CN104597242A - Biological chip for enriching tumor marker and application thereof - Google Patents
Biological chip for enriching tumor marker and application thereof Download PDFInfo
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- CN104597242A CN104597242A CN201510063576.7A CN201510063576A CN104597242A CN 104597242 A CN104597242 A CN 104597242A CN 201510063576 A CN201510063576 A CN 201510063576A CN 104597242 A CN104597242 A CN 104597242A
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Abstract
The invention discloses a biological chip for enriching a tumor marker. The biological chip comprises a substrate and a micro flow channel system; the micro flow channel system is distributed on the surface of the substrate and comprises a sample inlet, a channel, a reaction tank, a separated magnet area, a collection tank, a fixed magnet area and a sample outlet in sequence; the sample inlet is communicated with the reaction tank through the channel; the channel is divided into two branched channels after penetrating through the reaction tank; the first branched channel is communicated with the collection tank, and the second branched channel is communicated with the sample outlet; the separated magnet area is arranged on one side of the first branched channel; the fixed magnet area is arranged around the collection tank; magnetic nanoparticle marked antibodies which serve as probes are distributed in the reaction tank; and magnetic nanoparticles are connected with the magnetic nanoparticle marked antibodies through photofission molecules. The invention further discloses application of the biological chip for enriching the tumor marker. The biological chip disclosed by the invention is simple in structure, can be used for enriching the low-concentration tumor marker efficiently and can meet the application demand for sensitivity improvement of the detection field.
Description
Technical field
The present invention relates to a kind of biochip and the application thereof that realize tumor markers enrichment, belong to detection technique field.
Background technology
Malignant tumour is one of major disease threatening people healthy at present, and its incidence of disease in Chinese urban residents ranks first year after year, and early stage to it, quick, sensitive diagnosis is the important channel of saving patient vitals.Current industrial community and scientific circles have developed the detection that multiple strategy carries out tumour, and comprise ELISA method, electrochemical process, iconography detection etc., the detection wherein based on tumour-specific mark has been widely used, shows great using value.Generally speaking, tumor markers concentration is in blood lower, and in conventional sense process, inevitably use Sample pretreatment technology, as washed corpuscles, damping fluid dilution etc., cause the further reduction of tumor-marker substrate concentration, affect the sensitivity of subsequent detection means, limit simultaneously tumor invasion early stage time detection application demand.Therefore, develop a kind of quick, efficient tumor markers beneficiation technologies and there is important using value.
Summary of the invention
Goal of the invention: the deficiency existed for prior art, first object of the present invention is to provide a kind of biochip realizing tumor markers enrichment, can realize the efficiently concentrating of tumor associated marker, and simple to operate, with low cost.
Second object of the present invention there is provided the application of above-mentioned biochip.
For realizing the object of foregoing invention, the technical solution used in the present invention is: a kind of biochip realizing tumor markers enrichment, comprise substrate and micro sprue system, described micro sprue system is distributed in substrate surface, described micro sprue system comprises injection port successively, path, reaction tank, be separated magnetic area, collecting pit, fixing magnetic area and outlet, described injection port is communicated with reaction tank by path, described path separates two branch roads after reaction tank, wherein the first branch road is communicated with collecting pit, second branch road is communicated with outlet, described separation magnetic area is positioned at the side of the first branch road, described fixing magnetic area is around collecting pit, the antibody being distributed with magnetic nanoparticle mark in described reaction tank, as probe, wherein divides sub-connection by photo-cleavage between magnetic nanoparticle and antibody.
Above-mentioned biochip is transferred to substrate surface by micro sprue system, aims at also bonding and fixedly form.
The preparation of described substrate comprises the following steps:
(1) clean substrate, more evenly apply one deck photoresist at substrate surface;
(2) utilize ultraviolet photolithographic technology to prepare magnetic regions photoetching agent pattern, utilize film deposition techniques, magnetic membrane material is deposited on patterned surfaces by magnetron sputtering, and obtain magnetic regions in conjunction with lift-off technology, film thickness is 0.5 μm ~ 10 μm;
(3) evenly one deck photoresist is applied again at substrate surface, in conjunction with the photoetching agent pattern of ultraviolet photolithographic technology preparation feedback pond and collecting pit;
(4) use the technology etched substrate such as dark silicon etching or ion beam etching, obtain reaction tank and collecting pit structure; The degree of depth of reaction tank and collecting pit is 100-500 μm.
The preparation of described micro sprue system and integratedly to comprise the following steps:
(1) ultraviolet photolithographic technology is utilized to prepare fluid channel punch at silicon chip surface, projective structure height 50-500 μm;
(2) fluid channel material be cast in convex mould surface and solidify, after the demoulding, obtaining micro-channel structure;
(3) hydrophilic treatment is carried out to micro-channel structure surface, obtain the micro sprue system with Superhydrophilic matter.
Described substrate is that silicon chip or piezoid are made; Described micro sprue system, its material is dimethyl silicone polymer (PDMS) or SU-8 glue.
The material of described magnetic nanoparticle is Fe
3o
4nano particle, SiO
2the Fe of parcel
3o
4one in nano particle, FeCo nano particle, FePt nano particle and Co nano particle etc.; Be of a size of 5-50nm.
Described photo-cleavage molecule comprises adjacent nitrobenzyl derivatives, and the wavelength of described illumination is 200-400nm.
Described separation magnetic area and fixing magnetic area, by Fe
3o
4, any one composition in FeCo, CoPt material.
Described tumor markers include but not limited in alpha-fetoprotein and heteroplasmon (AFP, AFP-L3), carcinomebryonic antigen (CEA), interleukin-6 (IL-6), serum cancer antigen (CA199, CA742), cytokeratin (CK19) etc. one or more.
Realize an application for the biochip of tumor markers enrichment, comprise the following steps:
(1) first utilize syringe pump or peristaltic pump to be delivered to by micro sprue system by sample to be tested in the reaction tank (3) of chip and also stablize 1-12 hour, be fully combined with probe to make the target tumor mark in sample to be tested;
(2) continue to utilize syringe pump or peristaltic pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit by the attraction being separated magnetic area;
(3) in collecting pit, apply illumination, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers;
(4) collect the tumor markers solution of enrichment and carry out subsequent treatment; Subsequent treatment comprises extraction and the purifying of target tumor mark, can also carry out bio-sensing simultaneously and measure the process such as the content to detect tumor markers in sample.
Described sample is whole blood, serum or phosphate buffer (PBS);
The antibody being distributed with magnetic nanoparticle mark in reaction tank, as probe, wherein divides sub-connection by photo-cleavage between magnetic nanoparticle and antibody; Antibody can with target tumor mark generation specific binding; The separation magnetic area of chip surface can optionally attract magnetic nanoparticle to flow to collecting pit; The illumination applying certain wavelength in collecting pit can promote photo-cleavage molecular breakdown, and magnetic nanoparticle is separated with tumor markers, thus achieves the enrichment of tumor markers in sample.
Beneficial effect: compared with prior art, advantage of the present invention comprises:
(1) the present invention is by using magnetic nanoparticle in conjunction with the target tumor mark in sample, in conjunction with separation magnetic area, the shunting of magnetic nanoparticle in micro sprue system can be realized, optionally flow into the collecting pit of chip surface, facilitate the efficiently concentrating of tumor markers;
(2) utilize lighting to combine to be separated magnetic area and achieve being separated of magnetic nanoparticle and tumor markers, the magnetic nanoparticle effectively reduced in sample remains, and improves the tumor markers purity after enrichment;
(3) biochip involved in the present invention adapts to whole blood sample, can avoid the loss causing tumor markers because of Sample pretreatment process, can meet subsequent detection application demand.
Accompanying drawing explanation
The biochip pattern vertical view of Fig. 1 a kind of tumor markers enrichment involved in the present invention; Injection port in 1-micro sprue system, the path in 2-micro sprue system, the reaction tank of 3-substrate surface, the separation magnetic area of 4-substrate surface, the collecting pit of 5-substrate surface, the fixing magnetic area of 6-substrate surface, the outlet in 7-micro sprue system;
The sectional view of Fig. 2 biochip involved in the present invention: 8-magnetic nanoparticle probe, 9-substrate, 10-micro sprue system;
In Fig. 3 chip manufacture process of the present invention, substrate surface applies one deck photoresist profile figure: 11-photoresist;
In Fig. 4 chip manufacture process of the present invention, after ultraviolet photolithographic, the photoetching agent pattern sectional view of substrate surface, in figure with the separation magnetic area of chip surface for signal;
In Fig. 5 chip manufacture process of the present invention, the magnetic membrane material of substrate surface deposition, this figure is to be separated the material of magnetic area for signal;
In Fig. 6 chip manufacture process of the present invention, on the basis of Fig. 5, in the process of preparation feedback pond and collecting pit, evenly apply one deck photoresist profile figure at chip surface;
In Fig. 7 chip manufacture process of the present invention, after ultraviolet photolithographic, the photoetching agent pattern sectional view of substrate surface;
In Fig. 8 chip manufacture process of the present invention, etching technics is utilized to obtain reaction tank and collecting pit sectional view;
In Fig. 9 chip manufacture process of the present invention, the substrat structure sectional view obtained after degumming process;
Figure 10 prepares in micro sprue system process, first utilizes spin processes and ultraviolet photolithographic technique to obtain photoetching agent pattern sectional view on silicon chip substrate surface;
Figure 11 is prepared in micro sprue system process, the silicon chip punch obtained after degumming process;
Fluid channel material is cast in silicon chip convex mould surface schematic diagram by Figure 12,10-fluid channel material;
The fluid channel sectional view of Figure 13 after releasing process process;
Figure 14 utilizes card punch to define fluid channel import, outlet sections figure;
Figure 15 magnetic nanoparticle probe schematic diagram: 12-magnetic nanoparticle, 13-photo-cleavage molecule, 14-corresponds to the antibody of tumor markers.
Embodiment
Below in conjunction with the drawings and specific embodiments, illustrate the present invention further, the present embodiment is implemented under premised on technical solution of the present invention, should understand these embodiments and only be not used in for illustration of the present invention and limit the scope of the invention.
embodiment 1
A kind of biochip realizing tumor markers enrichment, comprise substrate 9 and micro sprue system 10, micro sprue system 10 is distributed in substrate 9 surface, micro sprue system 10 comprises injection port 1, path 2, reaction tank 3 successively, is separated magnetic area 4, collecting pit 5, fixing magnetic area 6 are communicated with reaction tank 3 by path 2 with outlet 7, injection port 1, path 2 separates two branch roads after reaction tank 3, wherein the first branch road is communicated with collecting pit 5, second branch road is communicated with outlet 7, be separated the side that magnetic area 4 is positioned at the first branch road, fixing magnetic area 6 is around collecting pit 5; The antibody being distributed with magnetic nanoparticle mark in reaction tank 3, as probe, wherein divides sub-connection by photo-cleavage between magnetic nanoparticle and antibody.This biochip is transferred to substrate 9 surface by micro sprue system 10, aims at also bonding and fixedly form.
The preparation of substrate 9 comprises the following steps:
(1) clean substrate, more evenly apply one deck photoresist at substrate surface;
(2) utilize ultraviolet photolithographic technology to prepare magnetic regions photoetching agent pattern, utilize film deposition techniques, magnetic membrane material is deposited on patterned surfaces by magnetron sputtering, and obtain magnetic regions in conjunction with lift-off technology, film thickness is 0.5 μm ~ 10 μm;
(3) evenly one deck photoresist is applied again at substrate surface, in conjunction with the photoetching agent pattern of ultraviolet photolithographic technology preparation feedback pond and collecting pit;
(4) use the technology etched substrate such as dark silicon etching or ion beam etching, obtain reaction tank and collecting pit structure; The degree of depth of reaction tank and collecting pit is 100-500 μm.
The preparation of micro sprue system 10 and integratedly to comprise the following steps:
(1) ultraviolet photolithographic technology is utilized to prepare fluid channel punch at silicon chip surface, projective structure height 50-500 μm;
(2) fluid channel material be cast in convex mould surface and solidify, after the demoulding, obtaining micro-channel structure;
(3) hydrophilic treatment is carried out to micro-channel structure surface, obtain the micro sprue system with Superhydrophilic matter.
the enrichment of embodiment 2 alpha-fetoprotein (AFP):
(1) preparation of biochip:
The preparation of substrat structure: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes for silicon chip first successively, utilize glue evenning table to apply one deck AZ5214 photoresist on surface, coating thickness is 1.6 μm, as shown in Figure 3; Utilize ultraviolet photolithographic technique to prepare photoetching agent pattern at silicon chip surface, define and be separated magnetic area and fixing magnetic area, as shown in Figure 4; Utilize magnetron sputtering apparatus deposited magnetic Fe further
3o
4film, deposit thickness 0.5 μm; And then use acetone, isopropyl alcohol to carry out stripping technology to remove AZ5214 glue.By the enforcement of this technique, can prepare at silicon chip surface and be separated magnetic area and fixing magnetic area, as shown in Figure 5.
Utilize glue evenning table at above-mentioned silicon chip surface coating AZ4620 photoresist further, coating thickness is 10 μm, as shown in Figure 6; Utilize ultraviolet photolithographic to prepare photoetching agent pattern at silicon chip surface, define reaction tank and collecting pit, as shown in Figure 7; Dark silicon etching technology is utilized to etch silicon, etching depth 100 μm, as shown in Figure 8; Use acetone, isopropyl alcohol cleaning silicon chip, after the AZ4620 photoresist of removing silicon chip surface, reaction tank and collecting pit structure can be prepared.By the enforcement of this technique, the substrat structure comprising reaction tank, collecting pit, separation magnetic area and collect magnetic area can be obtained, as shown in Figure 9.
The preparation of micro sprue system: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes to silicon chip first successively, glue evenning table is utilized to apply one deck AZ4620 photoresist on surface, coating thickness is 10 μm, utilizes ultraviolet photolithographic to obtain fluid channel photoetching agent pattern, as shown in Figure 10; Dark silicon etching is utilized to etch silicon chip, etching depth 50 μm, as shown in figure 11.Silicon fluoride process is carried out to silicon chip, makes surface presentation superhydrophobic property so that the stripping of further microfluidic road material.Dimethyl silicone polymer (PDMS) is coated to silicon chip surface and solidifies process, as shown in figure 12.Silicon chip surface is gone to strip down PDMS after solidification, as shown in figure 13.Further, utilize card punch in the punching of PDMS surface, obtain fluid channel import and outlet, region is therebetween then the path in micro sprue system, as shown in figure 14.
Micro sprue system integrated: first utilize oxygen plasma system to carry out surface treatment to micro sprue system and silicon chip substrate, obtain super hydrophilic surface, then aims at the two the preparation that bonding can complete biochip, as shown in Figure 2.
(2) enrichment of AFP
Use 5nm Fe
3o
4nano particle and adjacent nitrobenzyl derivatives molecule, AFP antibody prepare probe jointly, and are fixed in advance in the reaction tank of biochip by probe.In the process of AFP enrichment, first utilizing syringe pump by stablizing 1 hour in the reaction tank being delivered to chip containing the whole blood sample of 1 μ g/mLAFP by micro sprue system, being fully combined with probe to make AFP; Continue to utilize syringe pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit by the attraction being separated magnetic area; In collecting pit, apply the ultraviolet lighting that wavelength is 200nm, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers; Use pipettor to collect the AFP solution of enrichment, the AFP concentration utilizing Coomassie Brilliant Blue to detect institute's enrichment finds, final enriched concentration reaches 20 μ g/mL.
the common enrichment of embodiment 3 carcinomebryonic antigen (CEA), alpha-fetoprotein variant (AFP-L3) and interleukin-6 (IL-6):
(1) preparation of biochip:
The preparation of substrat structure: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes for silicon chip first successively, utilize glue evenning table to apply one deck AZ5214 photoresist on surface, coating thickness is 2 μm, as shown in Figure 3; Utilize ultraviolet photolithographic technique to prepare photoetching agent pattern at silicon chip surface, define and be separated magnetic area and fixing magnetic area, as shown in Figure 4; Utilize magnetron sputtering apparatus deposited magnetic FeCo film further, deposit thickness 1 μm; And then use acetone, isopropyl alcohol to carry out stripping technology to remove AZ5214 glue.By the enforcement of this technique, can prepare at silicon chip surface and be separated magnetic area and fixing magnetic area, as shown in Figure 5.
Utilize glue evenning table at above-mentioned silicon chip surface coating AZ4620 photoresist further, coating thickness is 30 μm, as shown in Figure 6; Utilize ultraviolet photolithographic to prepare photoetching agent pattern at silicon chip surface, define reaction tank and collecting pit, as shown in Figure 7; Dark silicon etching technology is utilized to etch silicon, etching depth 150 μm, as shown in Figure 8; Use acetone, isopropyl alcohol cleaning silicon chip, after the AZ4620 photoresist of removing silicon chip surface, reaction tank and collecting pit structure can be prepared.By the enforcement of this technique, the substrat structure comprising reaction tank, collecting pit, separation magnetic area and collect magnetic area can be obtained, as shown in Figure 9.
The preparation of micro sprue system: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes to silicon chip first successively, glue evenning table is utilized to apply one deck AZ4620 photoresist on surface, coating thickness is 30 μm, utilizes ultraviolet photolithographic to obtain fluid channel photoetching agent pattern, as shown in Figure 10; Dark silicon etching is utilized to etch silicon chip, etching depth 150 μm, as shown in figure 11.Silicon fluoride process is carried out to silicon chip, makes surface presentation superhydrophobic property so that the stripping of further microfluidic road material.SU-8 glue is coated to silicon chip surface and solidifies process, as shown in figure 12.Silicon chip surface is gone to strip down SU-8 after solidification, as shown in figure 13.Further, utilize card punch in the punching of SU-8 surface, obtain fluid channel import and outlet, region is therebetween then the path in micro sprue system, as shown in figure 14.
Micro sprue system integrated: first utilize oxygen plasma system to carry out surface treatment to micro sprue system and silicon chip substrate, obtain super hydrophilic surface, then aims at the two the preparation that bonding can complete biochip, as shown in Figure 2.
(2) enrichment of CEA, AFP-L3 and IL-6
Use 20nm SiO
2the Fe of parcel
3o
4nano particle and adjacent nitrobenzyl derivatives molecule, CEA, AFP-L3 and IL-6 antibody prepare probe respectively, and are fixed in advance in the reaction tank of biochip by probe.In the process of the common enrichment of CEA, AFP-L3 and IL-6, first utilizing peristaltic pump by stablizing 4 hours in the reaction tank being delivered to chip containing the serum sample of 1 μ g/mLCEA, 1 μ g/mLAFP-L3 and 1 μ g/mLIL-6 by micro sprue system, fully combining with probe to make CEA, AFP-L3 and IL-6; Continue to utilize peristaltic pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit by the attraction being separated magnetic area; In collecting pit, apply the illumination that wavelength is 365nm, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers; Pipettor is used to collect the CEA of enrichment, the solution of AFP-L3 and IL-6, utilize the two anti-fixedly separated protein concentration discoveries detecting institute's enrichment in conjunction with Coomassie Brilliant Blue respectively, final enriched concentration can reach 30 μ g/mL CEA, 20 μ g/mL AFP-L3 and 50 μ g/mL IL-6.
the enrichment of embodiment 4 serum cancer antigen (CA199):
(1) preparation of biochip:
The preparation of substrat structure: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes for silicon chip first successively, utilize glue evenning table to apply one deck AZ4620 photoresist on surface, coating thickness is 50 μm, as shown in Figure 3; Utilize ultraviolet photolithographic technique to prepare photoetching agent pattern at silicon chip surface, define and be separated magnetic area and fixing magnetic area, as shown in Figure 4; Utilize magnetron sputtering apparatus deposited magnetic CoPt film further, deposit thickness 10 μm; And then use acetone, isopropyl alcohol to carry out stripping technology to remove AZ4620 glue.By the enforcement of this technique, can prepare at silicon chip surface and be separated magnetic area and fixing magnetic area, as shown in Figure 5.
Utilize glue evenning table at above-mentioned silicon chip surface coating AZ4620 photoresist further, coating thickness is 50 μm, as shown in Figure 6; Utilize ultraviolet photolithographic to prepare photoetching agent pattern at silicon chip surface, define reaction tank and collecting pit, as shown in Figure 7; Dark silicon etching technology is utilized to etch silicon, etching depth 500 μm, as shown in Figure 8; Use acetone, isopropyl alcohol cleaning silicon chip, after the AZ4620 photoresist of removing silicon chip surface, reaction tank and collecting pit structure can be prepared.By the enforcement of this technique, the substrat structure comprising reaction tank, collecting pit, separation magnetic area and collect magnetic area can be obtained, as shown in Figure 9.
The preparation of micro sprue system: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes to silicon chip first successively, glue evenning table is utilized to apply one deck AZ4620 photoresist on surface, coating thickness is 50 μm, utilizes ultraviolet photolithographic to obtain fluid channel photoetching agent pattern, as shown in Figure 10; Dark silicon etching is utilized to etch silicon chip, etching depth 500 μm, as shown in figure 11.Silicon fluoride process is carried out to silicon chip, makes surface presentation superhydrophobic property so that the stripping of further microfluidic road material.Dimethyl silicone polymer (PDMS) is coated to silicon chip surface and solidifies process, as shown in figure 12.Silicon chip surface is gone to strip down PDMS after solidification, as shown in figure 13.Further, utilize card punch to bore mouth on PDMS surface, obtain fluid channel import and outlet, region is therebetween then the path in micro sprue system, as shown in figure 14.
Micro sprue system integrated: first utilize oxygen plasma system to carry out surface treatment to micro sprue system and silicon chip substrate, obtain super hydrophilic surface, then aims at the two the preparation that bonding can complete biochip, as shown in Figure 2.
(2) enrichment of CA199
Use 50nm FeCo nano particle and adjacent nitrobenzyl derivatives molecule, CA199 antibody jointly to prepare probe, and probe is fixed in the reaction tank of biochip in advance.In the process of CA199 enrichment, first utilizing syringe pump by stablizing 12 hours in the reaction tank being delivered to chip containing phosphate buffered solution (PBS) sample of 1 μ g/mLCA199 by micro sprue system, being fully combined with probe to make CA199; Continue to utilize syringe pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit by the attraction being separated magnetic area; In collecting pit, apply the illumination that wavelength is 400nm, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers; Use pipettor to collect the CA199 solution of enrichment, the protein concentration utilizing Coomassie Brilliant Blue to detect institute's enrichment finds, final enriched concentration can reach 100 μ g/mL CA199.
the enrichment of embodiment 5 serum cancer antigen (CA742):
(1) preparation of biochip:
The preparation of substrat structure: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes for piezoid first successively, utilize glue evenning table to apply one deck AZ5214 photoresist on surface, coating thickness is 1.6 μm, as shown in Figure 3; Utilize ultraviolet photolithographic technique to prepare photoetching agent pattern on piezoid surface, define and be separated magnetic area and fixing magnetic area, as shown in Figure 4; Utilize magnetron sputtering apparatus deposited magnetic Fe further
3o
4film, deposit thickness 0.5 μm; And then use acetone, isopropyl alcohol to carry out stripping technology to remove AZ5214 glue.By the enforcement of this technique, can prepare on piezoid surface and be separated magnetic area and fixing magnetic area, as shown in Figure 5.
Utilize glue evenning table at above-mentioned piezoid surface coating AZ4620 photoresist further, coating thickness is 10 μm, as shown in Figure 6; Utilize ultraviolet photolithographic to prepare photoetching agent pattern on piezoid surface, define reaction tank and collecting pit, as shown in Figure 7; Ion beam etching technology is utilized to etch quartz, etching depth 100 μm, as shown in Figure 8; Use acetone, isopropyl alcohol cleaning piezoid, after the AZ4620 photoresist on removing piezoid surface, reaction tank and collecting pit structure can be prepared.By the enforcement of this technique, the substrat structure comprising reaction tank, collecting pit, separation magnetic area and collect magnetic area can be obtained, as shown in Figure 9.
The preparation of micro sprue system: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes to silicon chip first successively, glue evenning table is utilized to apply one deck AZ4620 photoresist on surface, coating thickness is 10 μm, utilizes ultraviolet photolithographic to obtain fluid channel photoetching agent pattern, as shown in Figure 10; Dark silicon etching is utilized to etch silicon chip, etching depth 50 μm, as shown in figure 11.Silicon fluoride process is carried out to silicon chip, makes surface presentation superhydrophobic property so that the stripping of further microfluidic road material.Dimethyl silicone polymer (PDMS) is coated to silicon chip surface and solidifies process, as shown in figure 12.Silicon chip surface is gone to strip down PDMS after solidification, as shown in figure 13.Further, utilize card punch to bore mouth on PDMS surface, obtain fluid channel import and outlet, region is therebetween then the path in micro sprue system, as shown in figure 14.
Micro sprue system integrated: first utilize oxygen plasma system to carry out surface treatment to micro sprue system and piezoid substrate, obtain super hydrophilic surface, then aims at the two the preparation that bonding can complete biochip, as shown in Figure 2.
(2) enrichment of CA742
Use 10nm FePt nano particle and adjacent nitrobenzyl derivatives molecule, CA742 antibody jointly to prepare probe, and probe is fixed in the reaction tank of biochip in advance.In the process of CA742 enrichment, first utilizing syringe pump by stablizing 12 hours in the reaction tank being delivered to chip containing the whole blood sample of 1 μ g/mLCA742 by micro sprue system, being fully combined with probe to make CA742; Continue to utilize syringe pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit by the attraction being separated magnetic area; In collecting pit, apply the illumination that wavelength is 365nm, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers; Use pipettor to collect the CA742 solution of enrichment, the protein concentration utilizing Coomassie Brilliant Blue to detect institute's enrichment finds, final enriched concentration can reach 150 μ g/mL CA742.
the enrichment of embodiment 6 cytokeratin (CK19):
(1) preparation of biochip:
The preparation of substrat structure: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes for silicon chip first successively, utilize glue evenning table to apply one deck AZ5214 photoresist on surface, coating thickness is 1.6 μm, as shown in Figure 3; Utilize ultraviolet photolithographic technique to prepare photoetching agent pattern at silicon chip surface, define and be separated magnetic area and fixing magnetic area, as shown in Figure 4; Utilize magnetron sputtering apparatus deposited magnetic Fe further
3o
4film, deposit thickness 0.5 μm; And then use acetone, isopropyl alcohol to carry out stripping technology to remove AZ5214 glue.By the enforcement of this technique, can prepare at silicon chip surface and be separated magnetic area and fixing magnetic area, as shown in Figure 5.
Utilize glue evenning table at above-mentioned silicon chip surface coating AZ4620 photoresist further, coating thickness is 10 μm, as shown in Figure 6; Utilize ultraviolet photolithographic to prepare photoetching agent pattern at silicon chip surface, define reaction tank and collecting pit, as shown in Figure 7; Dark silicon etching technology is utilized to etch silicon, etching depth 100 μm, as shown in Figure 8; Use acetone, isopropyl alcohol cleaning silicon chip, after the AZ4620 photoresist of removing silicon chip surface, reaction tank and collecting pit structure can be prepared.By the enforcement of this technique, the substrat structure comprising reaction tank, collecting pit, separation magnetic area and collect magnetic area can be obtained, as shown in Figure 9.
The preparation of micro sprue system: use acetone, isopropyl alcohol and ultrapure water to carry out ultrasonic cleaning each 15 minutes to silicon chip first successively, glue evenning table is utilized to apply one deck AZ4620 photoresist on surface, coating thickness is 10 μm, utilizes ultraviolet photolithographic to obtain fluid channel photoetching agent pattern, as shown in Figure 10; Dark silicon etching is utilized to etch silicon chip, etching depth 50 μm, as shown in figure 11.Silicon fluoride process is carried out to silicon chip, makes surface presentation superhydrophobic property so that the stripping of further microfluidic road material.Dimethyl silicone polymer (PDMS) is coated to silicon chip surface and solidifies process, as shown in figure 12.Silicon chip surface is gone to strip down PDMS after solidification, as shown in figure 13.Further, utilize card punch to bore mouth on PDMS surface, obtain fluid channel import and outlet, region is therebetween then the path in micro sprue system, as shown in figure 14.
Micro sprue system integrated: first utilize oxygen plasma system to carry out surface treatment to micro sprue system and silicon chip substrate, obtain super hydrophilic surface, then aims at the two the preparation that bonding can complete biochip, as shown in Figure 2.
(2) enrichment of CK19
Use 20nm Co nano particle and adjacent nitrobenzyl derivatives molecule, CK19 antibody jointly to prepare probe, and probe is fixed in the reaction tank of biochip in advance.In the process of CK19 enrichment, first utilizing syringe pump by stablizing 4 hours in the reaction tank being delivered to chip containing the buffer solution sample of 1 μ g/mLCK19 by micro sprue system, being fully combined with probe to make CK19; Continue to utilize syringe pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit by the attraction being separated magnetic area; In collecting pit, apply the illumination that wavelength is 365nm, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers; Use pipettor to collect the CK19 solution of enrichment, the protein concentration utilizing Coomassie Brilliant Blue to detect institute's enrichment finds, final enriched concentration can reach 75 μ g/mL CK19.
The above, it is only preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, do not departing within the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be do not depart from technical solution of the present invention content, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (10)
1. one kind realizes the biochip of tumor markers enrichment, it is characterized in that, comprise substrate (9) and micro sprue system (10), described micro sprue system (10) is distributed in substrate (9) surface, and described micro sprue system (10) comprises injection port (1), path (2), reaction tank (3) successively, is separated magnetic area (4), collecting pit (5), fixes magnetic area (6) and outlet (7); Described injection port (1) is communicated with reaction tank (3) by path (2), described path (2) separates two branch roads after reaction tank (3), wherein the first branch road is communicated with collecting pit (5), second branch road is communicated with outlet (7), described separation magnetic area (4) is positioned at the side of the first branch road, and described fixing magnetic area (6) is around collecting pit (5); The antibody being distributed with magnetic nanoparticle mark in described reaction tank (3), as probe, wherein divides sub-connection by photo-cleavage between magnetic nanoparticle and antibody.
2. a kind of biochip realizing tumor markers enrichment according to claim 1, is characterized in that, described biochip is transferred to substrate (9) surface by micro sprue system (10), aims at also bonding and fixedly form.
3. a kind of biochip realizing tumor markers enrichment according to claim 1, is characterized in that, the preparation of described substrate (9) comprises the following steps:
(1) clean substrate, more evenly apply one deck photoresist at substrate surface;
(2) utilize ultraviolet photolithographic technology to prepare magnetic regions photoetching agent pattern, utilize film deposition techniques, magnetic membrane material is deposited on patterned surfaces by magnetron sputtering, and obtain magnetic regions in conjunction with lift-off technology, film thickness is 0.5 μm ~ 10 μm;
(3) evenly one deck photoresist is applied again at substrate surface, in conjunction with the photoetching agent pattern of ultraviolet photolithographic technology preparation feedback pond and collecting pit;
(4) use the technology etched substrate such as dark silicon etching or ion beam etching, obtain reaction tank and collecting pit structure; The degree of depth of reaction tank and collecting pit is 100-500 μm.
4. a kind of biochip realizing tumor markers enrichment according to claim 1, is characterized in that, the preparation of described micro sprue system (10) and integratedly to comprise the following steps:
(1) ultraviolet photolithographic technology is utilized to prepare fluid channel punch at silicon chip surface, projective structure height 50-500 μm;
(2) fluid channel material be cast in convex mould surface and solidify, after the demoulding, obtaining micro-channel structure;
(3) hydrophilic treatment is carried out to micro-channel structure surface, obtain the micro sprue system with Superhydrophilic matter.
5. a kind of biochip realizing tumor markers enrichment according to claim 1, described substrate (9) is made for silicon chip or piezoid; Described micro sprue system (10), its material is dimethyl silicone polymer (PDMS) or SU-8 glue.
6. a kind of biochip realizing tumor markers enrichment according to claim 1, is characterized in that, the material of described magnetic nanoparticle is Fe
3o
4nano particle, SiO
2the Fe of parcel
3o
4one in nano particle, FeCo nano particle, FePt nano particle and Co nano particle etc.; Be of a size of 5-50nm.
7. a kind of biochip realizing tumor markers enrichment according to claim 1, it is characterized in that, described photo-cleavage molecule comprises adjacent nitrobenzyl derivatives, and the wavelength of described illumination is 200-400nm.
8. a kind of biochip realizing tumor markers enrichment according to claim 1, is characterized in that, described separation magnetic area (4) and fixing magnetic area (6), by Fe
3o
4, any one composition in FeCo, CoPt material.
9. a kind of biochip realizing tumor markers enrichment according to claim 1, it is characterized in that, described tumor markers comprise in alpha-fetoprotein and heteroplasmon thereof, carcinomebryonic antigen, interleukin-6, serum cancer antigen, cytokeratin one or more.
10. a kind of application realizing the biochip of tumor markers enrichment described in any one of claim 1 ~ 9, is characterized in that, comprise the following steps:
(1) first utilize syringe pump or peristaltic pump to be delivered to by micro sprue system (10) by sample to be tested in the reaction tank (3) of chip and also stablize 1-12 hour, be fully combined with probe to make the target tumor mark in sample to be tested;
(2) continue to utilize syringe pump or peristaltic pump solution to be delivered to fluid channel outlet, in course of conveying, probe can enter in collecting pit (5) by the attraction being separated magnetic area (4);
(3) in collecting pit (5), apply illumination, promote the photo-cleavage molecular breakdown in probe, be separated magnetic nanoparticle and tumor markers;
(4) collect the tumor markers solution of enrichment and carry out subsequent treatment.
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