CN101157952B - Functionalized microfluidic chip and method for PCR product analysis by using same - Google Patents
Functionalized microfluidic chip and method for PCR product analysis by using same Download PDFInfo
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- CN101157952B CN101157952B CN2007100308099A CN200710030809A CN101157952B CN 101157952 B CN101157952 B CN 101157952B CN 2007100308099 A CN2007100308099 A CN 2007100308099A CN 200710030809 A CN200710030809 A CN 200710030809A CN 101157952 B CN101157952 B CN 101157952B
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Abstract
The invention discloses a functionalized microfluidic chip and a method for PCR product analysis, wherein the chip consists of a buffer solution pool, a buffer solution waste solution pool, a sample injection waste solution pool, a sample pool, a separation channel and a sample injection channel, wherein two ends of the separation channel are respectively connected with the buffer solution pool and the buffer solution waste solution pool, two ends of the sample injection channel are respectively connected with the sample pool and the sample injection waste solution pool, and the length of an effective sample injection channel is 0.5-20 cm. The method combines isokinetic electrophoretic preconcentration and screening electrophoretic separation, the buffer solution contains trailing dielectric medium and DNA screening medium, and the PCR product contains chloride-containing ions as leading ions. The chip only needs to introduce one buffer solution, reduces the number of required electrodes, simplifies the operation, has the advantages of sensitivity and rapid analysis, and can effectively reduce the PCR reaction time by improving the detection sensitivity. The invention can sensitively, quickly and simply analyze PCR products.
Description
Technical field
The invention belongs to the bioassay technique field, particularly a kind of PCR of being used for product analysis functionalization micro-flow control chip and be used for the method for PCR product analysis.
Background technology
Polymerase chain reaction (PCR) is one of technique means that is most widely used in the DNA research, the template DNA copy can be amplified 10 by this technology
6Doubly above to satisfy the analysis needs.The PCR product component is the dna fragmentation after the amplification, in the PCR damping fluid that is dissolved in.
Traditional PCR product analysis method is a gel electrophoresis separation and combination gel imaging, and the shortcoming of this method is that sensitivity is lower, need long proliferation time for the dna profiling of low copy.In addition, the method based on gel electrophoresis also has complex operation and the big shortcoming of sample consumption.Need a kind of highly sensitive, easy and simple to handle and PCR product analysis method that analysis speed is fast in the actually operating.
Micro-fluidic chip is a main platform of realizing microflow control technique, is meant integrated serial operating unit on chip, realizes chip operation through the microchannel network through total system with controlled fluid.Chip capillary cataphoresis is a kind of significant element operative technique of micro-fluidic chip, this technology can be on the micro-fluidic chip platform DNA isolation fragment quickly and efficiently.The sample feeding amount of traditional cross posture channel architecture micro-fluidic electrophoresis chip seldom though obtain high separation because sample introduction district band is narrow, is unfavorable for the further raising of micro-fluid control chip electrophoretic detection sensitivity less because of sample size.Under tradition district band screening electrophoretic separation condition, can improve sample size though increase the effective sample introduction of chip district strip length,, resolution do not possess actual application value because descending.
The isotachophoresis pre-concentration realizes by means of discontinuous damping fluid, promptly leading damping fluid and trail damping fluid, conducting medium and trail dielectric medium before containing respectively.In the isotachophoresis process, the sample area band is between leading ion and trailing ion.The rate of migration of leading ion is higher than all dna fragmentations in the PCR product and the trailing ion rate of migration is lower than all dna fragmentations in the PCR product.The process of isotachophoresis realizes according to the difference of rate of migration in the band of this mixing zone, and the difference of different zones field intensity is concentrated sample in the sample area band.Isotachophoresis can improve detection sensitivity by the sample pre-concentration, but in most cases needs leading and trail two kinds of damping fluids, operates comparatively loaded down with trivial details.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art part, primary and foremost purpose of the present invention is to provide a kind of functionalization micro-flow control chip of the PCR of being used for product analysis, this chip structurally has effective sample intake passage of remarkable lengthening, integrated isotachophoresis pre-concentration and screening electrophoretic separation.
Another object of the present invention is to provide the above-mentioned functions micro-fluidic chip to be used for the method for PCR product analysis, by in conjunction with micro-fluidic chip special and analytical reagent, only need a kind of damping fluid promptly can finish the isotachophoresis pre-concentration of PCR product and concentrate back screening electrophoretic separation, this method utilizes the chlorion that contains in the PCR product needn't additionally introduce leading damping fluid as the leading ion of isotachophoresis.
Purpose of the present invention is achieved through the following technical solutions: a kind of functionalization micro-flow control chip, integrated PCR product isotachophoresis pre-concentration and screening electrophoretic separation function, this chip is by being made up of 4 liquid storage tanks, split tunnel and sample intake passages, it is characterized in that, described liquid storage tank is respectively buffer pool, damping fluid waste liquid pool, sample introduction waste liquid pool and sample pool, the split tunnel two ends connect buffer pool and damping fluid waste liquid pool respectively, and the sample intake passage two ends connect sample pool and sample introduction waste liquid pool respectively; Split tunnel and sample intake passage overlapping region are effective sample intake passage, and the length range of described effective sample intake passage is 0.5～20 centimetre.
In order to realize the present invention better, described sample pool and sample introduction waste liquid pool are positioned at the homonymy or the heteropleural of split tunnel.
The described material that is used for the functionalization micro-flow control chip of PCR product analysis can be silicon, quartz, glass, plastics, as polymethylmethacrylate (PMMA), polycarbonate (PC), silica gel such as polydimethylsiloxane (PDMS) etc., the chip channel internal surface can modify or unmodified is crossed.
The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, comprises the steps:
(1) damping fluid is introduced chip: split tunnel, sample intake passage, buffer pool, sample introduction waste liquid pool and the damping fluid waste liquid pool of damping fluid being introduced functionalization micro-flow control chip by pressure;
(2) add the PCR product at sample pool;
(3) sample introduction: by electric sample introduction or pressure sample introduction the PCR product is full of sample intake passage, for electric sample introduction: apply voltage between sample pool and sample introduction waste liquid pool, field strength range is between 10V/cm～10000V/cm, and applying voltage time is 1～600 second; For the pressure sample introduction: with positive pressure in sample pool or with suction function in the sample introduction waste liquid pool, pressure action time is 1～180 second;
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field strength range is between 50V/cm～2000V/cm, and applying the voltage time length is 30～600 seconds; Effectively separation length is that the distance between effective sample intake passage end and the detector is 1～6cm, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.
Functionalization micro-flow control chip of the present invention places chip analyzer to use when being used for the PCR product analysis.Chip analyzer comprises high-voltage power supply, detector, data acquisition and processing (DAP) system.High-voltage power supply is connected with the chip liquid storage tank by electrode, and output voltage acts on passage between two liquid storage tanks of chip by time variable control.
The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, adopts methods such as laser induced fluorescence(LIF), ultraviolet to detect.
For laser-Induced Fluorescence Detection, used fluorescence dye can be covalent labeling dyestuff, non-covalent labeling dye.The fluorescence dye of DNA covalent labeling is selected from FITC series, BODIPY series, Cy series, rhodamine series, Alexa Fluor series, DABCYL, HEX, JOX, NAP, Oregon Green488, Pyrene, ROX, TAMARA, TET or Texas red etc.; The fluorescence dye of the non-covalent mark of DNA is selected from SYBR series, Cyanine series, SYTOX series, Thiazole series or ethidium bromide etc.
For ultraviolet detection, dna fragmentation can mark or mark not.
Chlorion in the described PCR product is a leading ion, and described damping fluid contains trails dielectric medium and DNA sieving medium, describedly trails dielectric anion transport speed and is lower than dna fragmentation.Trail dielectric medium and can be but be not limited to following composition: 3-(N-morphine quinoline) propanesulfonic acid (MOPS), N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES) or N-three (methylol) methyl-3-aminopropanesulfonicacid acid (TAPS); Described DNA sieving medium can be but be not limited to hydroxypropylcellulose, Natvosol, Vltra tears, polyvidone, polyvinyl alcohol, polyoxyethylene glycol, polyacrylamide, both can be single composition, also can be the combination of two kinds or two or more components.
When the above-mentioned functions micro-fluidic chip is used for the PCR product analysis, be leading ion with the chlorion that self contains in the PCR product, what contain in the damping fluid trails the dielectric medium negatively charged ion as trailing ion, by the dna fragmentation among the isotachophoresis method pre-concentration PCR.Dna fragmentation isotachophoresis pre-concentration in the PCR product and concentrated back screening electrophoretic separation are finished synchronously, and The whole analytical process only need be used a kind of damping fluid.
Increase effective sample intake passage length on the micro-fluidic chip of the present invention and can effectively improve the dna fragmentation detection sensitivity and do not lose resolution again, utilize isotachophoresis that sample area band in this interval is compressed the realization pre-concentration, sieve the electrophoretic separation dna fragmentation then.The above-mentioned functions micro-fluidic chip is used for the PCR product analysis method, utilizes the chlorion that contains in the PCR product as the leading ion of isotachophoresis and needn't additionally introduce leading damping fluid.
The present invention compared with prior art has following advantage and beneficial effect:
Functionalization micro-flow control chip of the present invention only need be introduced a kind of damping fluid, has compared with prior art reduced a kind of damping fluid, has also reduced required electrode number, thereby has simplified operation.The present invention has the advantage of sensitive and real-time analysis, can effectively reduce the PCR reaction times by improving detection sensitivity.This method can realize sensitivity, quick and easy PCR product analysis.Compare with traditional cross bar Flow Control electrophoresis chip structure that declines, functionalization micro-flow control chip of the present invention has improved sample size by effective sample introduction district band that extends, the isotachophoresis pre-concentration sieves electrophoretic separation then with PCR product boil down to stenosis area band, does not lose resolution when significantly improving detection sensitivity.By spectrogram among Fig. 4 and the table 1 and data, the long 50 μ m of the corresponding effectively sample intake passage of Fig. 1 chip structure, and the corresponding effectively long 1.9cm of sample intake passage of Fig. 3 chip structure.Fig. 3 structure chip does not lose resolution because of sample size increases, and compares its detectability with Fig. 1 structure chip and has reduced about 300 times (table 1).Compare with traditional isotachophoresis among Fig. 2 and screening electrophoresis integrated microfluidic chip structure, functionalization micro-flow control chip of the present invention reduces by a buffer pool, correspondingly reduces by an electrode in the control.The isotachophoresis pre-concentration of tradition isotachophoresis and screening electrophoresis integrated microfluidic chip is a lock out operation with the screening electrophoretic separation, needs to switch electrode in the operation; And the isotachophoresis pre-concentration of functionalization micro-flow control chip of the present invention carries out synchronously with the screening electrophoretic separation, does not need to switch electrode in the operation.
Description of drawings
Fig. 1 is traditional cross bar channel-type micro-fluidic electrophoresis chip structural representation.
Fig. 2 is traditional isotachophoresis and screening electrophoresis integrated microfluidic chip structural representation.
Fig. 3 is a functionalization micro-flow control chip structural representation of the present invention.
Fig. 4 is the decline electrophoresis spectrogram comparison diagram of Flow Control electrophoresis chip analytical standard DNA sample of functionalization micro-flow control chip of the present invention and traditional cross bar.
Fig. 5 is another structural representation of functionalization micro-flow control chip of the present invention.
Fig. 6 is the electrophoretogram that functionalization micro-flow control chip of the present invention is analyzed the PCR product.
Fig. 7 uses the analytical standard DNA sample electrophoresis spectrogram of effective sample intake passage length as the functionalization micro-flow control chip of 0.5cm, 10cm and 20cm respectively for the present invention.
Another DNA sample electrophoresis spectrogram that Fig. 8 obtains for functionalization micro-flow control chip analysis of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Fig. 1, Fig. 2, Fig. 3 are respectively decline Flow Control electrophoresis chip, traditional isotachophoresis and screening electrophoresis integrated microfluidic chip and functionalization micro-flow control chip structure iron of the present invention of traditional cross bar, be sample intake passage between sample pool 3 and the sample waste liquid pool 4, be split tunnel between buffer pool 1 and the damping fluid waste liquid pool 4, sample intake passage and split tunnel overlapping region are effective sample intake passage.Effective sample intake passage length of Fig. 1, Fig. 2, Fig. 3 correspondence is respectively 50 μ m, 1.9cm, 1.9cm.
Fig. 1 is traditional cross bar Flow Control electrophoresis chip that declines, passage is a cross structure, 4 liquid storage tanks are arranged, buffer pool 1, damping fluid waste liquid pool 2, sample introduction waste liquid pool 3 and sample pool 4, its analytical procedure is: (1) damping fluid is introduced chip: split tunnel, sample intake passage, buffer pool, sample introduction waste liquid pool and the damping fluid waste liquid pool of damping fluid being introduced chip by pressure; (2) add the PCR product at sample pool; (3) sample introduction: sample introduction waste liquid pool 3 connection electrode output voltages, sample pool 4 connection electrode ground connection; (4) separation and detection: damping fluid waste liquid pool 2 connection electrode output voltages, buffer pool 1 connection electrode ground connection.
Fig. 2 is traditional isotachophoresis and screening electrophoresis integrated microfluidic chip, 5 liquid storage tanks are arranged, be respectively buffer pool 1, damping fluid waste liquid pool 2, sample introduction waste liquid pool 3, sample pool 4 and leading buffer pool 5, its analytical procedure is: (1) damping fluid is introduced chip: split tunnel, sample intake passage, leading buffer pool, sample introduction waste liquid pool and the damping fluid waste liquid pool of leading damping fluid being introduced chip by pressure; (2) damping fluid is trailed in adding in the buffer pool; (3) add the PCR product at sample pool; (4) sample introduction: No. 3 pond connection electrode output voltages, No. 4 pond connection electrode ground connection; (5) damping fluid is trailed in perfusion: sample introduction waste liquid pool 3 connection electrode output voltages, buffer pool 1 connection electrode ground connection; (6) isotachophoresis pre-concentration: damping fluid waste liquid pool 2 connection electrode output voltages, buffer pool 1 connection electrode ground connection; (7) separate: damping fluid waste liquid pool 2 connection electrode output voltages, leading buffer pool 5 connection electrode ground connection.
Fig. 3 is a functionalization micro-flow control chip of the present invention, 4 liquid storage tanks are arranged, be respectively buffer pool 1, damping fluid waste liquid pool 2, sample introduction waste liquid pool 3 and sample pool 4, its analytical procedure is: (1) damping fluid is introduced chip: split tunnel, sample intake passage, buffer pool, sample introduction waste liquid pool and the damping fluid waste liquid pool of damping fluid being introduced functionalization micro-flow control chip by pressure; (2) add the PCR product at sample pool; (3) sample introduction: sample introduction waste liquid pool 3 connection electrode output voltages, sample pool 4 connection electrode ground connection; (4) isotachophoresis pre-concentration, separation and detection: damping fluid waste liquid pool 2 connection electrode output voltages, buffer pool 1 connection electrode ground connection.
Adopt the functionalization micro-flow control chip of PMMA material, configuration as shown in Figure 3, chip is made up of 4 liquid storage tanks, split tunnel and sample intake passages, 4 liquid storage tanks are respectively buffer pool 1, damping fluid waste liquid pool 2, sample pool 3 and sample introduction waste liquid pool 4.Sample pool 3 and sample introduction waste liquid pool 4 are positioned at the heteropleural of split tunnel.Split tunnel and sample intake passage overlapping region are effective sample intake passage.All channel sizes are width 50 μ m, the degree of depth 20 μ m, effective long 1.9cm of sample intake passage, chip channel internal surface unmodified.Trailing dielectric medium is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES), and the DNA sieving medium is 3% (quality volume percent, unit is g/100ml) Natvosol.Sample is Φ x174 HaeIII Digest, and total dna content is 2.5pg/ μ L, and 11 dna fragmentations of 72 to 1382 base pairs are arranged.Sample dissolution contains the 60mmol/L chlorion in 1 * PCR reaction buffer.Adopt Fig. 1 structure tradition cross bar Flow Control electrophoresis chip that declines to carry out conventional non gel sieving and separate in contrast, it is wide that channel size is all 50 μ m, 20 μ m are dark, the long 50 μ m of effective sample intake passage, sample is Φ x174 HaeIII Digest, total dna content is 500pg/ μ L, and sample dissolution contains the 60mmol/L chlorion in 1 * PCR reaction buffer.Adopting the dna marker dyestuff of two kinds of chip analysis methods all is SYBR Green, and its concentration in damping fluid is 1 μ mol/L.Laser excitation wavelength is 473nm, and the electrophoresis that obtains compares collection of illustrative plates as shown in Figure 4, and spectrogram A obtains for adopting Fig. 1 structure chip, and spectrogram B obtains for employing Fig. 3 structure chip.Compare with Fig. 1 tradition cross bar Flow Control electrophoresis chip that declines, integrated form micro-fluidic chip of the present invention does not lose resolution because of sample size increases, except that 271/281 base pair fragment, other fragment all reaches baseline separation, and its resolution is suitable with cross posture chip.Calculating (table 1 is the comparison that has or not isotachophoresis pre-concentration micro-fluidic chip Φ x174 HaeIII Digest dna fragmentation detectability among Fig. 4) by table 1, compare with traditional cross bar Flow Control electrophoresis chip that declines, this structure micro-fluidic chip detectability has reduced about 300 times.
The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, comprises the steps:
(1) damping fluid is introduced chip: by pressure damping fluid (trailing dielectric medium is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES), and the DNA sieving medium is 3% Natvosol) is introduced all parts of chip channel (split tunnel and sample intake passage), buffer pool, sample introduction waste liquid pool and damping fluid waste liquid pool.
(2) add the PCR product at sample pool.
(3) sample introduction: sample pool ground connection, the sample introduction waste liquid pool applies voltage, and field intensity 10000V/cm between the two applied voltage time 1 second.
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field intensity 500V/cm applied voltage time 500 seconds.Detector is arranged in split tunnel and detects PCR product signal, and effectively distance is 1cm between sample intake passage end and the detector, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.
The functionalization micro-flow control chip configuration of used glass material as shown in Figure 5, chip is made up of 4 liquid storage tanks, split tunnel and sample intake passages, 4 liquid storage tanks are respectively buffer pool 1, damping fluid waste liquid pool 2, sample pool 3 and sample introduction waste liquid pool 4.Sample pool 3 and sample introduction waste liquid pool 4 are positioned at the homonymy of split tunnel.Split tunnel and sample intake passage overlapping region are effective sample intake passage.Chip channel is of a size of width 50 μ m, the degree of depth 20 μ m, the effectively long 1.9cm of sample intake passage, effectively the width 50 μ m of sample intake passage.The chip channel internal surface is modified with linear polyacrylamide.Utilize the PCR product of one 120 base pair of this chip analysis, and with DL2000 DNA standard model as internal reference.Damping fluid contains 20mmol/L MOPS for trailing dielectric medium, and the 40mmol/L imidazoles, 1 μ mol/L SYBR Green as insert labeling dye and 2% hydroxypropyl first class Mierocrystalline cellulose as the DNA sieving medium.The DNA standard model comprises 100,250,500,750,1000, the 2000bp fragment.Adopt laser induced fluorescence detector, the electrophoretogram that obtains comprises that all fragments of PCR product and DNA standard model all obtain baseline separation as shown in Figure 6, and institute's reference numbers is the base pair number of DNA standard model among the figure.
The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, comprises the steps:
(1) damping fluid is introduced chip: by pressure damping fluid is introduced all parts of chip channel and buffer pool and damping fluid waste liquid pool.
(2) add the PCR product at sample pool.
(3) sample introduction: the sample introduction waste liquid pool connects negative pressure, 180 seconds time length.
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field intensity 1000V/cm applied voltage time 350 seconds.Detector is arranged in split tunnel and detects PCR product signal, and effectively distance is 3cm between sample intake passage end and the detector, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.
Adopt the functionalization micro-flow control chip of PDMS material, configuration as shown in Figure 3, chip is made up of 4 liquid storage tanks, split tunnel and sample intake passages, 4 liquid storage tanks are respectively buffer pool 1, damping fluid waste liquid pool 2, sample pool 3 and sample introduction waste liquid pool 4.Sample pool 3 and sample introduction waste liquid pool 4 are positioned at the heteropleural of split tunnel.Split tunnel and sample intake passage overlapping region are effective sample intake passage.All channel sizes are width 150 μ m, the degree of depth 50 μ m, and effectively sample intake passage length is respectively 0.5cm, 10cm and 20cm, and width is 150 μ m, and the chip channel internal surface is modified.Sample is Φ x174 HaeIII Digest, and total dna content is 2.5pg/ μ L, and 11 dna fragmentations of 72 to 1382 base pairs are arranged.Sample dissolution contains the 60mmol/L chlorion in the PCR reaction buffer.The dissociating buffer composition for 20mmol/L TAPS for trailing dielectric medium, and 40mmol/L imidazoles, 2% polyvinyl alcohol is as the DNA sieving medium.Adopt UV-detector.
The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, comprises the steps:
(1) damping fluid is introduced chip: by pressure damping fluid is introduced all parts of chip channel and buffer pool and damping fluid waste liquid pool.
(2) add the PCR product at sample pool.
(3) sample introduction: the sample introduction waste liquid pool connects negative pressure, 10 seconds time length.
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field intensity 50V/cm applied voltage time 600 seconds.Detector is arranged in split tunnel and detects PCR product signal, and effectively distance is 6cm between sample intake passage end and the detector, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.The electrophoresis spectrogram is followed successively by effective sample intake passage length from top to bottom and is respectively the electrophoresis spectrogram that 0.5cm, 10cm and 20cm chip obtain as shown in Figure 7.
Adopt the functionalization micro-flow control chip of PC material, configuration as shown in Figure 3, chip is made up of 4 liquid storage tanks, split tunnel and sample intake passages, 4 liquid storage tanks are respectively buffer pool 1, damping fluid waste liquid pool 2, sample pool 3 and sample introduction waste liquid pool 4.Sample pool 3 and sample introduction waste liquid pool 4 are positioned at the heteropleural of split tunnel.Split tunnel and sample intake passage overlapping region are effective sample intake passage.All channel sizes are 80 μ m * 30 μ m, effective long 2cm of sample intake passage, and the chip channel internal surface is modified.Sample is Φ x174 HaeIII Digest, and total dna content is 2.5pg/ μ L, and 11 dna fragmentations of 72 to 1382 base pairs are arranged.Sample dissolution contains the 60mmol/L chlorion in the PCR reaction buffer.The dissociating buffer composition is a 20mM TAPS/40mmol/L imidazoles, and pH value 7.2 contains 1 μ mol/L SYTOX and serves as a mark dyestuff and 1.8% hydroxypropylcellulose, 0.48% Natvosol as the DNA sieving medium in the damping fluid.Adopt laser induced fluorescence detector.
The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, comprises the steps:
(1) damping fluid is introduced chip: by pressure damping fluid is introduced all parts of chip channel and buffer pool and damping fluid waste liquid pool.
(2) add the PCR product at sample pool.
(3) sample introduction: the sample introduction waste liquid pool connects negative pressure, 10 seconds time length.
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field intensity 1000V/cm applied voltage time 350 seconds.Detector is arranged in split tunnel and detects PCR product signal, and effectively distance is 4cm between sample intake passage end and the detector, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.The electrophoresis spectrogram as shown in Figure 8.
Embodiment 5
The detectability of comparison diagram 2 and Fig. 3 structure micro-fluidic chip detects and all adopts laser-Induced Fluorescence Detection, separating distance 4cm.
Fig. 2 is traditional isotachophoresis and screening electrophoresis integrated microfluidic chip, and effectively the long 1.9cm of sample intake passage has 5 liquid storage tanks, is respectively buffer pool 1, damping fluid waste liquid pool 2, sample introduction waste liquid pool 3, sample pool 4 and leading buffer pool 5.
Sample is the Φ x174 HaeIII Digest of total dna content 2.5pg/ μ L, and 11 dna fragmentations of 72 to 1382 base pairs are arranged.Sample dissolution is in deionized water.
Its analytical procedure is:
(1) at first leading damping fluid is poured into chip channel, damping fluid waste liquid pool, sample introduction waste liquid pool and leading buffer pool, composition is preceding conducting medium for 15mmol/L hydrochloric acid, and the 36mmol/L imidazoles, 1 μ mol/L SYBR Green serves as a mark dyestuff and 2% polyvinyl alcohol as the DNA sieving medium;
(2) will trail damping fluid and add buffer pool, composition is 20mM HEPES as trailing dielectric medium, and the 40mmol/L imidazoles;
(3) the PCR product is added sample pool 4;
(4) sample introduction: sample introduction waste liquid pool 3 connection electrode output voltages, sample pool 4 connection electrode ground connection;
(5) damping fluid is trailed in perfusion: sample introduction waste liquid pool 3 connection electrode output voltages, buffer pool 1 connection electrode ground connection;
(6) isotachophoresis pre-concentration: damping fluid waste liquid pool 2 connection electrode output voltages, buffer pool 1 connection electrode ground connection;
(7) separation and detection: damping fluid waste liquid pool 2 connection electrode output voltages, leading buffer pool 5 connection electrode ground connection.
Fig. 3 is a functionalization micro-flow control chip structure iron of the present invention, and its effective sample intake passage length is 1.9cm, and this chip has 4 liquid storage tanks, is respectively buffer pool 1, damping fluid waste liquid pool 2, sample introduction waste liquid pool 3 and sample pool 4.Split tunnel and sample intake passage overlapping region are effective sample intake passage.Sample is the Φ x174 HaeIII Digest of total dna content 2.5pg/ μ L, and 11 dna fragmentations of 72 to 1382 base pairs are arranged.Sample dissolution contains the 60mmol/L chlorion in the PCR reaction buffer.
Analytical procedure is: (1) at first imports damping fluid chip channel and buffer pool and damping fluid waste liquid pool, composition is a 20mmol/L HEPES/40 mmol/L imidazoles, pH value 7.2 contains 1 μ mol/L SYBRGreen and serves as a mark dyestuff and 2% polyvinyl alcohol as the DNA sieving medium; (2) sample is added sample pool 4; (3) sample introduction: sample introduction waste liquid pool 3 connection electrode output voltages, sample pool 4 connection electrode ground connection; (4) isotachophoresis concentrates and separates: damping fluid waste liquid pool 2 connection electrode output voltages, buffer pool 1 connection electrode ground connection.
The detectability of Fig. 2 and Fig. 3 structure chip relatively sees Table 2.Fig. 3 is suitable with Fig. 2 structure micro-fluidic chip detectability, and reduces a kind of damping fluid, has simplified analysis operation.
Embodiment 6
Adopt the functionalization micro-flow control chip of glass material, configuration as shown in Figure 3, chip is made up of 4 liquid storage tanks, split tunnel and sample intake passages, 4 liquid storage tanks are respectively buffer pool 1, damping fluid waste liquid pool 2, sample pool 3 and sample introduction waste liquid pool 4.Sample pool 3 and sample introduction waste liquid pool 4 are positioned at the heteropleural of split tunnel.Split tunnel and sample intake passage overlapping region are effective sample intake passage.All channel sizes are width 30 μ m, the degree of depth 15 μ m, and effective long 0.5cm of sample intake passage, the chip channel internal surface is modified.Trailing dielectric medium is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES), and the DNA sieving medium is 3% (quality volume percent (g/100ml)) Natvosol.Sample is Φ x174 HaeIII Digest, and total dna content is 2.5pg/ μ L, and 11 dna fragmentations of 72 to 1382 base pairs are arranged.Sample dissolution contains the 60mmol/L chlorion in 1 * PCR reaction buffer.The dna marker dyestuff is SYBR Green, and its concentration in damping fluid is 1 μ mol/L.Laser excitation wavelength is 473nm, and the electrophoresis that obtains compares collection of illustrative plates as shown in Figure 8.The above-mentioned functions micro-fluidic chip is used for the method for PCR product analysis, comprises the steps:
(1) damping fluid is introduced chip: by pressure damping fluid (trailing dielectric medium is N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid (HEPES), and the DNA sieving medium is 3% Natvosol) is introduced all parts of chip channel (split tunnel and sample intake passage), buffer pool, sample introduction waste liquid pool and damping fluid waste liquid pool.
(2) add the PCR product at sample pool.
(3) sample introduction: sample pool 3 ground connection, sample introduction waste liquid pool 4 applies voltage, and field intensity 10000V/cm between the two applied voltage time 1 second.
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field intensity 2000V/cm applied voltage time 30 seconds.Detector is arranged in split tunnel and detects PCR product signal, and effectively distance is 1cm between sample intake passage end and the detector, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.
Table 1 has or not the comparison of isotachophoresis pre-concentration micro-fluidic chip Φ x174 HaeIII Digest dna fragmentation detectability
| Dna fragmentation | The total DNA:2.5 pg/ of integrated isotachophoresis pre-concentration micro-fluidic chip μ L | The total DNA:500pg/ μ of no pre-concentration function micro-fluidic electrophoresis chip L | ||||
| Concentration pg/ μ L | Signal to noise ratio | Detectability pg/ μ L | Concentration pg/ μ L | Signal to noise ratio | Detectability pg/ |
|
| 72 118 194 234 310 603 872 1078 1353 | 0.03 0.05 0.09 0.11 0.14 0.28 0.40 0.50 0.63 | 32.33 73.53 165.23 196.97 142.15 479.01 807.23 901.40 824.83 | 0.0031 0.0022 0.0016 0.0016 0.0030 0.0017 0.0015 0.0016 0.0022 | 6.7 10.9 18.0 21.7 28.8 55.9 80.9 100.1 125.6 | 33.5 64 83.5 91.5 95.5 252 433 498 567.5 | 0.598 0.513 0.647 0.712 0.904 0.666 0.561 0.603 0.664 |
| Mean value | 0.0021 | 0.652 | ||||
Table 2 Fig. 2 and Fig. 3 structure micro-fluidic chip are analyzed the comparison of Φ x174 HaeIII Digest series dna fragmentation detectability
| Dna fragmentation base logarithm (bp) | DNA fragment concentrations (pg/ μ L) | Fig. 2 structure micro-fluidic chip detectability (pg/ μ L) | Fig. 3 structure micro-fluidic chip detectability (pg/ μ L) |
| ?72?118?194?234?310?603?872?1078?1353 | ?0.03?0.05?0.09?0.11?0.14?0.28?0.40?0.50?0.63 | 0.0033 0.0023 0.0016 0.0016 0.0030 0.0018 0.0016 0.0015 0.0023 | 0.0031 0.0022 0.0016 0.0016 0.0030 0.0017 0.0015 0.0016 0.0022 |
| Mean value | 0.002 | 0.002 |
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1. one kind is used for the functionalization micro-flow control chip that the PCR product detects, described chip is by being made up of 4 liquid storage tanks, split tunnel and sample intake passages, it is characterized in that: described liquid storage tank is respectively buffer pool, damping fluid waste liquid pool, sample introduction waste liquid pool and sample pool, the split tunnel two ends connect buffer pool and damping fluid waste liquid pool respectively, and the sample intake passage two ends connect sample pool and sample introduction waste liquid pool respectively; Split tunnel and sample intake passage overlapping region are effective sample intake passage; The length range of described effective sample intake passage is 0.5～20 centimetre.
2. according to the described functionalization micro-flow control chip that is used for the detection of PCR product of claim 1, it is characterized in that: described sample pool and sample introduction waste liquid pool are positioned at the homonymy or the heteropleural of split tunnel.
3. according to the described functionalization micro-flow control chip that is used for the detection of PCR product of claim 1, it is characterized in that: the material of the micro-fluidic chip that the described PCR of being used for product detects is quartz, glass, plastics or silica gel.
4. according to the described functionalization micro-flow control chip that is used for the detection of PCR product of claim 3, it is characterized in that: described plastics are polymethylmethacrylate or polycarbonate, and described silica gel is polydimethylsiloxane.
5. the described functionalization micro-flow control chip of claim 1 is used for the method for PCR product analysis, it is characterized in that comprising the steps:
(1) damping fluid is introduced chip: split tunnel, sample intake passage, buffer pool, sample introduction waste liquid pool and the damping fluid waste liquid pool of damping fluid being introduced functionalization micro-flow control chip by pressure;
(2) add the PCR product at sample pool;
(3) sample introduction: by electric sample introduction or pressure sample introduction the PCR product is full of sample intake passage, for electric sample introduction: apply voltage between sample pool and sample introduction waste liquid pool, field strength range is between 10V/cm～10000V/cm, and applying voltage time is 1～600 second; For the pressure sample introduction: with positive pressure in sample pool or with suction function in the sample introduction waste liquid pool, pressure action time is 1～180 second;
(4) isotachophoresis concentrates, separates and detects: apply voltage between buffer pool and damping fluid waste liquid pool, field strength range is between 50V/cm～2000V/cm, and applying the voltage time length is 30～600 seconds; Effectively separation length is that the distance between effective sample intake passage end and the detector is 1～6cm, and detected PCR product signal is handled the output electrophoretogram after testing and promptly be can be used for the PCR product analysis.
6. functionalization micro-flow control chip according to claim 5 is used for the method for PCR product analysis, it is characterized in that: the chlorion in the described PCR product is a leading ion, described damping fluid contains trails dielectric medium and sieving medium, and the described dielectric medium of trailing comprises 3-(N-morphine quinoline) propanesulfonic acid, N-(second-hydroxyethyl) piperazine-N-2 ethane sulfonic acid or N-three (methylol) methyl-3-aminopropanesulfonicacid acid; Described DNA sieving medium comprises a kind of or two or more in hydroxypropylcellulose, Natvosol, Vltra tears, polyvidone, polyvinyl alcohol, polyoxyethylene glycol or the polyacrylamide.
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| CN102206571A (en) * | 2011-03-09 | 2011-10-05 | 武汉馨世生物科技有限公司 | Molecular diagnosis chip and preparation method and application thereof |
| IL228026B (en) * | 2013-08-19 | 2018-05-31 | Technion Res & Dev Foundation | Detection of genetic sequences using pna probes and isotachophoresis |
| CN104561286B (en) * | 2015-02-10 | 2016-11-16 | 同济大学 | A kind of novel polymeric enzyme chain reaction micro-fluid chip control system and preparation method thereof |
| WO2017087416A1 (en) | 2015-11-16 | 2017-05-26 | Revere Biosensors, Llc | Systems and methods for identifying and distinguishing genetic samples |
| CN105505763A (en) * | 2016-01-12 | 2016-04-20 | 上海理工大学 | Natural-convection-type PCR-electrophoresis integrated chip and detection method |
| CN110869512A (en) * | 2017-05-09 | 2020-03-06 | 雷瑞生物传感器有限责任公司 | System and method for identifying and differentiating genetic samples |
| CN110579527B (en) * | 2019-08-28 | 2021-08-13 | 中国科学院合肥物质科学研究院 | Electrophoresis microchip with ion online enrichment device and detection method |
| CN110567790B (en) * | 2019-09-11 | 2021-11-05 | 南京信息工程大学 | Micro-electrophoresis chip for online concentration and detection of charged small particles and detection method |
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