CN106353497A - Multi-tumor-marker detecting platform and method based on SERS detecting technology and micro-fluidic chip - Google Patents
Multi-tumor-marker detecting platform and method based on SERS detecting technology and micro-fluidic chip Download PDFInfo
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Abstract
The invention discloses a multi-tumor-marker detecting platform based on a SERS detecting technology and a micro-fluidic chip. A SERS substrate is formed in a micro-fluidic channel at first to improve detection sensitivity, and then a typical sandwich structure is formed on the SERS substrate to detect whether a to-be-detected reagent contains specific tumor markers or not. The substrate is connected with a specific capturing antibody by a PDMS chip at first, and then a placing position of a new PDMS chip is perpendicular to a previous placing position to achieve the effect of simultaneously detecting multiple tumor markers in an intersection region. A SERS immunoprobe which is connected with a second antibody and a Raman reporting molecule to achieve the effect of qualitatively and quantitatively detecting the tumor marks in the to-be-detected reagent, the second antibody can amplify signals, and therefore, detection sensitivity can be improved further. The whole platform can simultaneously detect the multiple tumor markers qualitatively and quantitatively, and provides favorable technical support for early diagnosis of cancer of cancerous persons and increasing of survival rate.
Description
Technical field
The invention belongs to spectroscopy and bioassay technique field are and in particular to a kind of be based on sers detection technique and miniflow
The Diagnostic Value of Several Serum Tumor Markers detection platform of control chip and method.
Background technology
In recent years, although current people increasingly understand the evolution process of breast carcinoma, medical mode also constantly improving,
But the mortality rate of patient with breast cancer is still very high.One of reason is exactly that patient with breast cancer is diagnosed with mammary gland
Cancer ratio is later, so the early diagnosiss accuracy improving patient with breast cancer seems most important, this can improve patient with breast cancer's
Survival rate.
Up to the present, substantial amounts of experiment has been had to prove that tumor markerses have for early diagnosis of cancer and monitoring very big
Potentiality.But only detect a kind of tumor markerses thus judging whether patient is often inaccurate with cancer, examine simultaneously
Surveying Diagnostic Value of Several Serum Tumor Markers can increase the accuracy of early diagnosis of cancer.As detected three kinds of breast cancer tumour marks simultaneously
(antigen 15-3) ca153, (antigen 12-5) ca125, (carcinoembryonic antigen) cea can improve
The accuracy of breast cancer diagnosis has been proved to, so we need a platform can detect Diagnostic Value of Several Serum Tumor Markers simultaneously.
Detection for cancer mark mainly has sers, elisa, lspr, spr, ria, cleia etc. at present.
Due to content in earlier stage cancer patients' body for the mark seldom, such as breast cancer tumour mark (ca153 < 30u/ml ca125 <
35u/ml cea≤5.9ng/ml), and sers can improve the sensitivity of detection, so using sers detection technique.It is based on
The detection method of sers is broadly divided into no marks and has mark detection, and no mark detection refers to that biomolecule is directly detected, and has mark
It is detected as the probe using connecting upper Raman reporter molecules and indirectly detect target molecule.Due to there being mark detection to compare no mark detection
The sensitivity of detection can be greatly improved, so there being mark detection method to be used.
In recent years, because micro-fluidic chip has, liquid flowing is controlled, it is few with reagent to consume sample, analyze speed becomes ten
Times hundreds of times ground improves, and analysis while can carrying out up to a hundred samples within even shorter time a few minutes, and
Can be with the advantage of the pretreatment of canbe used on line sample and analysis overall process etc. so that dna, protein be carried out based on micro-fluidic chip
Detection Deng trace substance has obtained increasingly being widely applied.Simultaneously micro-fluidic chip due to its small volume be easy to collect
Become, using sample and reagent few while can also obtain high channel, reagent the features such as in microfluidic channel, response speed is fast with
And obtain substantial amounts of use in biology, the field such as chemistry, medical science.In view of the portability in practical application and Patient Sample A's amount
The reason such as few, we have selected pdms chip.
Content of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, improve the accuracy of early-stage cancer diagnosis and sensitive
Degree, the present invention provides a kind of Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip, can be simultaneously
Detection Diagnostic Value of Several Serum Tumor Markers simultaneously can improve detection sensitivity, can greatly strengthen the detection sensitivity of tumor markerses, be
The diagnosis of early-stage cancer provides strong technical support.
Technical scheme: for achieving the above object, the technical solution used in the present invention is:
A kind of Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip, including by micro-fluidic
Microfluidic channel and its interior sers substrate that chip is made, and sers probe and detection antibody；
Described microfluidic channel is cruciform shape, and interconnection is interior to be described sers substrate, in vertical vertical passage
It is passed through described sers probe and detects antibody, intersection region is detection zone；
Described sers substrate is passed through in described microfluidic channel by metal nanoparticle and obtains, and described sers substrate connects
There is capture antibody；
Described sers probe by metal nanoparticle respectively with Raman reporter molecules, two anti-connections, then carry out gold with bsa
Filling up of metal surface space is made.
Further, described micro-fluidic chip is pdms chip.
Further, described capture antibody is no less than two kinds.
A kind of method of the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip, including with
Lower step:
First, prepare sers probe:
Step 1). prepare metal nanoparticle；
Step 2). in step 1) in the surfaces of metal nanoparticles that obtains connect Raman reporter molecules；
Step 3). in step 2) in that the surfaces of metal nanoparticles being connected with Raman reporter molecules is connected capture is anti-
Body；
Step 4). in step 3) form sers probe using the space that bsa fills up surfaces of metal nanoparticles afterwards；
Secondly, prepare sers substrate:
Step 5). pdms chip is fitted on hydroxylated sheet glass, forms microfluidic channel；
Step 6). it is passed through the solution of positively charged into described microfluidic channel；
Step 7). by step 1) in metal nanoparticle be passed through in described microfluidic channel, obtain sers substrate；
Step 8). repeat step 6) and step 7) two to three times, form multilamellar sers substrate；
Step 9). in step 8) connect upper capture antibody on the sers substrate that obtains；
Step 10). in step 9) on the basis of be passed through bsa and carry out filling up metal surface space；
Then, antibody test:
Step 11). after taking off the wait glass surface drying of pdms chip, fit in the direction with original pdms Chip Vertical
Upper new pdms chip；
Step 12). in step 11) on the basis of be passed through bsa；
Step 13). in step 12) after be passed through solution to be measured into new microchannel further；
Step 14). in step 13) on the basis of be passed through and step 9 into new microfluidic channel) corresponding detect anti-
Body；
Step 15). in step 14) after by step 4) prepared by sers probe be passed in new microfluidic channel；
Finally, sers light spectrum image-forming:
Step 16). to step 11) the passage intersection region that formed carries out sers light spectrum image-forming.
Further, described step 2), 3), 4), 9), 10), 12) in Raman reporter molecules, capture antibody, bsa pass through
Chemical bond is inserted into surfaces of metal nanoparticles.
Further, described step 6), 7), 8), 9), 10), 12), 13), 14), 15) in be passed through into microfluidic channel
The method of reagent is that the reagent in the syringe will be equipped with reagent by syringe pump connects draw point by pe pipe, then draw point insertion
Mode in described microfluidic channel is injected, by adjusting amount of reagent and the injection rate of the injection of syringe pump state modulator.
Further, described step 7) in described sers substrate be to be formed using the principle of Electrostatic Absorption.
Further, described step 13) and step 14) in, between tumor markerses in solution to be measured and capture antibody
It is connected as the specific recognition between antigen and antibody.
Further, described step 15) described in sers probe with detect antibody be identified as on sers probe two resist
With the described specific recognition detecting antibody.
Further, described step 13)～16) if described in contain tumor markerses in test agent, be passed through detection
Antibody, can form " sandwich " structure；Continue logical sers probe, you can whether detection should " sandwich " structure form.
Beneficial effect: the Diagnostic Value of Several Serum Tumor Markers detection based on sers detection technique and micro-fluidic chip that the present invention provides
Platform, has the advantage that
1st, designed platform can detect Diagnostic Value of Several Serum Tumor Markers simultaneously, improves the accuracy of early diagnosis of cancer；
2nd, the sers substrate that designed platform is used can improve the sensitivity of detection；
3rd, designed platform is resisted by being connected with two on the basis of antibody-antigen-antibody forms " sandwich " structure
Sers probe detected, due to two anti-can detect an anti-presence and amplify signal, and and the combination of sers technology can
With the great sensitivity increasing detection.
4th, because the antibody price related to tumor markerses is relatively all higher, and two anti-prevailing prices are relatively low, so two
Anti- use can be cost-effective while reaching compared with high detection sensitivity.
5th, designed platform is because micro-fluidic chip is using so that can also be obtained while few using sample and reagent
High flux, and have when reagent reacts in microfluidic channel response speed fast the features such as.
6th, designed platform size is compact is easy to carry and integrated.
Brief description
Fig. 1 is Cleaning Principle schematic diagram of the present invention；
Fig. 2 is that the one kind designed by the present invention detects Diagnostic Value of Several Serum Tumor Markers schematic diagram simultaneously.
Specific embodiment
Below in conjunction with the accompanying drawings the present invention is further described.
It is illustrated in figure 1 a kind of Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip,
The present invention is that one kind can detect kinds of tumors based on surface enhanced raman spectroscopy (sers) detection technique and micro-fluidic chip simultaneously
Mark simultaneously can improve the platform of detection sensitivity, and this platform improves inspection by first forming sers substrate in microfluidic channel
The sensitivity surveyed, then formed on sers substrate classical " sandwich " structure with detect whether contain in test agent specific
Tumor markerses.By first specific capture antibody being connected on substrate with pdms chip, then by new pdms chip
Placement location perpendicular to placement location before to reach the work that can detect Diagnostic Value of Several Serum Tumor Markers in intersection region simultaneously
With.It is connected with two anti-and Raman reporter molecules sers immunological probes to reach qualitative and quantitative detection to be tested by being passed through
The effect of tumor markerses in agent, due to the two anti-sensitivity that can amplify signal it is possible to further raising detects.Whole
Individual platform can qualitative and quantitative detection Diagnostic Value of Several Serum Tumor Markers simultaneously, go out with cancer to cancer patient's early diagnosiss and improve to deposit
Motility rate provides favourable technical support.
Sers probe as shown in Figure 1, this probe by metal nanoparticle respectively with Raman reporter molecules, two anti-connections,
Then carry out filling up of metal surface space with bsa to make.Fig. 1 show the present invention and is based on sers detection technique and micro-fluidic core
The Cleaning Principle of piece platform, forms sers substrate in microfluidic channel and is used for strengthening the sensitivity of detection, then on substrate
In connection, specific antibody is in order to capture the specific tumors mark in test agent, if containing specific tumor in test agent
Mark, then can form golden allusion quotation " sandwich " structure when next leading to and detecting antibody, if not containing spy in test agent
This kind " sandwich " structure then will not be formed during fixed tumor markerses.If then defining in follow-up logical sers probe
" sandwich " structure then can form 4 Rotating fields as shown in Figure 1 because two anti-identifications one are anti-, without then will not forming this
Plant special construction, can indirectly qualitatively judge whether to contain spy in test agent by the Raman molecular signal in detection probe
Fixed tumor markerses.
As shown in Fig. 2 by first specific capture antibody being connected on substrate with pdms chip, then by new pdms
The placement location of chip is perpendicular to placement location before, if logical different capture antibody in different microfluidic channels,
Diagnostic Value of Several Serum Tumor Markers can be detected in intersection region simultaneously.
The present invention devises one kind and can detect Diagnostic Value of Several Serum Tumor Markers based on sers detection technique and micro-fluidic chip simultaneously
And can improve the platform of detection sensitivity, the special environment being provided using micro-fluidic chip and the high signal enhancing of sers
Effect, more greatly increase the sensitivity of detection and can detect many simultaneously by forming sers substrate and sers probe
Plant tumor markerses.
One kind of the present invention is based on sers detection technique and micro-fluidic chip can detect Diagnostic Value of Several Serum Tumor Markers energy simultaneously
Improve the detection of platform procedure of detection sensitivity, specifically can be divided into following steps:
Step 1). prepare metal nanoparticle；
Step 2). in step 1) in the surfaces of metal nanoparticles that obtains connect Raman reporter molecules；
Step 3). in step 2) in will be connected with the surfaces of metal nanoparticles of Raman reporter molecules and connect upper specific capture
Antibody；
Step 4). in step 3) form sers probe using the space that bsa fills up surfaces of metal nanoparticles afterwards；
Step 5). pdms chip is fitted on the sheet glass that hydroxylating is crossed；
Step 6). it is passed through the solution of positively charged into microfluidic channel；
Step 7). using step 1) in metal nanoparticle be passed through in microfluidic channel, using the principle of Electrostatic Absorption
Form sers substrate；
Step 8). repeat step 6) and step 7) two to three times, form multilamellar sers substrate；
Step 9). in step 8) connect upper specific capture antibody on the sers substrate that obtains；
Step 10). in step 9) on the basis of be passed through bsa and carry out filling up metal surface space；
Step 11). take off pdms chip and wait glass surface to fit in the direction with original pdms Chip Vertical after being dried
Upper new pdms chip；
Step 12). in step 11) on the basis of be passed through bsa；
Step 13). in step 12) after be passed through solution to be measured into new microfluidic channel further；
Step 14). in step 13) on the basis of be passed through and step 9 into new microfluidic channel) corresponding particular probe
Antibody；
Step 15). in step 14) after by step 4) prepared by sers probe be passed in microfluidic channel；Probe with
Detect be identified as on probe the two anti-and specific specific recognition detecting antibody of antibody；
Step 16). to step 11) the passage intersection region that formed carries out sers light spectrum image-forming.
Wherein, step 2), 3), 4), 9), 10), 12) in Raman reporter molecules, antibody, bsa be inserted into by chemical bond
Surfaces of metal nanoparticles.
Wherein, step 6), 7), 8), 9), 10), 12), 13), 14), 15) in be passed through the side of reagent into microfluidic channel
Method is that reagent in the syringe will be equipped with reagent by syringe pump connects draw point by pe pipe, then draw point be inserted into micro-fluidic
Mode in passage is injected, and can control amount of reagent and the injection rate of injection by adjusting syringe pump parameter.
Step 13), 14) in connection between tumor markerses and specific antibodies be specific recognition between antigen and antibody.
Embodiment
As Fig. 1 and as shown in Fig. 2 the present invention a kind of can be detected based on sers detection technique and micro-fluidic chip simultaneously multiple
The process of tumor markerses the detection of platform that can improve detection sensitivity comprises the following steps:
1) preparation of Nano silver grain, first, by 390ml deionized water and 0.072g agno3With 130 DEG C in stirring shape
It is heated under state seething with excitement, is subsequently adding 10ml, 1% sodium citrate, continuing to be heated to solution is in yellow green, stops heating,
Solution is allowed to cool down, thus making elargol for future use under room temperature.
2) preparation of sers probe, is taken 6ml elargol centrifuge to be centrifuged 30 minutes with the speed of 11000rpm, removes upper strata
Clear liquid adds 4ml to remove particle water.Then, add the 4mba of 10mmol of 40 μ l in the fulmargin of 4ml and in stirring
Under allow its mixing at least two hours.Finally, after taking 1.5ml mixing, solution is centrifuged 20 minutes with the speed of 900rpm, remove on
Clear liquid is subsequently adding the bbs buffer of 1ml.The sheep anti-Mouse of the 1mg/ml of 50ul is added to the 1ml 4- handling well before
In the elargol of mba labelling, it is allowed to place 2h at 4 DEG C.Then, 12min is centrifuged with the speed of 6500rpm, removes in supernatant
Unnecessary antibody the bbs buffer to addition 1ml in precipitation.Then, add the bsa solution of 10 μ l 5% to fill up silver nanoparticle
The space that particle surface is not covered by 4-mba and sheep anti-Mouse, then places it in 1h at 4 DEG C.Finally with the speed of 6500rpm
Rate is centrifuged 12min, removes supernatant and adds the bbs buffer of 200 μ l.
3) sheet glass and pdms chip pretreatment, the glass being used is immersed in 98%h in advance2so4And 30%h2o2With body
In the long-pending solution than 3:1 mixing, use substantial amounts of deionized water rinsing when using, then dried up with nitrogen, be placed in temperature and set
For in 80 DEG C of baking ovens.The pdms chip being used is immersed in the ethanol that concentration is 99% in advance, and good seal be positioned over super
Surpass in sound machine and wash 2h, then pdms chip is taken out from ethanol and use substantial amounts of deionized water rinsing, then dried up with nitrogen,
Repeat the above steps at least twice, are subsequently placed in 2h in 80 DEG C of baking oven.Take out pdms chip and by itself and glass from baking oven
Piece is fitted it is ensured that not having bubble between chip and sheet glass.
4) 1% pdda is expelled to the speed of 0.8 μ l/min by immune detection as shown in Figure 2 first by syringe pump
25min in microfluidic channel on pdms chip, is then passed through with identical speed into microfluidic channel and concentrates 20 times
Elargol leads to 25min.Repeat the above steps.Then we lead to antibody rabbit with the speed of 0.8 μ l/ml into microfluidic channel and resist
The anti-ca125 of ca153, rabbit, rabbit anti-cea 25min, then standing 25min is so that antibody is sufficiently coupled on Nano silver grain, logical
The purpose of antibody is tumor markerses ca153, ca125, the cea in capture test agent.Then led to speed for 2 μ l/min
5%bsa continues 3min to wash out the antibody being not attached on nanoparticle, and then speed is changed to the logical 25min of 0.8 μ l/m in order to anti-
Only non-specific adsorption.Then, careful take down pdms chip and wait the liquid natural air drying at room temperature on sheet glass.From
Take out one piece of new pdms chip in baking oven and so that pdms chip is fitted with sheet glass, in the channel direction on pdms and sheet glass
Substrate direction vertical.Into microfluidic channel successively with injection infusion bsa, test agent, it is specifically corresponding that to detect antibody little
The anti-ca153 of Mus, the anti-ca125 of mice, mice anti-cea, sers probe, each reactant is all with the speed of 0.8 μ l/m, injects
Time is all 25min, and has injected and be all allowed to settle 25min, and all uses syringe pump after having stood with 2 μ l/m every time
Speed injection pbst continue 3 minutes.After all finishing etc. above-mentioned steps by the liquid air in microfluidic channel by its
Just can be measured with sers after discharge.
The result of above-described embodiment is: when having modified different capture antibody on substrate, containing anti-with capture on substrate
The test agent of the corresponding tumor markerses of body phase, when finally carrying out sers measurement, its sers signal is relatively strong, and to be measured
Contain then relatively weak with capture antibody its sers signal of not corresponding tumor markerses on substrate in reagent.The above results explanation
The described Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip and method can be very good to pass through
The measurement of sers signal judging whether contain specific breast cancer tumour mark in test agent, and this platform compare with
Prior art its using sample and reagent few while can also obtain high flux, and when reagent reacts in microfluidic channel
Have response speed fast the features such as, and due to two anti-using so that the cost of whole detection reduces.
The above be only the preferred embodiment of the present invention it should be pointed out that: for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip it is characterised in that: bag
Include the microfluidic channel being made up of micro-fluidic chip and its interior sers substrate, and sers probe and detection antibody；
Described microfluidic channel is cruciform shape, and interconnection is interior to be described sers substrate, is passed through in vertical vertical passage
Described sers probe and detection antibody, intersection region is detection zone；
Described sers substrate is passed through in described microfluidic channel by metal nanoparticle and obtains, and described sers substrate is connected with and catches
Obtain antibody；
Described sers probe by metal nanoparticle respectively with Raman reporter molecules, two anti-connections, then carry out metal watch with bsa
Filling up of face space is made.
2. the Diagnostic Value of Several Serum Tumor Markers detection based on sers detection technique and micro-fluidic chip according to claim 1 is flat
Platform it is characterised in that: described micro-fluidic chip be pdms chip.
3. the Diagnostic Value of Several Serum Tumor Markers detection based on sers detection technique and micro-fluidic chip according to claim 1 is flat
Platform it is characterised in that: described capture antibody be no less than two kinds.
4. a kind of method of the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip, its feature exists
In: comprise the following steps:
First, prepare sers probe:
Step 1). prepare metal nanoparticle；
Step 2). in step 1) in the surfaces of metal nanoparticles that obtains connect Raman reporter molecules；
Step 3). in step 2) in will be connected with Raman reporter molecules surfaces of metal nanoparticles connect on capture antibody；
Step 4). in step 3) form sers probe using the space that bsa fills up surfaces of metal nanoparticles afterwards；
Secondly, prepare sers substrate:
Step 5). pdms chip is fitted on hydroxylated sheet glass, forms microfluidic channel；
Step 6). it is passed through the solution of positively charged into described microfluidic channel；
Step 7). by step 1) in metal nanoparticle be passed through in described microfluidic channel, obtain sers substrate；
Step 8). repeat step 6) and step 7) two to three times, form multilamellar sers substrate；
Step 9). in step 8) connect upper capture antibody on the sers substrate that obtains；
Step 10). in step 9) on the basis of be passed through bsa and carry out filling up metal surface space；
Then, antibody test:
Step 11). after taking off the wait glass surface drying of pdms chip, new on fitting in the direction with original pdms Chip Vertical
Pdms chip；
Step 12). in step 11) on the basis of be passed through bsa；
Step 13). in step 12) after be passed through solution to be measured into new microchannel further；
Step 14). in step 13) on the basis of be passed through and step 9 into new microfluidic channel) corresponding detection antibody；
Step 15). in step 14) after by step 4) prepared by sers probe be passed in new microfluidic channel；
Finally, sers light spectrum image-forming:
Step 16). to step 11) the passage intersection region that formed carries out sers light spectrum image-forming.
5. the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip according to claim 4
Method it is characterised in that: described step 2), 3), 4), 9), 10), 12) in Raman reporter molecules, capture antibody, bsa lead to
Cross chemical bond and be inserted into surfaces of metal nanoparticles.
6. the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip according to claim 4
Method it is characterised in that: described step 6), 7), 8), 9), 10), 12), 13), 14), 15) in be passed through into microfluidic channel
The method of reagent is that the reagent in the syringe will be equipped with reagent by syringe pump connects draw point by pe pipe, then draw point insertion
Mode in described microfluidic channel is injected, by adjusting amount of reagent and the injection rate of the injection of syringe pump state modulator.
7. the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip according to claim 4
Method it is characterised in that: described step 7) in described sers substrate be to be formed using the principle of Electrostatic Absorption.
8. the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip according to claim 4
Method it is characterised in that: described step 13) and step 14) in, between tumor markerses in solution to be measured and capture antibody
It is connected as the specific recognition between antigen and antibody.
9. the Diagnostic Value of Several Serum Tumor Markers detection platform based on sers detection technique and micro-fluidic chip according to claim 4
Method it is characterised in that: described step 15) described in sers probe with detect antibody be identified as on sers probe two resist
With the described specific recognition detecting antibody.
10. the Diagnostic Value of Several Serum Tumor Markers detection based on sers detection technique and micro-fluidic chip according to claim 4 is flat
The method of platform it is characterised in that: described step 13)～16) if described in contain tumor markerses in test agent, be passed through spy
Survey antibody, " sandwich " structure can be formed；Continue logical sers probe, you can whether detection should " sandwich " structure form.
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