CN109856278A - A method of the screening active constituent based on three-phase laminar flow micro-fluidic chip - Google Patents
A method of the screening active constituent based on three-phase laminar flow micro-fluidic chip Download PDFInfo
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Abstract
本发明公开了一种基于三相层流微流控芯片的筛选活性成分的方法，本发明可以将亲和分子和游离分子在短距离的微通道内进行快速分离，提高效率；而且自动化程度高，重现性好，游离小分子也可以同时富集浓缩；而且一步就可以同时完成大分子和小分子的无膜有效分离，以及分子萃取的多步样品前处理操作，省时省事；降低经济成本，大力推动中药现代化的进程。The invention discloses a method for screening active ingredients based on a three-phase laminar flow microfluidic chip. The invention can rapidly separate affinity molecules and free molecules in a short-distance microchannel, thereby improving efficiency; and the degree of automation is high. , good reproducibility, free small molecules can also be enriched and concentrated at the same time; moreover, the membraneless effective separation of macromolecules and small molecules, and the multi-step sample pretreatment operation of molecular extraction can be completed at the same time in one step, saving time and trouble; reducing economic cost, and vigorously promote the process of modernization of traditional Chinese medicine.
Description
Technical field
The invention belongs to technical field of traditional Chinese medicines, in particular to a kind of screening activity based on three-phase laminar flow micro-fluidic chip at
The method divided.
Background technique
Traditional Chinese medicine ingredients are sufficiently complex, and physical chemical differences are very big, and the active constituent of Chinese medicine is unclear, Ren Menzhen
Its definite curative effect is difficult to existing index, therefore the active detection of Chinese traditional medicine biology has become the research of Chinese medicine Quality Control technology
Hot spot.
The research of Chinese drugs mostly uses extraction and separation technology (such as thin-layer chromatography, low middle compression leg chromatography, adverse current color
Spectrum, preparative chromatography) various traditional Chinese medicine ingredients are separated, its effective component is then determined after cell, the animal pharmacology active testing.
Wherein the separation process of ingredient is cumbersome, and the period is long, the compound being finally recovered may be because without activity or simultaneously
It is not needed for researcher, and consumes a large amount of manpower and material resources.Therefore, in order to solve existing technical problem, one kind is found
For the purpose of simplifying sample pre-treatments, the method for filtering out active constituent (effective substance), if being a worth further investigated
Topic.
Summary of the invention
The primary purpose of the present invention is that providing a kind of side of screening active constituent based on three-phase laminar flow micro-fluidic chip
Method.
The technical solution used in the present invention is:
A kind of three-phase laminar flow micro-fluidic chip, including main channel, three injection ports and three outlets, three sample introductions
Mouth is connected to one end of main channel, and three outlets are connected to the other end of main channel；Three injection ports are respectively
Sample to be tested injection port (1), blank buffer solution injection port (2) and organic extraction solution injection port (3)；Three outlets
Respectively there are large biological molecule outlet (4), large biological molecule and the free drug small molecule of affinity interaction with bio-target molecule
Mixture outlet (5) and organic solvent outlet (6)；A length of 2~the 3cm in main channel, width are 300~325 μm, deeply 30
~35 μm.
Further, chip is bonded by dry plate glass and cover glass, and slot is made in the dry plate glass end punching, institute
Stating cover glass is polished silicon wafer.
Further, the sample to be tested injection port (1), blank buffer solution injection port (2) and organic extraction solution into
Sample mouth (3) is connected with syringe respectively；The large biological molecule outlet (4), large biological molecule and the mixing of free drug small molecule
Object outlet (5) and organic solvent outlet (6) are connected with test tube respectively.
A method of screening or assisting sifting active constituent based on three-phase laminar flow micro-fluidic chip, the three-phase layer
Stream micro-fluidic chip is three-phase laminar flow micro-fluidic chip described above, comprising the following steps:
1) after being incubated for sample to be tested I, purpose bio-target molecule and blank buffer solution, it is micro-fluidic to be injected into three-phase laminar flow
Chip sample to be tested injection port (1), while blank buffer solution is introduced into blank buffer solution injection port (2) and will be organic
Solvent is introduced into organic extraction solvent injection port (3)；
2) after being passed through 16~30min, the organic solvent ie in solution II of organic solvent outlet (6) is collected as experimental group,
It volatilizes and is dissolved with methanol, carry out HPLC-MS separation identification；
3) separately three-phase laminar flow micro-fluidic chip is taken to do blank control experiment, sample to be tested I and blank buffer solution is mixed
Afterwards, it is injected into three-phase laminar flow micro-fluidic chip sample to be tested injection port (1), blank buffer solution is introduced into blank buffer solution
Injection port (2), organic solvent are introduced into organic extraction solution injection port (3)；
4) the organic solvent ie in solution III for collecting organic solvent outlet (6) as a control group, is volatilized and is dissolved with methanol,
Carry out HPLC-MS separation identification；
5) step 2) solution II and step 4) solution III are made HPLC-MS separation identification to compare, if peak area is reduced
Ingredient be then with purpose bio-target molecule have affinity interaction active constituent.
Further, step 1) and step 3) the sample to be tested I include the compound of Chinese medical extract, compound library.
Further, step 1) the purpose bio-target molecule is tetra- serobila of G-, haemocyanin, alpha-glucosidase.
Further, the step 1) incubation time is 60min.
Further, the step 1) organic solvent be methylene chloride, chloroform, n-butanol, ether, ethyl acetate at least
It is a kind of.
Application of the three-phase laminar flow micro-fluidic chip described in any of the above embodiments in active ingredient screening.
The beneficial effects of the present invention are:
Micro-fluidic chip three-phase laminar-flow technique of the invention can be by affinity molecule and free molecule short-range micro- logical
Without UF membrane in road, the absorption of film is avoided, reduces the error of weak affine ingredient, improves separative efficiency；Being integrated with will divide
Free drug molecule from after is extracted to the operation of organic solvent, reduces pre-treatment step；It automates and integration degree is high,
Favorable reproducibility, it is chip used reusable, it is time saving to save trouble；Economic cost is reduced, pushes the process of the modernization of Chinese medicine energetically.
Detailed description of the invention
Fig. 1 is schematic diagram when three-phase micro-fluidic chip of the present invention is in running order；Syringe pump refers in figure
It is syringe pump, Eppendorf tube is to collect test tube；
Fig. 2 is the structural schematic diagram of three-phase micro-fluidic chip of the present invention；
Fig. 3 is that sample to be tested is compared using the chromatogram of micro-fluidic chip method of the present invention and ultrafiltration.
In figure: 1, sample to be tested injection port；2, blank buffer solution injection port；3, organic extraction solvent injection port；4, with
Bio-target molecule has the large biological molecule outlet of affinity interaction；5, large biological molecule and free drug small-molecule mixture go out sample
Mouthful；6, organic solvent outlet.
Specific embodiment
Embodiment 1
A kind of three-phase laminar flow micro-fluidic chip (see Fig. 1 and Fig. 2), including main channel, three injection ports and three outlets,
Three injection ports are connected to one end of main channel, and three outlets are connected to the other end of main channel；Three injection ports are respectively as follows:
Sample to be tested injection port 1, blank buffer solution injection port 2 and organic extraction solvent injection port 3；Three outlets are respectively as follows:
There are large biological molecule outlet 4, large biological molecule and the free drug small-molecule mixture of affinity interaction to go out with bio-target molecule
Sample mouth 5 and organic solvent outlet 6 (outlet of free drug small molecule of the extraction without affinity interaction).Wherein, chip material
Material is glass material, and chip overall length 6.3cm, wide 2.1cm, (length of main channel can be according to biological target by a length of 2~3cm in main channel
Dispersal behavior and the extraction yield of free drug be adjusted), width is 300~325 μm, 30~35 μm deep.Three-phase laminar flow is micro-
Fluidic chip is bonded by dry plate glass and cover glass, and dry plate glass passes through chemical etching method etched channels, dry plate glass
Slot is made in channel end punching, and cover glass is polished silicon wafer.
Main channel section is equal between organic extraction solvent injection port 3, organic solvent outlet 6, injection port 3 and outlet 6
There is surface selective modification, inner surface has hydrophobic property, makes organic solvent mobile freely.Glass and pump between connecting tube be
Polyfluortetraethylene pipe.
Embodiment 2
The step of screening or assisting sifting active constituent method are as follows:
By the methanol extract liquid 60ml of macleaya cordata, it is evaporated water redissolution, obtains solution I.
By tetra- serobila of bio-target molecule G- and 600 μ L blank buffer solution (50mM of 200 μ L solution Is and 200 μ L mesh
KCl,10mM KH2PO4,and 1mM K2EDTA, pH 7.4) it mixes 60 minutes, 95 DEG C of incubation 5min, after cooled to room temperature
The sample to be tested injection port 1 of three-phase laminar flow micro-fluidic chip of the present invention is introduced, blank buffer solution injection port 2 introduces 1mL simultaneously
Blank buffer solution (50mM KCl, 10mM KH2PO4,and 1mM K2EDTA, pH 7.4) as isolation solution, make biological target
Molecule discord extraction is directly contacted with organic solvent such as methylene chloride, in case biological target activity is affected.Organic extraction solvent
Injection port 3 introduces dichloromethane solution, has the large biological molecule of affinity interaction to pass through 4 row of large biological molecule outlet with biological target
Out, large biological molecule and free drug small-molecule mixture pass through large biological molecule and free drug small-molecule mixture outlet
5 discharges.The organic solvent for collecting free drug small molecule of the extraction without affinity interaction that organic solvent outlet 6 obtains, volatilizes
After again with methanol dissolution, as solution II is added to HPLC-MS and carries out separation identification.
Sample introduction product are to collecting the organic of obtained free drug small molecule of the extraction without affinity interaction of organic solvent outlet 6
Solvent, whole experiment process maintains 16~30min, in this period, injection port 1~3 according to the solution that describes of the present invention and
Experiment condition introduces solution incessantly.1 flow velocity of injection port is 6 μ L/min, and 2 flow velocity of injection port is 6 μ L/min, and injection port 3 flows
Speed is 12 μ L/min.
Blank control separately is done with three-phase laminar flow micro-fluidic chip of the invention, 800 μ are added in the solution I of another 200 μ L
L blank buffer solution introduces sample to be tested injection port 1 as placebo solution, and blank buffer solution injection port 2 introduces simultaneously
Blank buffer solution introduces methylene chloride as isolation solution, organic extraction solvent injection port 3, collects organic solvent outlet 6
Dichloromethane solution, as solution III volatilizes again with methanol dissolution, is added to liquid chromatography-mass spectrography (HPLC-MS) and separated
Detection.
Liquid phase chromatogram condition: YMC-Pack ODS-AQ (150mm × 2.0mm, 3 μm)；Mobile phase A is acetonitrile, Mobile phase B
For 0.2% formic acid water；Gradient elution: 0 → 5min, 11% → 11%A；5 → 45min, 11% → 50%A；45 → 50min,
50% → 85%A；50 → 55min, 85% → 85%A；55 → 60min, 85% → 11%A；60 → 65min, 11% → 11%
A；Flow velocity: 0.2mL/min；Sample volume: 2 μ L, column temperature: 35 DEG C.
Mass Spectrometry Conditions: electric spray ion source (ESI)；Using positive ion detection mode；Scanning range m/z 150~1000；
Dry temperature degree: 350 DEG C；Dry gas stream speed: 8L/min；Atomizing pressure: 45psi；Capillary voltage: 3500V；Desolventizing gas and
Atomization gas is nitrogen.
Extraction is at least one of methylene chloride, chloroform, n-butanol, ether, ethyl acetate with organic solvent.
Solution II is compared with the result of solution III, if the ingredient that peak area becomes smaller is can be with bio-target molecule
There is the active constituent of affinity interaction.
Comparative example
Hyperfiltration process: 300 μ L placebo solutions and sample solution are respectively placed in centrifugal ultrafiltration pipe, and (shut off molecular weight
In 3KD), and it is centrifuged 20min at 10000r/min, the centrifugation of 300 μ L PBS solutions is added into chimney filter, washes for repeated washing 3 times
Remove unbonded ingredient.Blank control ultrafiltrate and sample ultrafiltrate are collected respectively, are extracted with 200 μ L methylene chloride, by dichloromethane
The solution of alkane part is sucked out with liquid-transfering gun, volatilizes solvent, is redissolved solvent with methanol, is passed through HPLC and is carried out separation detection.
Three-phase laminar flow micro-fluidic chip principle prepared by the present invention are as follows: in flow process, have affinity interaction with biological target
Large biological molecule and free drug small-molecule mixture from the free drug small molecule of three-phase laminar flow micro-fluidic chip mix
Object outlet 5 flows out, and does not have the free drug small molecule of affinity interaction to enter organic solvent extraction across PBS phase with biological target
Phase finally flows out and collects from the organic solvent outlet 6 of free drug small molecule of the extraction without affinity interaction, obtains experimental group
Free drug small molecule of the extraction without affinity interaction organic solvent outlet 6 flow out dichloromethane solution II, be added to
Separation identification is carried out in HPLC-MS, compared with the dichloromethane solution III that blank control group organic solvent outlet 6 is collected,
By comparing the chromatogram of experimental group and blank control group, the determining active constituent for having affinity interaction with biological target.
In chip microscale channel, solution is flowed with laminar flow.Solute in solution flow process, in each fluid
Molecule is because concentration difference is spread.Diffusion velocity is related to molecular size.The big diffusion velocity of molecule is slow, the small expansion of molecule
It is fast to dissipate speed.In three-phase laminar flow micro-fluidic chip prepared by the present invention, there are the drug of affinity interaction and biology big with biological target
Molecular diffusion rates are slow, mainly flow out from the large biological molecule outlet 4 for having affinity interaction with bio-target molecule；Do not have with biological target
There is the free drug small molecule diffusion velocity of affinity interaction fast, passes through the isolation of intermediate buffering solution and mutually enter organic solvent extraction phase
(the i.e. organic extraction solvent such as methylene chloride that organic extraction solvent injection port 3 introduces in Fig. 2, finally from extraction without affinity interaction
Free drug small molecule organic solvent outlet 6 flow out and collect, volatilize again with methanol dissolution after, in HPLC-MS into
Row separation identification, by comparing the chromatogram of experimental group and blank control group, determines the ingredient of chromatographic peak area significant change i.e.
To there is the active constituent of affinity interaction with biological target.
This chip pre-treating method is integrated with large biological molecule and separation of small molecuies, free drug small molecule extracting and enriching two
A functional unit is not required to be centrifuged, and easily integrated with other separation methods etc., the time is short, favorable reproducibility, high-efficient.Compare traditional experiment
The room hyperfiltration process time shortens 3 times or more.
Further detection is made to the effect of above-described embodiment.
Bio-target molecule of the present invention can be greater than 8000 for tetra- serobila of G-, haemocyanin, alpha-glucosidase, molecular weight
Nucleic acid and protein.Sample to be tested I includes the compound of Chinese medical extract, compound library.It is micro- using three-phase laminar flow of the invention
Fluidic chip screens the effect of active constituent, also with following example: the ligand one affine with tetra- serobila of G- in screening macleaya cordata
Sample, significant effect.
With three-phase laminar flow micro-fluidic chip prepared by the present invention screening macleaya cordata in G- tetra- serobilas (anti-tumor biological target
Point) affine ingredient as a result, as shown in Figure 3.
As a result: to ultrafiltration result and three-phase laminar flow micro-fluidic chip prepared by the present invention use respectively fingerprint spectrum method into
The comparison of row peak area.It is found that in 10~45 minutes, the peak area difference RSD that chip method of the present invention obtains is greater than table 1
20% has 46 peaks, and hyperfiltration process has 33 peaks；In the difference peak of RSD > 20%, peak area accounts for all chromatographic peak gross areas
Percentage is greater than 5%, and chip method of the present invention has 4 ingredients, and hyperfiltration process only has 2 ingredients；Chip method of the present invention
Peak area accounts in all chromatographic peak gross area percentages, and minimum chip is 0.019%, and minimum hyperfiltration process is 0.010%.
This demonstrate that chip method of the present invention is compared with hyperfiltration process, because the absorption problem of film, sized molecules are not present in it
Separation it is more significant, diffusion is mild, reduces error of the second kind.
The comparison of 1 ultrafiltration of table and chip method effect of the present invention
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
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Cited By (4)
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| CN111185029A (en) * | 2020-01-09 | 2020-05-22 | 昆明理工大学 | Multi-metal ion extraction device and method based on micro-fluidic chip and extraction rate calculation method |
| CN111921572A (en) * | 2020-07-15 | 2020-11-13 | 广东药科大学 | Microfluidic chip and multi-target active ingredient screening method |
| CN112858177A (en) * | 2019-11-26 | 2021-05-28 | 武汉理工大学 | Heavy metal ion on-line measuring chip based on micro-fluidic extraction technique |
| CN114450077A (en) * | 2019-09-26 | 2022-05-06 | 香奈儿香水美妆品公司 | Method for microfluidic extraction from vegetable oils |
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