 1-（3- dimethylamino-propyls）- 3- ethyl carbodiimides（EDC）, place a period of time at room temperature.

（11）By step（10）The rGO substrates for obtaining are placed in the phosphate buffer of 1 Streptavidin, are carried out under room temperature Substrate is taken out and is cleaned by reaction；Obtain HA-rGO.

（12）By step（11）The HA-rGO substrates for obtaining add antibody（Anti-EpCAM）, placing response under room temperature obtains To Anti-EpCAM-rGO substrates.

The concentration of redox graphene substrate graphene oxide solution used in preparation process is 0 ~ 7mg/ ml。

The Graphene chip that cancerous cell is captured for whole blood of the present invention has preparation cost cheap, simple to operate, Yi Gong The advantages of industry metaplasia is produced, and clinical experiment has been done, with very sensitive cellular affinity and capture rate.The life of the present invention Thing chip is particularly well-suited to carcinoma of prostate, breast carcinoma, colon and rectum carcinoma, malignant melanoma, alveolar soft part sarcoma Past tumor cell in etc. the blood for causing transfer has good concentration effect.The present invention's is enriched with for circulating tumor cell With detection biochip preparation process without harmful substance of being always or usually as specified, environmental friendliness, good stability.

Description of the drawings

Fig. 1. the preparation method of the Graphene biochip of the whole blood cancerous cell capture used by the present invention.

Fig. 2. the photo in kind of the Graphene chip that cancerous cell is captured for whole blood prepared by the present invention.

Fig. 3. the environment after cell has been captured of the Graphene chip that cancerous cell is captured for whole blood prepared by the present invention Stereoscan photograph.

Fig. 4. the Graphene chip for capturing cancerous cell for whole blood prepared by embodiment of the present invention 1-3,5 captures five kinds thin Born of the same parents system（MCF7、PC3、Hela、Daudi、Jurkat）Quantitative data.

Fig. 5. the human body blood of the Graphene chip capture that cancerous cell is captured for whole blood prepared by embodiment of the present invention 6-13 The trichrome stain figure of MCF7 cancerous cell and normal plasma cell in liquid.

Fig. 6. the clinic captured by the Graphene chip for capturing cancerous cell for whole blood prepared by embodiment of the present invention 6-13 The quantitative data of the cancerous cell in patient blood.

Specific embodiment

Embodiment 1.

（1）Graphite powder is added in the flask containing fuming nitric aicd and concentrated sulphuric acid, is added in the case of zero degrees celsius Potassium chlorate, all of process is all in the case of logical nitrogen.Then the object more than 80 hours in flask is stirred at room temperature.

（5）By step（4）The powder for obtaining is weighed, and is made into GO solution.

（7）By step（6）The thin film for obtaining is put into vacuum and carries out heating reduction, and the temperature of heating is more than 100 degrees Celsius.

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, room temperature is anti- Should, obtain PCA-rGO substrates.

（10）By step（9）The rGO substrates for obtaining are placed in six orifice plates, add N- hydroxy thiosuccinimides （NHS）With 1-（3- dimethylamino-propyls）- 3- ethyl carbodiimides（EDC）, placing response at room temperature.

（11）By step（10）The rGO substrates for obtaining are placed in the phosphate buffer of Streptavidin, are carried out under room temperature Substrate is taken out and is cleaned by reaction；Obtain HA-rGO.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in 6 orifice plates, add 3 milliliters Concentration is 1x10⁵The breast cancer cell MCF7 suspension of individual/ml, is placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minutes, more preferably 45 minutes）, breast cancer cell MCF7 suspension is sucked, biochip is abundant in phosphate buffer Rinse, then the paraformaldehyde phosphate buffer that the breast cancer cell MCF7 mass concentration that biochip is captured is 4% is consolidated It is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then delayed with the DAPI phosphate of 10 μ g/ml After rushing liquid dyeing, dried up with air draught after 3 times are rinsed in PBS, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively （Each at least 3 substrates, each substrate choose middle 10 different positions）, and the breast carcinoma to being captured on biochip Cell MCF7 is counted, and calculates capture rate.Breast cancer cell MCF7 captures quantitative data such as Fig. 3, shown in Fig. 4.

（14）As a control group 1, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The prostate gland cancer cell PC3 suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out 0 ~ 90 minute time of reaction, more preferably 45 minutes）, prostate gland cancer cell PC3 suspensions are sucked, by biochip in phosphoric acid Fully rinse in buffer, then by biochip capture prostate gland cancer cell PC3 mass concentrations be 4% paraformaldehyde After phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the prostate gland cancer cell PC3 that captured counted, calculate capture rate.Prostate gland cancer cell PC3 captures quantitative data is such as Shown in Fig. 4.

（15）As a control group 2, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵Human lymphoma cell's Jurkat suspensions of individual/ml, are placed in cell culture incubator（It is preferred that The time reacted is 0 ~ 90 minute, more preferably 45 minutes）, human lymphoma cell's Jurkat suspensions are sucked, by biology Chip is fully rinsed in phosphate buffer, then by the human lymphoma cell Jurkat that biochip is captured with mass concentration is After 4% paraformaldehyde phosphate buffer is fixed, then worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration Film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, dried up with air draught after 3 times are rinsed in PBS, use Taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate choose in the middle of 10 it is different Position）, and the lymphoma cell Jurkat to being captured on biochip counts, and calculates capture rate.Lymphoma cell Jurkat capture quantitative datas are as shown in Figure 4.

（16）As a control group 3, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵People's lymphocytic B cells oncocyte Daudi suspensions of individual/ml, are placed in cell culture incubator （It is preferred that the time reacted is 0 ~ 90 minute, more preferably 45 minutes）, suck people's lymphocytic B cells oncocyte Daudi suspensions Liquid, biochip is fully rinsed in phosphate buffer, the people's lymphocytic B cells oncocyte for then capturing biochip Daudi mass concentrations are that 4% paraformaldehyde phosphate buffer is fixed, then with the Trition X-100 of 0.2% volumetric concentration Phosphate buffer wears film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, uses air after rinsing 3 times in PBS Air-flow is dried up, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate is chosen middle 10 different positions）, and the people lymphocytic B cells oncocyte Daudi to being captured on biochip counts, and calculates capture Efficiency.People's lymphocytic B cells oncocyte Daudi capture quantitative datas are as shown in Figure 4.

（17）As a control group 4, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The uterine cancer cells Hela suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 45 minutes）, uterine cancer cells Hela suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture uterine cancer cells Hela mass concentrations be 4% paraformaldehyde After phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the uterine cancer cells Hela that captured counted, calculate capture rate.Uterine cancer cells Hela captures quantitative data such as Fig. 4 It is shown.

Test result indicate that, the breast cancer cell of the biochip for being enriched with for circulating tumor cell and detecting of the present invention The capture rate of MCF7 is 90.2%, and the capture rate of prostate gland cancer cell PC3 is 83.2%, and human lymphoma cell Jurkat's catches It is 0.01% to obtain efficiency, and the capture rate of people lymphocytic B cells oncocyte Daudi is 0.01%, and the capture of uterine cancer cells Hela is imitated Rate is 0.02%.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high and clever Quick capture rate, and non-specific adsorption is extremely low.

Embodiment 2.

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, room temperature is put Reaction is put, PCA-rGO substrates are obtained.

（12）By step（11）The HA-rGO substrates for obtaining add antibody（Anti-EpCAM）, place under room temperature, obtain Anti-EpCAM-rGO substrates.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in 6 orifice plates, add 3 milliliters Concentration is 1x10⁵The breast cancer cell MCF7 suspension of individual/ml, is placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minutes, more preferably 60 minutes）, breast cancer cell MCF7 suspension is sucked, biochip is abundant in phosphate buffer Rinse, then the paraformaldehyde phosphate buffer that the breast cancer cell MCF7 mass concentration that biochip is captured is 4% is consolidated After fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with the DAPI phosphate of 10 μ g/ml After buffer dyeing, dried up with air draught after 3 times are rinsed in PBS, with being clapped under 10 times of Nikon inverted fluorescence microscopes respectively According to（Each at least 3 substrates, each substrate choose middle 10 different positions）, and the mammary gland to being captured on biochip Cancerous cell MCF7 is counted, and calculates capture rate.Breast cancer cell MCF7 captures quantitative data such as Fig. 3, shown in Fig. 4.

（14）As a control group 1, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The prostate gland cancer cell PC3 suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 60 minutes）, prostate gland cancer cell PC3 suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture prostate gland cancer cell PC3 mass concentrations be 4% poly first After aldehyde phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then use 10 μ After the DAPI phosphate buffers dyeing of g/ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence is inverted with Nikon and is shown Taken pictures under 10 times of micro mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biological core The prostate gland cancer cell PC3 captured on piece is counted, and calculates capture rate.Prostate gland cancer cell PC3 captures quantitative data As shown in Figure 4.

（15）As a control group 2, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵Human lymphoma cell's Jurkat suspensions of individual/ml, are placed in cell culture incubator（It is preferred that The time reacted is 0 ~ 90 minute, more preferably 60 minutes）, human lymphoma cell's Jurkat suspensions are sucked, by biology Chip is fully rinsed in phosphate buffer, then by the human lymphoma cell Jurkat that biochip is captured with mass concentration is After 4% paraformaldehyde phosphate buffer is fixed, then worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration Film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, dried up with air draught after 3 times are rinsed in PBS, use Taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate choose in the middle of 10 it is different Position）, and the lymphoma cell Jurkat to being captured on biochip counts, and calculates capture rate.Lymphoma cell Jurkat capture quantitative datas are as shown in Figure 4.

（16）As a control group 3, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵People's lymphocytic B cells oncocyte Daudi suspensions of individual/ml, are placed in cell culture incubator （It is preferred that the time reacted is 0 ~ 90 minute, more preferably 60 minutes）, suck people's lymphocytic B cells oncocyte Daudi suspensions Liquid, biochip is fully rinsed in phosphate buffer, the people's lymphocytic B cells oncocyte for then capturing biochip Daudi mass concentrations are after 4% paraformaldehyde phosphate buffer is fixed, then with the Trition X- of 0.2% volumetric concentration 100 phosphate buffers wear film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, rinse 3 times afterwards with sky in PBS Gas air-flow is dried up, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, during each substrate is chosen Between 10 different positions）, and the people lymphocytic B cells oncocyte Daudi to being captured on biochip counts, calculating is caught Obtain efficiency.People's lymphocytic B cells oncocyte Daudi capture quantitative datas are as shown in Figure 4.

（17）As a control group 4, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The uterine cancer cells Hela suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 60 minutes）, uterine cancer cells Hela suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture uterine cancer cells Hela mass concentrations be 4% paraformaldehyde After phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the uterine cancer cells Hela that captured counted, calculate capture rate.Uterine cancer cells Hela captures quantitative data such as Fig. 4 It is shown.

Test result indicate that, the breast cancer cell of the biochip for being enriched with for circulating tumor cell and detecting of the present invention The capture rate of MCF7 is 91.3%, and the capture rate of prostate gland cancer cell PC3 is 82.5%, and human lymphoma cell Jurkat's catches It is 0.02% to obtain efficiency, and the capture rate of people lymphocytic B cells oncocyte Daudi is 0.01%, and the capture of uterine cancer cells Hela is imitated Rate is 0.03%.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high and clever Quick capture rate, and non-specific adsorption is extremely low.

Embodiment 3.

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, under room temperature Reaction, obtains PCA-rGO substrates.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in 6 orifice plates, add 3 milliliters Concentration is 1x10⁵The breast cancer cell MCF7 suspension of individual/ml, is placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minutes, more preferably 90 minutes）, breast cancer cell MCF7 suspension is sucked, biochip is abundant in phosphate buffer Rinse, then the paraformaldehyde phosphate buffer that the breast cancer cell MCF7 mass concentration that biochip is captured is 4% is consolidated After fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with the DAPI phosphate of 10 μ g/ml After buffer dyeing, dried up with air draught after 3 times are rinsed in PBS, with being clapped under 10 times of Nikon inverted fluorescence microscopes respectively According to（Each at least 3 substrates, each substrate choose middle 10 different positions）, and the mammary gland to being captured on biochip Cancerous cell MCF7 is counted, and calculates capture rate.Breast cancer cell MCF7 captures quantitative data such as Fig. 3, shown in Fig. 4.

（14）As a control group 1, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The prostate gland cancer cell PC3 suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 90 minutes）, prostate gland cancer cell PC3 suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture prostate gland cancer cell PC3 mass concentrations be 4% poly first Aldehyde phosphate buffer is fixed, then wears film with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the prostate gland cancer cell PC3 that captured counted, calculate capture rate.Prostate gland cancer cell PC3 captures quantitative data is such as Shown in Fig. 4.

（15）As a control group 2, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵Human lymphoma cell's Jurkat suspensions of individual/ml, are placed in cell culture incubator（It is preferred that The time reacted is 0 ~ 90 minute, more preferably 90 minutes）, human lymphoma cell's Jurkat suspensions are sucked, by biology Chip is fully rinsed in phosphate buffer, then by the human lymphoma cell Jurkat that biochip is captured with mass concentration is After 4% paraformaldehyde phosphate buffer is fixed, then worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration Film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, dried up with air draught after 3 times are rinsed in PBS, use Taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate choose in the middle of 10 it is different Position）, and the lymphoma cell Jurkat to being captured on biochip counts, and calculates capture rate.Lymphoma cell Jurkat capture quantitative datas are as shown in Figure 4.

（16）As a control group 3, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵People's lymphocytic B cells oncocyte Daudi suspensions of individual/ml, are placed in cell culture incubator （It is preferred that the time reacted is 0 ~ 90 minute, more preferably 90 minutes）, suck people's lymphocytic B cells oncocyte Daudi suspensions Liquid, biochip is fully rinsed in phosphate buffer, the people's lymphocytic B cells oncocyte for then capturing biochip Daudi mass concentrations are that 4% paraformaldehyde phosphate buffer is fixed, then with the Trition X-100 of 0.2% volumetric concentration Phosphate buffer wears film, then is dyeed with the DAPI phosphate buffers of 10 μ g/ml, uses air gas in PBS after rinsing 3 times Stream is dried up, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate choose middle 10 Individual different position）, and the people lymphocytic B cells oncocyte Daudi to being captured on biochip counts, and calculates capture effect Rate.People's lymphocytic B cells oncocyte Daudi capture quantitative datas are as shown in Figure 4.

（17）As a control group 4, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The uterine cancer cells Hela suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 90 minutes）, uterine cancer cells Hela suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture uterine cancer cells Hela mass concentrations be 4% paraformaldehyde After phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the uterine cancer cells Hela that captured counted, calculate capture rate.Uterine cancer cells Hela captures quantitative data such as Fig. 4 It is shown.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture rate is 92.2%, and the capture rate of prostate gland cancer cell PC3 is 84.2%, the capture rate of human lymphoma cell Jurkat For 0.01%, the capture rate of people lymphocytic B cells oncocyte Daudi is 0.02%, and the capture rate of uterine cancer cells Hela is 0.02%.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high and sensitive Capture rate, and non-specific adsorption is extremely low.

Embodiment 4.

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, under room temperature Place, obtain PCA-rGO substrates.

（12）By step（11）The HA-rGO substrates for obtaining add 10 μ g/ml antibody（Anti-EpCAM）, room temperature decentralization Put, obtain Anti-EpCAM-rGO substrates.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in 6 orifice plates, add 3 milliliters Concentration is 1x10⁵The breast cancer cell MCF7 suspension of individual/ml, is placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minutes, more preferably 60 minutes）, breast cancer cell MCF7 suspension is sucked, biochip is abundant in phosphate buffer Rinse, then the paraformaldehyde phosphate buffer that the breast cancer cell MCF7 mass concentration that biochip is captured is 4% is consolidated After fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with the DAPI phosphate of 10 μ g/ml After buffer dyeing, dried up with air draught after 3 times are rinsed in PBS, with being clapped under 10 times of Nikon inverted fluorescence microscopes respectively According to（Each at least 3 substrates, each substrate choose middle 10 different positions）, and the mammary gland to being captured on biochip Cancerous cell MCF7 is counted, and calculates capture rate.

（14）As a control group 1, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The prostate gland cancer cell PC3 suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 60 minutes）, prostate gland cancer cell PC3 suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture prostate gland cancer cell PC3 mass concentrations be 4% poly first After aldehyde phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then use 10 μ The DAPI phosphate buffers dyeing of g/ml, is dried up with air draught after rinsing 3 times, be inverted fluorescence microscopy with Nikon in PBS Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the prostate gland cancer cell PC3 that captured counted, calculate capture rate.

（15）As a control group 2, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵Human lymphoma cell's Jurkat suspensions of individual/ml, are placed in cell culture incubator（It is preferred that The time reacted is 0 ~ 90 minute, more preferably 60 minutes）, human lymphoma cell's Jurkat suspensions are sucked, by biology Chip is fully rinsed in phosphate buffer, then by the human lymphoma cell Jurkat that biochip is captured with mass concentration is After 4% paraformaldehyde phosphate buffer is fixed, then worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration Film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, dried up with air draught after 3 times are rinsed in PBS, use Taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate choose in the middle of 10 it is different Position）, and the lymphoma cell Jurkat to being captured on biochip counts, and calculates capture rate.

（16）As a control group 3, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵People's lymphocytic B cells oncocyte Daudi suspensions of individual/ml, are placed in cell culture incubator （It is preferred that the time reacted is 0 ~ 90 minute, more preferably 60 minutes）, suck people's lymphocytic B cells oncocyte Daudi suspensions Liquid, biochip is fully rinsed in phosphate buffer, the people's lymphocytic B cells oncocyte for then capturing biochip Daudi mass concentrations are after 4% paraformaldehyde phosphate buffer is fixed, then with the Trition X- of 0.2% volumetric concentration 100 phosphate buffers wear film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, rinse 3 times afterwards with sky in PBS Gas air-flow is dried up, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, during each substrate is chosen Between 10 different positions）, and the people lymphocytic B cells oncocyte Daudi to being captured on biochip counts, calculating is caught Obtain efficiency.

（17）As a control group 4, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The uterine cancer cells Hela suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 60 minutes）, uterine cancer cells Hela suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture uterine cancer cells Hela mass concentrations be 4% paraformaldehyde After phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the uterine cancer cells Hela that captured counted, calculate capture rate.

Test result indicate that, the breast cancer cell of the biochip for being enriched with for circulating tumor cell and detecting of the present invention The capture rate of MCF7 is 91.5%, and the capture rate of prostate gland cancer cell PC3 is 81.4%, and human lymphoma cell Jurkat's catches It is 0.01% to obtain efficiency, and the capture rate of people lymphocytic B cells oncocyte Daudi is 0.01%, and the capture of uterine cancer cells Hela is imitated Rate is 0.02%.These as shown by data, the present invention's capture the Graphene chip of cancerous cell with very high and sensitive for whole blood Capture rate, and non-specific adsorption is extremely low.

Embodiment 5.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in 6 orifice plates, add 3 milliliters Concentration is 1x10⁵The breast cancer cell MCF7 suspension of individual/ml, is placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minutes, more preferably 75 minutes）, breast cancer cell MCF7 suspension is sucked, biochip is abundant in phosphate buffer Rinse, then the paraformaldehyde phosphate buffer that the breast cancer cell MCF7 mass concentration that biochip is captured is 4% is consolidated After fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with the DAPI phosphate of 10 μ g/ml After buffer is dyeed 15 minutes, dried up with air draught after 3 times are rinsed in PBS, with 10 times of Nikon inverted fluorescence microscopes Take pictures respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to being captured on biochip Breast cancer cell MCF7 counted, calculate capture rate.Breast cancer cell MCF7 captures quantitative data such as Fig. 3, Fig. 4 institutes Show.

（14）As a control group 1, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates It is middle to add 3 milliliters of concentration to be 1x10⁵The prostate gland cancer cell PC3 suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 75 minutes）, prostate gland cancer cell PC3 suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture prostate gland cancer cell PC3 mass concentrations be 4% poly first After aldehyde phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then use 10 μ After the DAPI phosphate buffers dyeing of g/ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence is inverted with Nikon and is shown Taken pictures under 10 times of micro mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biological core The prostate gland cancer cell PC3 captured on piece is counted, and calculates capture rate.Prostate gland cancer cell PC3 captures quantitative data As shown in Figure 4.

（15）As a control group 2, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵Human lymphoma cell's Jurkat suspensions of individual/ml, are placed in cell culture incubator（It is preferred that The time reacted is 0 ~ 90 minute, more preferably 75 minutes）, human lymphoma cell's Jurkat suspensions are sucked, by biology Chip is fully rinsed in phosphate buffer, then by the human lymphoma cell Jurkat that biochip is captured with mass concentration is After 4% paraformaldehyde phosphate buffer is fixed, then worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration Film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, dried up with air draught after 3 times are rinsed in PBS, use Taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate choose in the middle of 10 it is different Position）, and the lymphoma cell Jurkat to being captured on biochip counts, and calculates capture rate.Lymphoma cell Jurkat capture quantitative datas are as shown in Figure 4.

（16）As a control group 3, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵People's lymphocytic B cells oncocyte Daudi suspensions of individual/ml, are placed in cell culture incubator （It is preferred that the time reacted is 0 ~ 90 minute, more preferably 75 minutes）, suck people's lymphocytic B cells oncocyte Daudi suspensions Liquid, biochip is fully rinsed in phosphate buffer, the people's lymphocytic B cells oncocyte for then capturing biochip Daudi mass concentrations are after 4% paraformaldehyde phosphate buffer is fixed, then with the Trition X- of 0.2% volumetric concentration 100 phosphate buffers wear film, then after being dyeed with the DAPI phosphate buffers of 10 μ g/ml, rinse 3 times afterwards with sky in PBS Gas air-flow is dried up, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, during each substrate is chosen Between 10 different positions）, and the people lymphocytic B cells oncocyte Daudi to being captured on biochip counts, calculating is caught Obtain efficiency.People's lymphocytic B cells oncocyte Daudi capture quantitative datas are as shown in Figure 4.

（17）As a control group 4, by step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed on 6 orifice plates In, add 3 milliliters of concentration to be 1x10⁵The uterine cancer cells Hela suspensions of individual/ml, are placed in cell culture incubator（It is preferred that carry out The time of reaction is 0 ~ 90 minute, more preferably 75 minutes）, uterine cancer cells Hela suspensions are sucked, by biochip in phosphorus Fully rinse in acid buffer, then by biochip capture uterine cancer cells Hela mass concentrations be 4% paraformaldehyde After phosphate buffer is fixed, then film is worn with the Trition X-100 phosphate buffers of 0.2% volumetric concentration, then with 10 μ g/ After the DAPI phosphate buffers dyeing of ml, dried up with air draught after 3 times are rinsed in PBS, fluorescence microscopy is inverted with Nikon Taken pictures under 10 times of mirror respectively（Each at least 3 substrates, each substrate choose middle 10 different positions）, and to biochip On the uterine cancer cells Hela that captured counted, calculate capture rate.Uterine cancer cells Hela captures quantitative data such as Fig. 4 It is shown.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture rate is 92.0%, and the capture rate of prostate gland cancer cell PC3 is 84.6%, the capture rate of human lymphoma cell Jurkat For 0.01%, the capture rate of people lymphocytic B cells oncocyte Daudi is 0.01%, and the capture rate of uterine cancer cells Hela is 0.03%.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high and sensitive Capture rate, and non-specific adsorption is extremely low.

Embodiment 6.

（10）By step（9）The rGO substrates for obtaining are placed in six orifice plates, add N- hydroxy thiosuccinimides （NHS）With 1-（3- dimethylamino-propyls：）- 3- ethyl carbodiimides（EDC）, placing response at room temperature.

（11）By step（10）The rGO substrates for obtaining are placed in the phosphate buffer of the Streptavidin of 10 μ g/ml, Reacted under room temperature, substrate is taken out and cleaned；Obtain HA-rGO.

（12）By step（11）The HA-rGO substrates for obtaining add 10 μ g/ml antibody（Anti-EpCAM）, place under room temperature 1 hour, obtain Anti-EpCAM-rGO substrates.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in special culture dish, plus Enter the blood of 1 milliliter of breast cancer patients, be placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minute）, suck many Remaining blood, biochip is fully rinsed in phosphate buffer, the breast cancer cell MCF7 for then capturing biochip Fixed after 20 minutes with the paraformaldehyde phosphate buffer that mass concentration is 4%, then with the Trition X- of 0.2% volumetric concentration 100 phosphate buffers wear film, with air draught are dried up after rinsing 3 times in PBS, add 200 microlitres of blocker（5% Sanguis caprae seu ovis Clearly, 0.1% polysorbas20, the phosphate buffer of 3% bovine serum albumin）, a hour is placed at room temperature.It is subsequently adding 200 microlitres Fluorescently-labeled antibody-solutions（The initial concentration of 20 μ g/ml, antibody are respectively anti-cytokeratin PE and FITC anti-human CD45）Then substrate is preserved overnight for 4 degrees Celsius under conditions of night shading.Phosphate buffer is added to enter Row cleaning.DAPI phosphate buffers are added afterwards（10μg/ml）, under room temperature, stand 5 minutes.Then carried out clearly with phosphate buffer Wash 3 times.Substrate is inverted, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, each substrate 10 different positions in the middle of choosing）, and the breast cancer cell MCF7 to being captured on biochip counts, and calculates capture Efficiency.Capture data such as Fig. 5 of breast cancer cell MCF7, shown in Fig. 6.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 3.These as shown by data, the present invention for circulating tumor cell enrichment capture chip have very it is high and Sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results are notable.

Embodiment 7.

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, room temperature is put Put, obtain PCA-rGO substrates.

（12）By step（11）The HA-rGO substrates for obtaining add 10 μ g/ml antibody（Anti-EpCAM）, place under room temperature React within 1 hour, obtain Anti-EpCAM-rGO substrates.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 4.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high With sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results are notable.

Embodiment 8.

（10）By step（9）The rGO substrates for obtaining are placed in six orifice plates, add N- hydroxy thiosuccinimides （NHS）With 1-（3- dimethylamino-propyls：）- 3- ethyl carbodiimides（EDC）, standing and reacting at room temperature.

（11）By step（10）The rGO substrates for obtaining are placed in the phosphate buffer of the Streptavidin of 10 μ g/ml, room Reacted under temperature, substrate is taken out and cleaned;Obtain HA-rGO.

（12）By step（11）The HA-rGO substrates for obtaining add 10 μ g/ml antibody（Anti-EpCAM）, under room temperature, place 1 Hour, obtain Anti-EpCAM-rGO substrates.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in special culture dish, plus Enter the blood of 1 milliliter of normal person, be placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minute）, it is unnecessary to suck Blood, biochip is fully rinsed in phosphate buffer, the breast cancer cell MCF7 matter for then capturing biochip Amount concentration is after 4% paraformaldehyde phosphate buffer fixes 20 minutes, then with the TritionX-100 phosphorus of 0.2% volumetric concentration Phthalate buffer wears film, with air draught is dried up after rinsing 3 times in PBS, adds 200 microlitres of blocker（5% sheep blood serum, 0.1% polysorbas20, the phosphate buffer of 3% bovine serum albumin）, a hour is placed at room temperature.It is subsequently adding 200 microlitres of fluorescence The antibody-solutions of labelling（The initial concentration of 20 μ g/ml, antibody are respectively anti-cytokeratinPE and FITCanti- humanCD45）Then substrate is preserved overnight for 4 degrees Celsius under conditions of night shading.Phosphate buffer is added to be cleaned. DAPI phosphate buffers are added afterwards（10μg/ml）, under room temperature, stand 5 minutes.Then cleaning 3 times is carried out with phosphate buffer. Substrate is inverted, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, during each substrate is chosen Between 10 different positions）, and the breast cancer cell MCF7 to being captured on biochip counts, and calculates capture rate. Capture data such as Fig. 5 of breast cancer cell MCF7, shown in Fig. 6.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 0.These as shown by data, the present invention for circulating tumor cell enrichment capture chip have very it is high and Sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results are notable.

Embodiment 9.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 2.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high With sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results are notable.

Embodiment 10.

（13）By step（12）The Anti-EpCAM-rGO substrates for obtaining face-up are placed in special culture dish, plus Enter the blood of 1 milliliter of breast cancer patients, be placed in cell culture incubator（It is preferred that the time reacted is 0 ~ 90 minute）, suck many Remaining blood, biochip is fully rinsed in phosphate buffer, the breast cancer cell MCF7 for then capturing biochip Fixed after 20 minutes with the paraformaldehyde phosphate buffer that mass concentration is 4%, then with the TritionX- of 0.2% volumetric concentration 100 phosphate buffers wear film, with air draught are dried up after rinsing 3 times in PBS, add 200 microlitres of blocker（5% Sanguis caprae seu ovis Clearly, 0.1% polysorbas20, the phosphate buffer of 3% bovine serum albumin）, a hour is placed at room temperature.It is subsequently adding 200 microlitres Fluorescently-labeled antibody-solutions（The initial concentration of 20 μ g/ml, antibody are respectively anti-cytokeratinPE and FITCanti- humanCD45）Then substrate is preserved overnight for 4 degrees Celsius under conditions of night shading.Phosphate buffer is added to be cleaned. DAPI phosphate buffers are added afterwards（10μg/ml）, under room temperature, stand 5 minutes.Then cleaning 3 times is carried out with phosphate buffer. Substrate is inverted, with being taken pictures under 10 times of Nikon inverted fluorescence microscopes respectively（Each at least 3 substrates, during each substrate is chosen Between 10 different positions）, and the breast cancer cell MCF7 to being captured on biochip counts, and calculates capture rate. Capture data such as Fig. 5 of breast cancer cell MCF7, shown in Fig. 6.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 2.These as shown by data, the present invention for circulating tumor cell enrichment capture chip have very it is high and Sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results are notable.

Embodiment 11.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 6.These as shown by data, the chip of the capture for circulating tumor cell enrichment of the present invention is with very high With sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results are notable.

Embodiment 12.

Test result indicate that, the breast cancer cell MCF7 of the Graphene chip that cancerous cell is captured for whole blood of the present invention Capture number is 5.The Graphene chip data of these whole bloods capture cancerous cell shows, the present invention for circulating tumor cell The chip of the capture of enrichment has very high and sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results Significantly.

Embodiment 13.

Claims

1. a kind of preparation method of the Graphene biochip for being enriched with for whole blood circulating tumor cell and detecting, is characterized in that, The biochip has the substrate with petal-like micro nano structure formed by grapheme material, the grapheme material For improved redox graphene material, the preparation method is comprised the following steps：

（1）Graphite powder is added in the flask containing fuming nitric aicd and concentrated sulphuric acid, chloric acid is added in the case of zero degrees celsius Potassium, all of process all, in the case of logical nitrogen, are then stirred at room temperature the object in flask；

（2）By step（1）The product for obtaining adds hydrochloric acid, washes sulfate radical elimination；

（3）By step（2）It is neutral that the product high purity water for obtaining cleans to pH value；

（4）By step（3）The method of the product high temperature drying for obtaining, obtains powder；

（5）By step（4）The powder for obtaining is weighed, and is made into the GO solution of the concentration of different ratio；

（6）By step（5）The solution for obtaining carries out vacuum filtration film forming, makes the thin film of graphene oxide；Prepare graphite oxide The solution concentration during thin film of alkene is 0 ~ 7mg/ml；

（7）By step（6）The thin film for obtaining is put into vacuum and carries out heating reduction, and the temperature of heating is more than 100 degrees Celsius；

（8）By step（7）The redox graphene for obtaining（rGO）Film be put in the solution of a pyrene formic acid, obtain PCA-rGO Thin film；

（9）By step（8）The PCA-rGO thin film for obtaining is fixed on the glass sheet, obtains rGO substrates；

（10）By step（9）The rGO substrates for obtaining are placed in six orifice plates, add N- hydroxy thiosuccinimides and 1-（3- Dimethylamino-propyl）- 3- ethyl carbodiimides, are placed a period of time at room temperature；

（11）By step（10）The rGO substrates for obtaining are placed in the phosphate buffer of Streptavidin, are reacted under room temperature, Substrate is taken out and cleaned；Obtain SA-rGO substrates；

（12）By step（11）The SA-rGO substrates for obtaining add the antibody of the specific recognition tumor cell of biotin labeling Anti-EpCAM, placing response under room temperature obtain Anti-EpCAM-rGO substrates.

2. the graphite for being enriched with for whole blood circulating tumor cell and detecting that prepared by a kind of preparation method according to claim 1 Alkene biochip.