CN101334377A - A method for rapid screening of chiral selectors and a dedicated array microfluidic chip - Google Patents

Abstract

The invention discloses an array micro fluidic chip which is used for quickly filtering chiral selecting agents and is characterized in that the chip consists of 2 to 100 groups of electrophoretic units (1) that are parallel arranged, and each electrophoretic unit (1) comprises a sample introducing unit (2) and a separating unit (3). Compared with the prior art, the array micro fluidic chip of the invention is characterized by direct, quick and simple operations and less sample usage, and has predictable enormous scientific and economic values.

Description

A method for rapid screening of chiral selectors and a dedicated array microfluidic chip

Technical field:

The present invention relates to chemistry and physical science, and specifically relates to chip electrophoresis chiral separation technology; in particular, it provides a method for rapidly screening chiral selectors based on an array microfluidic chip and a special microfluidic chip.

Background technique:

Driven by asymmetric catalysis and combinatorial chemistry technologies, the number of candidate chiral compounds in pharmaceutical development has multiplied, and the demand for resolution and analysis of chiral compounds continues to grow. The most important factor affecting chiral resolution is to find a suitable selector with chiral recognition ability for the compound to be analyzed. Since the screening of chiral selectors is a time-consuming and labor-intensive process, it is of great significance to develop methods and platforms for rapid screening of chiral selectors for the development of chiral drugs.

In the prior art, there are mainly the following methods and platforms for screening chiral selectors.

1. Liquid chromatography (document Analytical Chemistry, 2000, 72, 5201-5205). This method is to immobilize the two pure optical isomers of the target compound on silica gel to form two stationary phases, then incubate the two stationary phases with an equal amount of chiral selector mixture for 30 minutes, and centrifuge to discard the unadsorbed The components adsorbed on the stationary phase are eluted, collected and concentrated, and then the eluent is separated and detected by reversed-phase liquid chromatography. Comparing the chromatographic analysis results of the two eluents, it is considered that those components with the same retention time but with significant changes in peak height are effective chiral selectors for the target compound, and then identify the chiral selector based on the results of liquid chromatography-mass spectrometry. The structure of the agent. This method is suitable for screening a large number of chiral selector mixture libraries or unknown chiral selector mixtures, but it is not suitable for systems where pure optical isomers of target compounds are difficult to collect or prepare, and the process is cumbersome.

2. Capillary electrophoresis (Chromatographia, 1997, 44, 313-319; Electrophoresis, 2004, 25, 2772-2785). This method generally screens suitable chiral selectors and electrophoresis operating conditions by measuring the effect of changes or combinations of parameters such as the type and concentration of chiral selectors, buffer pH and concentration, on the resolution of target chiral compounds. This method is suitable for the screening of chiral selectors that exist independently and can be investigated separately.

3. Combination of liquid chromatography and capillary electrophoresis (document Analytical Chemistry. 1998, 70, 4967-4973). This method uses a combinatorial library of cyclohexyl peptides as chiral selectors. Firstly, reversed-phase liquid chromatography was used to collect the peptide library segmented according to the difference in the hydrophobicity of cyclohexyl peptide, and then the collected peptide segment was used as a chiral selector to resolve a series of racemic nitrophenyl amino acids. According to the degree of separation of nitrophenyl amino acids, determine the appropriate peptide and the amino acid composition that plays a role in the resolution of the peptide. This method is applicable to the situation that a specific combinatorial library is used as a chiral selector, and the spectral suitability is not strong.

4. NMR method (Journal of Chromatography A, 1998, 797, 149-164; Journal of Chromatography A, 961 (2002) 257-276). The method is to use the chiral selector as a chiral shift reagent, and judge the compound and select according to the chemical shift and spin-lattice relaxation time changes of certain atoms after the compound racemate and the chiral selector are mixed under different conditions. The strength and mode of action of the agent can be used to screen chiral selectors. This method has high requirements on instruments and is not easy to popularize. Capillary electrophoresis or liquid chromatography is often required to verify the screening results.

5. Microplate method (Journal of the American Chemical Society, 2006, 128, 2208-2209; Analytical Chemistry. 2002, 74, 5212-5216; Analytical Chemistry. 1999, 71, 4178-4182). In this method, the chiral selectors to be screened are respectively fixed on the organic phase or the resin, and the chiral compound is dissolved in the water phase. Add an equal amount of the aqueous phase to the microplate, and add an equal amount of the organic phase or resin to each well. If a selector is selective for chiral compounds, a certain amount of chiral compounds will be adsorbed. After incubation for a certain period of time, the organic phase or organic film is removed from the microwell, the absorbance of the aqueous phase is measured, and the chiral compounds in the aqueous phase are separated by circular dichroism or liquid chromatography. The chiral selector on the organic phase corresponding to microwells whose absorbance value, circular dichroism chromatogram or enantiomeric chromatographic peaks are significantly different before and after the addition of the organic phase may be a suitable selector. For the case of adding chiral compounds in the form of racemates, measuring the absorbance value alone is not enough to determine the chiral selector, and capillary electrophoresis or connecting the chiral selector to liquid chromatography fillers is required to verify the chiral selector Whether chiral compounds can be resolved. In addition, this method is time-consuming, often requiring several hours of incubation time to achieve partition equilibrium between the organic and aqueous phases of the chiral compound.

It is expected to obtain a method for rapidly screening chiral selectors based on an array microfluidic chip with better technical effects and its special equipment.

Invention content:

The purpose of the present invention is to provide a method for rapidly screening chiral selectors based on an array microfluidic chip with better technical effect and its special equipment-microfluidic chip.

The invention provides an array microfluidic chip for rapid screening of chiral selective agents, characterized in that: the chip is composed of 2 to 100 groups of electrophoresis units 1 arranged in parallel, and each electrophoresis unit 1 includes a unit 2 and a split unit 3. The "electrophoresis unit" refers to a microchannel system that can provide directional movement of analytes under a high-voltage direct current electric field, and separate and analyze analytes according to their migration speeds in the electric field. "Chip electrophoresis" is usually composed of a sampling unit 2 and a separation unit 3. From the perspective of a single electrophoresis unit 1, there is no essential difference between the electrophoresis unit 1 mentioned in this paper and the "electrophoresis unit" described in other documents or patents. .

In the array microfluidic chip for rapid screening of chiral selective agents, the sampling unit 2 is composed of a sample pool 4, a sampling channel 5 and a sample waste liquid pool 6;

The sampling unit 2 is composed of a linear channel with a length of 0.5 to 1 cm, a sample pool 4 and a sample waste pool 6, and the electrophoresis unit 1 is composed of a separation channel 8 perpendicular to the sampling channel 5 and a buffer reservoir. A liquid pool 7 and a buffer solution waste pool 9 are formed;

The separation unit 3 includes a buffer storage tank 7 and a separation channel 8 , and each separation unit 3 also has a buffer waste tank 9 used independently or together with other separation units 3 .

In the array microfluidic chip used for rapid screening of chiral selectors according to the present invention, each sampling unit 2 and separation unit 3 in each electrophoresis unit 1 have the same structure or mirror-image symmetry, and the separation of each separation unit 3 The channels 8 are concentrated in the detection area to facilitate detection. The width and depth of the sampling channel 5 and/or the separation channel 8 are in the range of 10-100 microns.

The present invention is a method for quickly screening chiral selectors using an array microfluidic chip, according to the resolution ability of each chiral selector for chiral compounds, that is, the separation degree of two or more isomers of chiral compounds Determine the best chiral selector for this chiral compound. It is characterized in that the array microfluidic chip used is composed of 2 to 100 groups of electrophoresis units 1 arranged in parallel, and each electrophoresis unit 1 includes a sampling unit 2 and a separation unit 3 .

In the method for quickly screening chiral selectors using an array microfluidic chip, the sampling unit 2 of the array microfluidic chip used is composed of a sample pool 4, a sampling channel 5, and a sample waste liquid pool 6; the separation unit 3 includes a buffer storage pool 7, a separation channel 8, and each separation unit 3 also has a buffer waste pool 9 that is used independently or jointly with other separation units 3; multiple parallel electrophoresis units 1 share a buffer waste pool Pool 7 is for the convenience of operation, and this design is adopted in all the experiments we conducted. The common buffer liquid waste pool 7 is used to store waste liquid, and has no other special purpose. The object of the present invention can also be achieved by adopting an independent buffer liquid waste pool;

In each electrophoresis unit, each sampling unit 2 and separation unit 3 have the same structure or are symmetrical in mirror image, and the separation channels 8 of each separation unit 3 are concentratedly arranged in the detection area to facilitate detection;

The width and depth of the sampling channel 5 and/or the separation channel 8 are in the range of 10-100 microns. The optimum size of the sampling channel 5 or the separation channel 8 is 10-15 microns deep. If the depth is too shallow, the injection volume is too small, which requires high sensitivity of the detector, so it is not suitable for matching; if the depth is too deep, the injection volume is too large, the chiral compounds are not easy to separate, and it is difficult to evaluate the chiral resolving agent.

Optimum width Theoretically speaking, the narrower the injection channel 5 or the separation channel 8, the better. The narrower the channel, the more conducive to the separation of the target operable substances. However, narrow channels are not suitable for fabrication and are easy to block, generally 40~ The width of 50 microns is better, which can meet the separation requirements and does not require special equipment for chip production.

The method for rapidly screening chiral selectors using an array microfluidic chip described in the present invention is specifically:

Put different types of chiral selective agents that need to be screened into the array microfluidic chip buffer reservoir 7, put the buffer without chiral selective agents into the buffer waste liquid pool 9 and The sample waste liquid pool 6 is a sample pool 4 into which a certain concentration of chiral compounds are respectively placed;

Then a voltage is applied between the sample pool 4 and the sample waste liquid pool 6 to carry out sample injection; Active compounds enter the separation channel 8 for electrophoretic separation and detection.

The buffer used in the different separation units in the array microfluidic chip is usually the same buffer that can dissolve all the chiral selection agents that need to be screened. Some buffers are commonly used as running solutions during electrophoresis, such as phosphate buffer, borax buffer and acetate buffer, etc. The same buffer refers to one of them, such as phosphate buffer.

When a point-focused laser-induced fluorescence detector is used for detection, the appropriate concentration range for chiral compounds is: 10 ^-6 ~ 10 ^-7 moles/liter; when a line-focused laser-induced fluorescence detector is used, the chiral compound The more suitable concentration range is 10 ^-4 ~ 10 ^-5 mol/liter.

The electrophoretic field strength used by each electrophoretic unit 1 is basically the same, and the specific field strength range is: 150-1000 V/cm. Generally, the field strength used is determined successively from low to high. A further preferred range is 300-600 V/cm, and generally the peak shape is better.

The detector used in the method of the present invention detects multiple channels at the same time; specifically, the signals are collected simultaneously at the same position of each separation channel, such as using a charge-coupled device image sensor (CCD) collection system, or using a photomultiplier tube to collect signals ; can also scan between channels at 500 rpm or faster.

Microfluidic chip technology is the current research hotspot in instrument analysis. This technology is mainly based on analytical chemistry and biochemistry. Micro-electromechanical processing technology is used to process 10-100 micron micro-chips on the surface of silicon, glass, quartz, and polymers. The channel network is mainly driven by electroosmotic flow and electrophoretic flow. By changing the driving voltage, the flow direction and rate of fluid in the microchannel network are controlled, which not only facilitates the sampling, dilution, enrichment, extraction, and mixing of target analytes. , reaction, separation, detection and other operations, and it is easy to array the operation unit. So far, there are few reports on the screening of chiral selectors using multiple sets of parallel electrophoretic units. The invention creatively designs an array microfluidic chip for screening of chiral selectors, which can realize the simultaneous screening of multiple chiral selectors. The whole process is completed in minutes and the sample consumption is in microliters. Compared with liquid chromatography, nuclear magnetic resonance, micro-orifice plates and other chiral selective agent screening methods, the present invention has the advantages of directness, rapidity, simple operation and less sample consumption.

In a word, the present invention can complete the simultaneous screening of multiple chiral selectors in a short period of time on a chip, glass or quartz chip of several square centimeters. Compared with the prior art, the present invention has the characteristics of directness, rapidity, simple operation and less sample consumption. It has foreseeable huge scientific value and economic value.

Description of drawings:

Fig. 1 is a structural diagram of an array microfluidic chip of the present invention (containing four electrophoresis units);

Fig. 2 is the electrophoresis spectrogram of chiral selector screening in embodiment 1, and chiral compound is baclofen;

Fig. 3 is the electrophoretic chromatogram of chiral selector screening in Example 2, and the chiral compound is norfenefrine.

Detailed ways:

Example 1

Chiral selector screening for the racemic compound baclofen. The chip used in this experiment is a glass chip containing four electrophoresis units 1 as shown in FIG. 1 , with a channel width of 50 μm and a depth of 15 μm. The experiment uses a laser-induced fluorescence detector for detection, so the compound must be fluorescently labeled. The process is as follows: Dissolve the fluorescent dye fluorescein isothiocyanate (FITC) in a small amount of acetone into a 20mM solution, and take 20μL and 40μL of 10mM baclofen solution. Mix, react at room temperature for 12 hours, and dilute to 60 μM with buffer. Four cyclodextrin-based chiral selectors (α-CD, β-CD, γ-CD, DM-β-CD) were dissolved in buffer and diluted to 2 mM. Put various cyclodextrin solutions into the buffer storage pool 7 respectively, put the balofen solution labeled with fluorescein isothiocyanate into the sample pool 4, put the blank buffer solution into each sample waste pool 6 and the common buffer Waste liquid pool9. Apply a voltage (field strength 400V) between the sample cell 4 and the sample waste liquid cell 6 for 15 seconds, then switch the voltage to between the buffer liquid reservoir 7 and the buffer liquid waste liquid cell 9 (field strength 300V), and the sample Pool 4 and sample waste pool 6 apply suppression voltage to prevent sample leakage, and detect at a distance of 5.8 cm from the sample injection point. The results are shown in Figure 2. It can be seen from Figure 2 that among the four cyclodextrins, DM-β-CD can baseline-separate the enantiomers of balofen and is a suitable chiral selector. The whole analysis process is completed within 1 minute.

Example 2

Screening of chiral selectors against the racemic compound norfenefrine. The implementation process of this example is basically the same as that of Example 1, except that the type of cyclodextrin screened is different from that of Example 1. The cyclodextrins used in this example include HP-α-CD, HP-β-CD, HP-γ-CD, and DM-β-CD. The screening results are shown in Figure 3. It can be seen from Figure 3 that among the four CDs screened, both HP-α-CD and DM-β-CD showed obvious chiral selectivity to the norfenefrine racemate, and the two enantiomers could be baseline separated . The whole analysis process is completed within 1 minute.

Note: In the above two examples, Tris-HCl buffer with pH 9.2 was used.

Claims (10)

1, a kind of array micro-fluidic chip that is used for quickly screening chiral selection agent is characterized in that: described chip is made up of 2～100 groups of electrophoretic cells that are arranged in parallel (1), and each electrophoretic cell (1) comprises a sample injection unit (2) and a separative element (3).

2, according to the described array micro-fluidic chip that is used for quickly screening chiral selection agent of claim 1, it is characterized in that:

Described sample injection unit (2) is made up of sample cell (4), sample intake passage (5) and sample waste liquid pool (6);

Separative element (3) includes damping fluid liquid storage tank (7), split tunnel (8), each separative element (3) also have independent or with the common damping fluid waste liquid pool (9) that uses of other separative element (3).

3, according to the described array micro-fluidic chip that is used for quickly screening chiral selection agent of claim, it is characterized in that:

Each sample injection unit (2) is identical with separative element (3) structure or become the mirror image symmetry in described each electrophoretic cell (1), and the split tunnel (8) of each separative element (3) is concentrated at surveyed area and arranged;

The width of described sample intake passage (5) and/or split tunnel (8) and the degree of depth are in 10～100 microns scope.

4, a kind of method of using the array micro-fluidic chip quickly screening chiral selection agent, it is characterized in that: employed array micro-fluidic chip is made up of 2～100 groups of electrophoretic cells that are arranged in parallel (1), and each electrophoretic cell (1) comprises a sample injection unit (2) and a separative element (3).

5, according to the method for the described use array micro-fluidic chip of claim 4 quickly screening chiral selection agent, it is characterized in that:

The sample injection unit of employed array micro-fluidic chip (2) is made up of sample cell (4), sample intake passage (5) and sample waste liquid pool (6); Separative element (3) includes damping fluid liquid storage tank (7), split tunnel (8), the damping fluid waste liquid pool (9) that each separative element (3) also has independence or uses jointly with other separative element (3);

Each sample injection unit (2) is identical with separative element (3) structure or become the mirror image symmetry in described each electrophoretic cell, and the split tunnel (8) of each separative element (3) is concentrated at surveyed area and arranged;

The width of described sample intake passage (5) and/or split tunnel (8) and the degree of depth are in 10～100 microns scope.

6, according to the method for claim 4 or 5 described use array micro-fluidic chip quickly screening chiral selection agents, it is characterized in that: described method is:

The chiral selector that dissimilar various needs screen is put into described array micro-fluidic chip damping fluid liquid storage tank (7), the damping fluid that does not contain chiral selector is put into damping fluid waste liquid pool (9) and sample waste liquid pool (6), the sample cell (4) that certain density chipal compounds is put into respectively;

Carry out sample introduction to applying voltage between sample cell (4) and the sample waste liquid pool (6) then; After sample introduction is finished, between damping fluid liquid storage tank (7) and damping fluid waste liquid pool (9), apply voltage, carry out electrophoretic separation and detection.

7, according to the method for the described use array micro-fluidic chip of claim 6 quickly screening chiral selection agent, it is characterized in that: employed damping fluid is the damping fluid that can dissolve all chiral selectors that need screen with a kind of in the different separative element in the described array micro-fluidic chip.

8, according to the method for the described use array micro-fluidic chip of claim 6 quickly screening chiral selection agent, it is characterized in that: when adopting the laser induced fluorescence detector of point focusing formula to detect, chipal compounds than the suitable concentration scope be: 10 ^-6～10 ^-7Mol; Adopt the laser induced fluorescence detector of line focus formula, chipal compounds be 10 than the suitable concentration scope ^-4～10 ^-5Mol.

9, according to the method for the described use array micro-fluidic chip of claim 6 quickly screening chiral selection agent, it is characterized in that: the electrophoresis field intensity unanimity that described each electrophoretic cell (1) uses, concrete field strength range is: 150～1000 volts/centimetre.

10, according to the method for the described use array micro-fluidic chip of claim 9 quickly screening chiral selection agent, it is characterized in that: the detecting device that described method is used detects a plurality of passages simultaneously; Specifically be in the same position while of each split tunnel acquired signal, also can be with 500 rev/mins or faster speed scanning between passage.

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