CN104714013A

CN104714013A - Graphene chip for capturing cancer cells in whole blood and preparation method thereof

Info

Publication number: CN104714013A
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: 2015-06-17
Anticipated expiration: 2035-04-12
Also published as: CN104714013B

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 whole blood Graphene chip of catching cancer cell and preparation method thereof

Technical field

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

Background technology

The detection of circulating tumor cell (CTCs), identification and be separated, have very great significance to the identification of stem cell cancer marker thing and understanding.Although CTCs quantity in blood considerably less (several to up to a hundred every milliliter) is entrained in a large amount of haemocyte (10 ⁹individual every milliliter) in, but at inspection cancer metastasis, the diagnosis of prediction patient, in the result of monitor therapy, but plays very important effect.Illustrate, CTCs separates the different organ being then transferred to health by blood vessel from primary tumor, the derivative epithelial cell of these tumours lived is probably the root of cancer metastasis, and this phenomenon obtains confirmation in cancer patient epithelium blood.That is, CTCs not only can replace the biopsy of intrusive mood to find, check cancer, and his number is of great importance for clinical treatment.In the last few years, there is a lot of platform, such as microfluid, immunomagnetic beadses etc. grow up, and these are all absolutely necessary to the inspection of early-stage cancer and diagnosis.But, now to the separation of CTCs and the method for counting all very complicated, and efficiency is very low.

Summary of the invention

The object of the invention is to provide a kind of Graphene chip of catching cancer cell for whole blood.

Another object of the present invention is to provide a kind of preparation cost cheap, simple and convenient and has the extremely highly sensitive preparation method catching the Graphene chip of cancer cell for whole blood.

The present invention is by selecting novel grapheme material, make the surface of different base pattern, interacted by biotin/biocompatible element (biotin/streptavidin) and connect upper specific antibody anti-EpCAM. in the effect of substrate topology, hardness effect and the collaborative lower of antibody specific recognition effect realize catching the efficient specificity of cancer cell.And in patient blood, reach higher capture rate, for later clinical detection provides simple and feasible method.

Graphene chip of catching cancer cell for whole blood of the present invention is the substrate with petal design utilizing grapheme material to be formed, and described graphenic surface is modified with the antibody (Anti-EpCAM) of specific recognition tumour cell.

The material of described Graphene is the redox graphene material improved, and is oxidation stripping method in the method that the process of carrying out being oxidized is used.

Described oxidation graphene film is by vacuum-thermal reduction, and reduction temperature is more than 100 DEG C, vacuum.

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

The concentration of the graphene oxide solution that described redox graphene substrate is used in preparation process is 0 ~ 7mg/m.

Graphene chip of catching cancer cell for whole blood of the present invention has petal-like micro nano structure, produced by the topological structure of micro nano structure and circulating tumor cell and strengthen adhesion effect, in addition, its distinctive hardness close to living things system is that the adhesion of cell provides the condition of more mating, the film of the redox graphene simultaneously has Superhydrophilic, for it has the condition that ultralow background adsorbs the indispensability provided.

Experimental result shows, Graphene chip of catching cancer cell for whole blood of the present invention to the bioaccumulation efficiency of specific circulating tumor cell (as breast cancer cell MCF7, prostate gland cancer cell PC3) up to more than 90%, and nonspecific clone (as JurkatT lymphocyte, Daudi lymphocyte, Hela uterine cancer cells) but seldom sticks on biochip, thus can be implemented in catching of the efficient circulation tumour cell in biological sample (blood).Such as, patient blood (only needing 1 milliliter) is instilled on biochip of the present invention, can realize the efficient of circulating tumor cell in blood sample and highly sensitive enrichment, and normal cell seldom sticks on biochip.Biochip of the present invention, in clinical trial, can reach extraordinary capture rate and the absorption of extremely low background.

The preparation method catching the Graphene chip of cancer cell for whole blood of the present invention comprises the following steps:

(1) joined by dag in the flask containing fuming nitric aicd and the concentrated sulphuric acid, add potash chlorate when zero degrees celsius, all processes are all when logical nitrogen.Then the object at room temperature in stirred flask.

(2) product that step (1) obtains is added hydrochloric acid, wash sulfate radical and eliminate.

(3) the product high purity water that step (2) obtains is cleaned to pH value neutrality.

(4) method of product high temperature drying step (3) obtained, obtains powder.

(5) powder that step (4) obtains is weighed, be made into the GO solution of the concentration of different ratio.

(6) solution that step (5) obtains is carried out vacuum filtration film forming, make the film of graphene oxide.

(7) film that step (6) obtains is put into vacuum and carry out heat reduction, the temperature of heating is more than 100 DEG C.

(8) film of the redox graphene (rGO) step (7) obtained puts into the solution of a pyrene formic acid, obtains PCA-rGO substrate.

(9) PCA-rGO film step (8) obtained is fixing on the glass sheet, obtains rGO substrate.

(10) the rGO substrate that step (9) obtains is placed in six orifice plates, adds N-hydroxy thiosuccinimide (NHS) and EDC (EDC), at room temperature place a period of time.

(11) the rGO substrate that step (10) obtains is placed in the phosphate buffer of 1 Streptavidin, reacts under room temperature, substrate is taken out and cleans; Obtain HA-rGO.

(12) the HA-rGO substrate that step (11) obtains is added antibody (Anti-EpCAM), ambient temperatare puts reaction, obtains Anti-EpCAM-rGO substrate.

The concentration of the graphene oxide solution that described redox graphene substrate is used in preparation process is 0 ~ 7mg/ml.

It is cheap, simple to operate that Graphene chip of catching cancer cell for whole blood of the present invention has preparation cost, the advantages such as easy suitability for industrialized production, and done clinical trial, has very sensitive cellular affinity and capture rate.Biochip of the present invention is specially adapted to cause the past tumour cell in the blood of transfer all to have good concentration effect to prostate cancer, breast cancer, colon and rectum carcinoma, malignant mela noma, alveolar soft part sarcoma etc.The preparation process of the biochip for circulating tumor cell enrichment and detection of the present invention without objectionable impurities of being always or usually as specified, environmental friendliness, good stability.

Accompanying drawing explanation

Fig. 1. the preparation method of the Graphene biochip that the present invention's whole blood cancer cell used is caught.

Fig. 2. the photo in kind of catching the Graphene chip of cancer cell for whole blood prepared by the present invention.

Fig. 3. the environmental scanning electronic microscope photo after having caught cell of catching the Graphene chip of cancer cell for whole blood prepared by the present invention.

Fig. 4. the Graphene chip of catching cancer cell for whole blood of embodiment of the present invention 1-3,5 preparations catches the quantitative data of five kinds of clones (MCF7, PC3, Hela, Daudi, Jurkat).

Fig. 5. the MCF7 cancer cell in the blood of human body that the Graphene chip of catching cancer cell for whole blood prepared by embodiment of the present invention 6-13 is caught and the trichrome stain figure of normal plasma cell.

Fig. 6. the quantitative data of the cancer cell in the clinical patient blood that the Graphene chip of catching cancer cell for whole blood prepared by embodiment of the present invention 6-13 is caught.

Embodiment

Embodiment 1.

(1) joined by dag in the flask containing fuming nitric aicd and the concentrated sulphuric acid, add potash chlorate when zero degrees celsius, all processes are all when logical nitrogen.Then the object more than 80 hours at room temperature in stirred flask.

(3) it is neutral for the product high purity water that step (2) obtains being cleaned to pH value.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(7) film that step (6) obtains is put into vacuum and carry out heat reduction, the temperature of heating is more than 100 degrees Celsius.

(8) film of the redox graphene (rGO) step (7) obtained puts into the solution of a pyrene formic acid, and room temperature reaction, obtains PCA-rGO substrate.

(10) the rGO substrate that step (9) obtains is placed in six orifice plates, adds N-hydroxy thiosuccinimide (NHS) and EDC (EDC), at room temperature placing response.

(11) the rGO substrate that step (10) obtains is placed in the phosphate buffer of Streptavidin, reacts under room temperature, substrate is taken out and cleans; Obtain HA-rGO.

(13) faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the breast cancer cell MCF7 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 45 minutes), suck breast cancer cell MCF7 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.Breast cancer cell MCF7 catches quantitative data as shown in Fig. 3, Fig. 4.

(14) as a control group 1, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the prostate gland cancer cell PC3 suspending liquid of individual/ml, be placed in cell culture incubator and (preferably carry out 0 ~ 90 minute time of reacting, be more preferably 45 minutes), suck prostate gland cancer cell PC3 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the prostate gland cancer cell PC3 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the prostate gland cancer cell PC3 that biochip is caught is counted, calculate capture rate.Prostate gland cancer cell PC3 catches quantitative data as shown in Figure 4.

(15) as a control group 2, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the human lymphoma cell Jurkat suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 45 minutes), suck human lymphoma cell Jurkat suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the human lymphoma cell Jurkat mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the lymphoma cell Jurkat that biochip is caught is counted, calculate capture rate.Lymphoma cell Jurkat catches quantitative data as shown in Figure 4.

(16) as a control group 3, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵people's lymphocytic B cells oncocyte Daudi suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 45 minutes), suck people's lymphocytic B cells oncocyte Daudi suspending liquid, biochip is fully rinsed in phosphate buffer and washes, people's lymphocytic B cells oncocyte Daudi mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and people's lymphocytic B cells oncocyte Daudi that biochip is caught is counted, calculate capture rate.People's lymphocytic B cells oncocyte Daudi catches quantitative data as shown in Figure 4.

(17) as a control group 4, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the uterine cancer cells Hela suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 45 minutes), suck uterine cancer cells Hela suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the uterine cancer cells Hela mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the uterine cancer cells Hela that biochip is caught is counted, calculate capture rate.Uterine cancer cells Hela catches quantitative data as shown in Figure 4.

Experimental result shows, the capture rate of the breast cancer cell MCF7 of the biochip for circulating tumor cell enrichment and detection of the present invention is 90.2%, the capture rate of prostate gland cancer cell PC3 is 83.2%, the capture rate of human lymphoma cell Jurkat is 0.01%, the capture rate of people's lymphocytic B cells oncocyte Daudi is 0.01%, and the capture rate of uterine cancer cells Hela is 0.02%.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.

Embodiment 2.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(8) film of the redox graphene (rGO) step (7) obtained puts into the solution of a pyrene formic acid, and room temperature placing response, obtains PCA-rGO substrate.

(12) the HA-rGO substrate that step (11) obtains is added antibody (Anti-EpCAM), ambient temperatare is put, and obtains Anti-EpCAM-rGO substrate.

(13) faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the breast cancer cell MCF7 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck breast cancer cell MCF7 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.Breast cancer cell MCF7 catches quantitative data as shown in Fig. 3, Fig. 4.

(14) as a control group 1, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the prostate gland cancer cell PC3 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck prostate gland cancer cell PC3 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the prostate gland cancer cell PC3 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the prostate gland cancer cell PC3 that biochip is caught is counted, calculate capture rate.Prostate gland cancer cell PC3 catches quantitative data as shown in Figure 4.

(15) as a control group 2, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the human lymphoma cell Jurkat suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck human lymphoma cell Jurkat suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the human lymphoma cell Jurkat mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the lymphoma cell Jurkat that biochip is caught is counted, calculate capture rate.Lymphoma cell Jurkat catches quantitative data as shown in Figure 4.

(16) as a control group 3, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵people's lymphocytic B cells oncocyte Daudi suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck people's lymphocytic B cells oncocyte Daudi suspending liquid, biochip is fully rinsed in phosphate buffer and washes, people's lymphocytic B cells oncocyte Daudi mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and people's lymphocytic B cells oncocyte Daudi that biochip is caught is counted, calculate capture rate.People's lymphocytic B cells oncocyte Daudi catches quantitative data as shown in Figure 4.

(17) as a control group 4, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the uterine cancer cells Hela suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck uterine cancer cells Hela suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the uterine cancer cells Hela mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the uterine cancer cells Hela that biochip is caught is counted, calculate capture rate.Uterine cancer cells Hela catches quantitative data as shown in Figure 4.

Experimental result shows, the capture rate of the breast cancer cell MCF7 of the biochip for circulating tumor cell enrichment and detection of the present invention is 91.3%, the capture rate of prostate gland cancer cell PC3 is 82.5%, the capture rate of human lymphoma cell Jurkat is 0.02%, the capture rate of people's lymphocytic B cells oncocyte Daudi is 0.01%, and the capture rate of uterine cancer cells Hela is 0.03%.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.

Embodiment 3.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(8) film of the redox graphene (rGO) step (7) obtained puts into the solution of a pyrene formic acid, reacts, obtain PCA-rGO substrate under room temperature.

(13) faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the breast cancer cell MCF7 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 90 minutes), suck breast cancer cell MCF7 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.Breast cancer cell MCF7 catches quantitative data as shown in Fig. 3, Fig. 4.

(14) as a control group 1, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the prostate gland cancer cell PC3 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 90 minutes), suck prostate gland cancer cell PC3 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the prostate gland cancer cell PC3 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the prostate gland cancer cell PC3 that biochip is caught is counted, calculate capture rate.Prostate gland cancer cell PC3 catches quantitative data as shown in Figure 4.

(15) as a control group 2, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the human lymphoma cell Jurkat suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 90 minutes), suck human lymphoma cell Jurkat suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the human lymphoma cell Jurkat mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the lymphoma cell Jurkat that biochip is caught is counted, calculate capture rate.Lymphoma cell Jurkat catches quantitative data as shown in Figure 4.

(16) as a control group 3, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵people's lymphocytic B cells oncocyte Daudi suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 90 minutes), suck people's lymphocytic B cells oncocyte Daudi suspending liquid, biochip is fully rinsed in phosphate buffer and washes, people's lymphocytic B cells oncocyte Daudi mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, dye with the DAPI phosphate buffer of 10 μ g/ml again, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and people's lymphocytic B cells oncocyte Daudi that biochip is caught is counted, calculate capture rate.People's lymphocytic B cells oncocyte Daudi catches quantitative data as shown in Figure 4.

(17) as a control group 4, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the uterine cancer cells Hela suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 90 minutes), suck uterine cancer cells Hela suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the uterine cancer cells Hela mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the uterine cancer cells Hela that biochip is caught is counted, calculate capture rate.Uterine cancer cells Hela catches quantitative data as shown in Figure 4.

Experimental result shows, capture rate of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 92.2%, the capture rate of prostate gland cancer cell PC3 is 84.2%, the capture rate of human lymphoma cell Jurkat is 0.01%, the capture rate of people's lymphocytic B cells oncocyte Daudi is 0.02%, and the capture rate of uterine cancer cells Hela is 0.02%.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.

Embodiment 4.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(8) film of the redox graphene (rGO) step (7) obtained puts into the solution of a pyrene formic acid, and ambient temperatare is put, and obtains PCA-rGO substrate.

(12) the HA-rGO substrate that step (11) obtains is added 10 μ g/ml antibody (Anti-EpCAM), ambient temperatare is put, and obtains Anti-EpCAM-rGO substrate.

(13) faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the breast cancer cell MCF7 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck breast cancer cell MCF7 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.

(14) as a control group 1, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the prostate gland cancer cell PC3 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck prostate gland cancer cell PC3 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the prostate gland cancer cell PC3 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, dye with the DAPI phosphate buffer of 10 μ g/ml again, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the prostate gland cancer cell PC3 that biochip is caught is counted, calculate capture rate.

(15) as a control group 2, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the human lymphoma cell Jurkat suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck human lymphoma cell Jurkat suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the human lymphoma cell Jurkat mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the lymphoma cell Jurkat that biochip is caught is counted, calculate capture rate.

(16) as a control group 3, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵people's lymphocytic B cells oncocyte Daudi suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck people's lymphocytic B cells oncocyte Daudi suspending liquid, biochip is fully rinsed in phosphate buffer and washes, people's lymphocytic B cells oncocyte Daudi mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and people's lymphocytic B cells oncocyte Daudi that biochip is caught is counted, calculate capture rate.

(17) as a control group 4, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the uterine cancer cells Hela suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 60 minutes), suck uterine cancer cells Hela suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the uterine cancer cells Hela mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the uterine cancer cells Hela that biochip is caught is counted, calculate capture rate.

Experimental result shows, the capture rate of the breast cancer cell MCF7 of the biochip for circulating tumor cell enrichment and detection of the present invention is 91.5%, the capture rate of prostate gland cancer cell PC3 is 81.4%, the capture rate of human lymphoma cell Jurkat is 0.01%, the capture rate of people's lymphocytic B cells oncocyte Daudi is 0.01%, and the capture rate of uterine cancer cells Hela is 0.02%.These data show, Graphene chip of catching cancer cell for whole blood of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.

Embodiment 5.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(13) faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the breast cancer cell MCF7 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 75 minutes), suck breast cancer cell MCF7 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, after dyeing 15 minutes with the DAPI phosphate buffer of 10 μ g/ml again, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.Breast cancer cell MCF7 catches quantitative data as shown in Fig. 3, Fig. 4.

(14) as a control group 1, being faced up by the Anti-EpCAM-rGO substrate that step (12) obtains is placed in 6 orifice plates that to add 3 ml concns be 1x10 ⁵the prostate gland cancer cell PC3 suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 75 minutes), suck prostate gland cancer cell PC3 suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the prostate gland cancer cell PC3 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the prostate gland cancer cell PC3 that biochip is caught is counted, calculate capture rate.Prostate gland cancer cell PC3 catches quantitative data as shown in Figure 4.

(15) as a control group 2, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the human lymphoma cell Jurkat suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 75 minutes), suck human lymphoma cell Jurkat suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the human lymphoma cell Jurkat mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the lymphoma cell Jurkat that biochip is caught is counted, calculate capture rate.Lymphoma cell Jurkat catches quantitative data as shown in Figure 4.

(16) as a control group 3, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵people's lymphocytic B cells oncocyte Daudi suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 75 minutes), suck people's lymphocytic B cells oncocyte Daudi suspending liquid, biochip is fully rinsed in phosphate buffer and washes, people's lymphocytic B cells oncocyte Daudi mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and people's lymphocytic B cells oncocyte Daudi that biochip is caught is counted, calculate capture rate.People's lymphocytic B cells oncocyte Daudi catches quantitative data as shown in Figure 4.

(17) as a control group 4, faced up by the Anti-EpCAM-rGO substrate that step (12) obtains and be placed in 6 orifice plates, adding 3 ml concns is 1x10 ⁵the uterine cancer cells Hela suspending liquid of individual/ml, (time of preferably carrying out reacting is 0 ~ 90 minute to be placed in cell culture incubator, be more preferably 75 minutes), suck uterine cancer cells Hela suspending liquid, biochip is fully rinsed in phosphate buffer and washes, the uterine cancer cells Hela mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fixing after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, again with after the DAPI phosphate buffer dyeing of 10 μ g/ml, rinse in PBS after washing 3 times and dry up with air draught, with (at least 3 substrates at every turn of taking pictures respectively under Nikon inverted fluorescence microscope 10 times, different position, 10, centre chosen by each substrate), and the uterine cancer cells Hela that biochip is caught is counted, calculate capture rate.Uterine cancer cells Hela catches quantitative data as shown in Figure 4.

Experimental result shows, capture rate of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 92.0%, the capture rate of prostate gland cancer cell PC3 is 84.6%, the capture rate of human lymphoma cell Jurkat is 0.01%, the capture rate of people's lymphocytic B cells oncocyte Daudi is 0.01%, and the capture rate of uterine cancer cells Hela is 0.03%.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.

Embodiment 6.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(10) the rGO substrate that step (9) obtains is placed in six orifice plates, adds N-hydroxy thiosuccinimide (NHS) and 1-(3-dimethylamino-propyl :)-3-ethyl carbodiimide (EDC), at room temperature placing response.

(11) the rGO substrate that step (10) obtains is placed in the phosphate buffer of the Streptavidin of 10 μ g/ml, reacts under room temperature, substrate is taken out and cleans; Obtain HA-rGO.

(12) the HA-rGO substrate that step (11) obtains is added 10 μ g/ml antibody (Anti-EpCAM), ambient temperatare puts 1 hour, obtains Anti-EpCAM-rGO substrate.

(13) the Anti-EpCAM-rGO substrate that step (12) obtains is faced up be placed in special double dish, add the blood of 1 milliliter of breast cancer patients, be placed in cell culture incubator (time of preferably carrying out reacting is 0 ~ 90 minute), suck unnecessary blood, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix 20 minutes after, film is worn again with the Trition X-100 phosphate buffer of 0.2% volumetric concentration, rinse in PBS after washing 3 times and dry up with air draught, add blocking agent (5% sheep blood serum of 200 microlitres, 0.1% polysorbas20, the phosphate buffer of 3% bovine serum albumin), at room temperature place one hour.Then add the fluorescently-labeled antibody-solutions of 200 microlitre (initial concentration of 20 μ g/ml, antibody is respectively anti-cytokeratin PE and FITC anti-human CD45) then substrate 4 degrees Celsius of preservations under the condition of shading at night to be spent the night.Add phosphate buffer to clean.Add DAPI phosphate buffer (10 μ g/ml) afterwards, left at room temperature 5 minutes.Then cleaning is carried out 3 times with phosphate buffer.Substrate is reversed, with take pictures respectively under Nikon inverted fluorescence microscope 10 times (at least 3 substrates at every turn, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.The capture-data of breast cancer cell MCF7 is as shown in Fig. 5, Fig. 6.

Experimental result shows, number of catching of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 3.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results is remarkable.

Embodiment 7.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(8) film of the redox graphene (rGO) step (7) obtained puts into the solution of a pyrene formic acid, and room temperature is placed, and obtains PCA-rGO substrate.

(12) the HA-rGO substrate that step (11) obtains is added 10 μ g/ml antibody (Anti-EpCAM), ambient temperatare puts reaction in 1 hour, obtains Anti-EpCAM-rGO substrate.

Experimental result shows, number of catching of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 4.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results is remarkable.

Embodiment 8.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(10) the rGO substrate that step (9) obtains is placed in six orifice plates, adds N-hydroxy thiosuccinimide (NHS) and 1-(3-dimethylamino-propyl :)-3-ethyl carbodiimide (EDC), at room temperature leave standstill reaction.

(13) the Anti-EpCAM-rGO substrate that step (12) obtains is faced up be placed in special double dish, add the blood of 1 milliliter of normal person, be placed in cell culture incubator (time of preferably carrying out reacting is 0 ~ 90 minute), suck unnecessary blood, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix 20 minutes after, film is worn again with the TritionX-100 phosphate buffer of 0.2% volumetric concentration, rinse in PBS after washing 3 times and dry up with air draught, add blocking agent (5% sheep blood serum of 200 microlitres, 0.1% polysorbas20, the phosphate buffer of 3% bovine serum albumin), at room temperature place one hour.Then add the fluorescently-labeled antibody-solutions of 200 microlitre (initial concentration of 20 μ g/ml, antibody is respectively anti-cytokeratinPE and FITCanti-humanCD45) then substrate 4 degrees Celsius of preservations under the condition of shading at night to be spent the night.Add phosphate buffer to clean.Add DAPI phosphate buffer (10 μ g/ml) afterwards, left at room temperature 5 minutes.Then cleaning is carried out 3 times with phosphate buffer.Substrate is reversed, with take pictures respectively under Nikon inverted fluorescence microscope 10 times (at least 3 substrates at every turn, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.The capture-data of breast cancer cell MCF7 is as shown in Fig. 5, Fig. 6.

Experimental result shows, number of catching of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 0.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results is remarkable.

Embodiment 9.

(5) powder that step (4) obtains is weighed, be made into GO solution.

Experimental result shows, number of catching of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 2.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results is remarkable.

Embodiment 10.

(5) powder that step (4) obtains is weighed, be made into GO solution.

(13) the Anti-EpCAM-rGO substrate that step (12) obtains is faced up be placed in special double dish, add the blood of 1 milliliter of breast cancer patients, be placed in cell culture incubator (time of preferably carrying out reacting is 0 ~ 90 minute), suck unnecessary blood, biochip is fully rinsed in phosphate buffer and washes, the breast cancer cell MCF7 mass concentration of then biochip being caught be 4% paraformaldehyde phosphate buffer fix 20 minutes after, film is worn again with the TritionX-100 phosphate buffer of 0.2% volumetric concentration, rinse in PBS after washing 3 times and dry up with air draught, add blocking agent (5% sheep blood serum of 200 microlitres, 0.1% polysorbas20, the phosphate buffer of 3% bovine serum albumin), at room temperature place one hour.Then add the fluorescently-labeled antibody-solutions of 200 microlitre (initial concentration of 20 μ g/ml, antibody is respectively anti-cytokeratinPE and FITCanti-humanCD45) then substrate 4 degrees Celsius of preservations under the condition of shading at night to be spent the night.Add phosphate buffer to clean.Add DAPI phosphate buffer (10 μ g/ml) afterwards, left at room temperature 5 minutes.Then cleaning is carried out 3 times with phosphate buffer.Substrate is reversed, with take pictures respectively under Nikon inverted fluorescence microscope 10 times (at least 3 substrates at every turn, different position, 10, centre chosen by each substrate), and the breast cancer cell MCF7 that biochip is caught is counted, calculate capture rate.The capture-data of breast cancer cell MCF7 is as shown in Fig. 5, Fig. 6.

Embodiment 11.

(5) powder that step (4) obtains is weighed, be made into GO solution.

Experimental result shows, number of catching of catching the breast cancer cell MCF7 of the Graphene chip of cancer cell for whole blood of the present invention is 6.These data show, the chip of catching for circulating tumor cell enrichment of the present invention has very high and sensitive capture rate, and non-specific adsorption is extremely low.Clinical trial results is remarkable.

Embodiment 12.

(5) powder that step (4) obtains is weighed, be made into GO solution.

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

Embodiment 13.

(5) powder that step (4) obtains is weighed, be made into GO solution.

Claims

1. the Graphene biochip for the enrichment of whole blood circulating tumor cell and detection, it is characterized in that: described biochip has the substrate with petal-like micro nano structure that grapheme material is formed, described graphenic surface is modified with the antibody (Anti-EpCAM) of specific recognition tumour cell.

2. according to claim 1 for the Graphene biochip of the enrichment of whole blood circulating tumor cell and detection, it is characterized in that: described grapheme material is the redox graphene material improved, is oxidation stripping method in the method that the process of carrying out being oxidized is used.

3., according to claim 1 for the Graphene biochip of the enrichment of whole blood circulating tumor cell and detection, it is characterized in that: described redox graphene is formed by vacuum-thermal reduction.

4., according to claim 1 for the Graphene biochip of the enrichment of whole blood circulating tumor cell and detection, it is characterized in that: the finishing of described redox graphene has the antibody (Anti-EpCAM) of specific recognition tumour cell.

5., according to claim 1 for the Graphene biochip of the enrichment of whole blood circulating tumor cell and detection, it is characterized in that: solution concentration when preparing the film of graphene oxide is 0 ~ 7mg/ml.

6. according to Claims 1 to 5 any one for a preparation method for the Graphene biochip of the enrichment of whole blood circulating tumor cell and detection, it is characterized in that, described preparation method comprises the following steps:

(1) prepare the raw material of graphene oxide, then utilize the method for vacuum filtration to prepare the graphene oxide membrane of 0 ~ 7mg/ml variable concentrations;

(2) the graphene oxide membrane high temperature reduction under the condition of vacuum step (1) obtained;

(3) film of redox graphene step (2) obtained carries out the modification of a pyrene formic acid, obtains the graphene film PCA-rGO containing a pyrene formic acid;

(4) the PCA-rGO film that step (3) obtains is bonded on glass sheet facilitates following operation;

(5) PCA-rGO substrate activation step (4) obtained is placed in the phosphate buffer of Streptavidin, reacts, taken out by substrate and clean under room temperature;

(6) phosphate buffer of the antibody (Anti-EpCAM) of specific recognition tumour cell is added drop-wise on substrate that step (5) obtains, room temperature is placed, antibody fully absorbed and captures at substrate surface, obtaining the described Graphene biochip for the enrichment of whole blood circulating tumor cell and detection.

7. described preparation method according to claim 6, is characterized in that: the temperature of described vacuum reduction is more than 100 DEG C.

8. described preparation method according to claim 6, is characterized in that: the process preparing the raw material of graphene oxide in step (1) comprises the steps: again

Dag joins in the flask containing fuming nitric aicd and the concentrated sulphuric acid by <1>, potash chlorate is added when zero degrees celsius, all processes all when logical nitrogen, the object then at room temperature in stirred flask;

The product that step <1> obtains by <2> adds hydrochloric acid, washes sulfate radical and eliminates;

The product high purity water that step <2> obtains by <3> cleans to pH value neutrality;

The method of the product high temperature drying that step <3> obtains by <4>, obtains powder.

9. described preparation method according to claim 6, is characterized in that: described in step (5), the process of activation comprises the steps: again

The PCA-rGO substrate that step (4) obtains is placed in six orifice plates, adds N-hydroxy thiosuccinimide (NHS) and EDC (EDC), at room temperature place a period of time.

10. the Graphene biochip for the enrichment of whole blood circulating tumor cell and detection that according to claim 6-9 any one prepared by preparation method.