CN110618184A - Efficient capillary electrophoresis detection system and application - Google Patents

Efficient capillary electrophoresis detection system and application Download PDF

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CN110618184A
CN110618184A CN201910987314.8A CN201910987314A CN110618184A CN 110618184 A CN110618184 A CN 110618184A CN 201910987314 A CN201910987314 A CN 201910987314A CN 110618184 A CN110618184 A CN 110618184A
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detection system
hpce
capillary
tciddc
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CN110618184B (en
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凌笑梅
梁春苏
李菁
李聪
田珂馨
杨希蓓
朱凯
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Peking University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/4473—Arrangements for investigating the separated zones, e.g. localising zones by electric means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples

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Abstract

The invention provides a high-efficiency capillary electrophoresis detection system, a detection method and application. The detection system comprises: the device comprises a separation buffer liquid bottle, two-end electrodes, a front-end capillary tube, a connector, a rear-end capillary tube, a high-voltage power supply, a signal amplification device, a current detector, an ultraviolet detector, a diode array detector or a laser-induced fluorescence detector. The detection system is suitable for qualitative and quantitative analysis, intermolecular interaction, drug screening and the like of substances such as inorganic compounds, amino acids, saccharides, peptides, proteins, lipids, DNA, RNA, organic small molecules and the like under the condition of using one or more detectors simultaneously.

Description

Efficient capillary electrophoresis detection system and application
Technical Field
The invention relates to the technical field of detection, relates to research on a new construction and detection method of a new efficient capillary electrophoresis mode and application thereof, and particularly relates to application of the new system in qualitative and quantitative analysis, qualitative and quantitative analysis of intermolecular interaction, drug screening and the like of inorganic compounds, amino acids, saccharides, peptides, proteins, lipids, DNA, RNA, organic small molecules and the like which do not have ultraviolet absorption or have weak ultraviolet absorption under the condition that a current detector and an ultraviolet detector/a diode array detector/a laser-induced fluorescence detector are simultaneously used.
Background
Increasingly complex biological samples and diverse qualitative and quantitative analysis demands present ever-increasing challenges to analytical chemistry and instrumental analysis. Among many analytical methods, High Performance Capillary Electrophoresis (HPCE) has been widely used because of its advantages of rapidness, High efficiency, automation, High sensitivity, and low sample consumption. In the existing HPCE detectors, the ultraviolet detector is most widely applied, and a detected object can be captured by the detector due to good ultraviolet absorption; the fluorescence detector is the most sensitive and needs the measured object to be able to emit fluorescence; the mass spectrum detector has the widest spectrum, but the wide application of the mass spectrum detector is limited due to the huge instrument, high price and the like; the conventional electrochemical detector needs the tested object to be subjected to oxidation-reduction reaction, and although the conventional electrochemical detector is sensitive, the conventional electrochemical detector has the defects that the detector is easy to be polluted and the like. At present, the most commonly configured detectors of the HPCE apparatus are an ultraviolet detector, a secondary array tube detector and a fluorescence detector, and when a substance to be measured lacks good ultraviolet absorption, the three detectors cannot be used for direct measurement, but a derivatization process is performed in advance, and signals can be captured and generated by the detectors after ultraviolet absorption groups or fluorescent groups are carried on. However, the derivatization process is complicated to operate, and the structure of the analyte is changed after derivatization, so that real information of the analyte cannot be obtained. Therefore, it is important to research and develop a new detection method so that substances with poor or no ultraviolet absorption can be directly qualitatively and quantitatively analyzed without derivatization.
The HPCE technology is widely applied in the field of biomedicine, and particularly shows good application prospect in the aspects of drug screening and the research of intermolecular interaction. In the work of screening antitumor drugs, a predecessor extracts and separates a specific protein receptor on the surface of a tumor cell, then uses the specific protein receptor as a stationary phase, and adopts HPCE to carry out intermolecular interaction research, so as to successfully screen a lead compound which has interaction with the specific protein receptor. However, in practical work, hydrophobic transmembrane receptor proteins on the surface of tumor cells are not easily obtained, and protein denaturation is easy to occur in the processes of extraction, separation and purification so as to change the original spatial structure and biological activity of the proteins, which often causes false positive and false negative results in subsequent drug screening work. If the specific receptor protein which keeps the original conformation can be directly obtained without the processes of extraction, separation and purification of the receptor protein, great advantages are brought to the subsequent drug screening work. In addition, the traditional Chinese medicine plays an important role in treating tumors. Through years of clinical practice, the traditional Chinese medicine can play an anti-tumor role in multiple aspects of improving the immune function of the organism, relieving the toxic and side effects of radiotherapy and chemotherapy, inhibiting the proliferation of tumor cells, reversing multidrug resistance and the like. However, the traditional Chinese medicine preparation has various and complicated components and insufficiently accurate and clear effective components, so that the traditional Chinese medicine preparation always faces a small challenge in clinical application. If the effective components of the Chinese medicinal mixture can be clarified, the ineffective components or toxic components in the preparation can be simplified, and new development can be brought to a plurality of Chinese medicinal mixed preparations.
Disclosure of Invention
The invention provides a high-efficiency capillary electrophoresis detection system, a detection method and application. The invention creates a brand new HPCE detection system by building a serial Connection interface (TCIDDC) of capillaries with Different inner diameters without a sieve or a serial Connection interface (TCIDDCM) of capillaries with Different inner diameters with a sieve, and combining a Current Detector (CRD), an ultraviolet-visible light Detector (UV Detector, UVD), a Diode Array Detector (DAD), and a Laser Induced Fluorescence Detector (LIF).
The high-efficiency capillary electrophoresis detection system of the invention can be combined into 12 systems by adopting the detectors, which are respectively a TCIDDC-CRD-HPCE detection system, a TCIDDC-CRD-UVD-HPCE detection system, a TCIDDC-CRD-DAD-HPCE detection system, a TCIDDC-CRD-LIF-HPCE detection system, a TCIDDC-CRD-UVD-LIF-HPCE detection system and a TCIDDC-CRD-DAD-LIF-HPCE detection system, the system comprises a TCIDDCM-CRD-HPCE detection system, a TCIDDCM-CRD-UVD-HPCE detection system, a TCIDDCM-CRD-DAD-HPCE detection system, a TCIDDCM-CRD-LIF-HPCE detection system, a TCIDDCM-CRD-UVD-LIF-HPCE detection system and a TCIDDCM-CRD-DAD-LIF-HPCE detection system.
Due to the different inner diameters of the front end capillary and the rear end capillary, the current of the whole system can be changed when a sample passes through the interface. The current fluctuation is related to the state of the sample, has good linear relation with the change of the concentration of the sample, and can be used as a detection signal. This is used to clarify the CRD for the detection principle. The invention adopts CRD to detect on HPCE instrument for the first time, and can be used for detecting substances which are not suitable for ultraviolet-visible light detector, such as substances without ultraviolet absorption or with poor ultraviolet absorption (inorganic compounds, amino acids, saccharides, organic small molecules and the like).
The invention constructs TCIDDC or TCIDDCM, so that the new system can be widely applied to the fields of pharmacy, biology, chemistry and the like. TCIDDC mode can place an entire piece of biological tissue into a capillary. The TCIDDCM mode is that silicon dioxide powder is processed to prepare a solid material with controllable pore diameter, and biological materials such as biological tissues, cells, cell membranes, cell nucleuses, endoplasmic reticulum, mitochondria, Golgi bodies, ribosomes, mesenchymal cells, solid matters and the like can be put into capillaries and intercepted. Through the two modes, the novel system can be applied to the aspects of drug screening, interaction of bioactive macromolecules and active ligands, drug component analysis, purification and identification of drug active components and the like. When TCIDDCM is adopted, the biological material is injected from the end part of the capillary with larger inner diameter and is intercepted in the capillary by the capillary or the column sieve with smaller inner diameter and a detection window, so that the detection of a detector is not influenced; the biological material can be updated at any time by reverse-direction punching, so that the situation that the target is occupied by a compound with strong interaction to inactivate the biological material to cause false positive or false positive results is avoided.
The invention has the advantages that: (1) the current detector can be used alone or in combination with other detectors. The single sample introduction is carried out, and the CRD is used independently to provide a current-electrophoresis chart; the use of CRD-UVD provides a current-electropherogram and a UV-electropherogram; the use of CRD-DAD provides current-electropherograms, UV-electropherograms, and full-wavelength scanning UV spectrograms; current-electropherograms and fluorescence-electropherograms can be provided using CRD-LIF; the CRD-UVD-LIF can be used for providing a current-electrophoretogram, a ultraviolet-electrophoretogram and a fluorescence-electrophoretogram; using CRD-DAD-LIF, a galvanophorogram, a UV-electropherogram, a full-wavelength scanning UV spectrogram, and a fluorescence-electropherogram can be provided. (2) CRD is unique in that it can resolve multiple ionic states in solution compared to other detectors. (3) Two interfaces, TCIDDC or TCIDDCM, were invented. (4) When biological tissues, cells, cell membranes, cell nucleuses, endoplasmic reticulum, mitochondria, Golgi bodies, ribosomes, interstitial cells, solid matters and the like are directly used for drug screening and molecular interaction research by adopting TCIDDCM, cell electrotransformation and protein purification are not needed, and biological materials can be recycled.
In one aspect, the invention creates a new HPCE detection method using current as a detector by using a new system. The method can quantitatively detect substances without ultraviolet absorption or poor ultraviolet absorption such as inorganic compounds, amino acids, saccharides, peptides, proteins, lipids, DNA, RNA, organic small molecules and the like with high sensitivity, and solves the problems that the conventional HPCE has poor sensitivity and cannot detect substances without ultraviolet absorption and the like.
The HPCE system in TCIDDC mode includes: the device comprises a separation buffer liquid bottle, two-end electrodes, a front-end capillary tube, a connector, a rear-end capillary tube, a high-voltage power supply, a signal amplification device, a current detector and other detectors. The HPCE method for quantitatively detecting the substances without ultraviolet absorption or poor ultraviolet absorption by taking CR-EG as a detection signal comprises the following steps:
(1) preparing TCIDDC or TCIDDCM comprising:
acid, alkali and organic solvent resistance experiments are carried out on TCIDDC or TCIDDCM, and the internal diameter ratio and the lengths of front and back capillary tubes are optimized. The capillaries of different inner diameters are connected by a connector. Pretreating the capillary tubes connected in series; the capillary is washed by methanol, ultrapure water, 0.1M HCl, ultrapure water, 0.1M NaOH and ultrapure water in sequence;
(2) mounting the cartridge into a capillary electrophoresis apparatus;
(3) optimizing the type, concentration, PH, additive, sample introduction pressure, sample introduction time, separation voltage and separation pressure of a background buffer solution, and determining the optimal conditions;
(4) and carrying out a system applicability experiment and a detector performance index test experiment on the sample, wherein the experiments comprise detecting the noise, sensitivity, precision, detector, linear range and the like of the detector, comparing the response values of the sample in CR-EG and UV-EG, clarifying the complementary advantages of simultaneous use of CRD and UVD, and further carrying out qualitative analysis and quantitative analysis on the sample.
In another aspect, the present invention provides a new multi-detector-combination HPCE system for directly intercepting biological materials (e.g., biological tissues, cells, cell membranes, cell nuclei, endoplasmic reticulum, mitochondria, Golgi apparatus, ribosomes, mesenchymal cells, solids, etc.) in capillaries by using TCIDDC or TCIDDCM model, including TCIDDC-CRD-HPCE detection system, TCIDDC-CRD-UVD-HPCE detection system, TCIDDC-CRD-DAD-HPCE detection system, TCIDDC-CRD-LIF-HPCE detection system, TCIDDC-CRD-UVD-LIF-HPCE detection system, TCIDDC-CRD-DAD-LIF-HPCE detection system, TCIDDC-CRD-LIF-HPCE detection system, TCIDDCM-CRD-UVD-HPCE detection system, TCIDDCM-CRD-HPCE detection system, TCIDDC-CRD-, TCIDDCM-CRD-LIF-HPCE detection system, TCIDDCM-CRD-UVD-LIF-HPCE detection system, and TCIDDCM-CRD-DAD-LIF-HPCE detection system.
And a new HPCE platform for researching the interaction between molecules and screening drugs by taking biological materials as targets is established. The platform has the advantages that the processes of extracting, separating and purifying cell receptor proteins can be omitted, and the three-dimensional space structure and the three-dimensional microenvironment of specific receptor proteins are maintained; the detection of the detector is not influenced by effectively intercepting the biological materials; the biological material can be updated at any time by reverse-direction punching, thereby avoiding the defects that the biological material is inactivated by the compound station with the strongly combined target spot to cause false positive or false positive results and the like. In addition, the method is used for analyzing the combination effect of the traditional Chinese medicine mixed component Addie injection (ADI) and tumor tissues, further defines and proves the effective components of the Addie injection, and lays a foundation for finding potential anti-tumor candidate compounds, reducing the adverse reaction of clinical medication, enhancing the effect of the medicine and the like.
The TCIDDC-CRD-HPCE detection system, the TCIDDC-CRD-UVD-HPCE detection system, the TCIDDC-CRD-DAD-HPCE detection system, the TCIDDC-CRD-LIF-HPCE detection system, the TCIDDC-CRD-UVD-LIF-HPCE detection system, the TCIDDC-CRD-DAD-LIF-HPCE detection system, the TCIDDCM-CRD-UVD-HPCE detection system, the TCIDDCM-CRD-DAD-HPCE detection system, the TCIDDCM-CRD-LIF-HPCE detection system, the TCIDDCM-CRD-UVD-LIF-HPCE detection system, the TCIDDCM-CRD-DAD-LIF-HPCE detection system and the TCIDDCM-CRD-DAD-LIF-HPCE detection system comprise: the device comprises a sample injection system, a high-voltage power supply, electrodes, a buffer liquid tank, capillaries connected in series with different inner diameters, a CRD, a DAD, a UVD, a LIF, a control system and a data processing system. The HPCE for researching the interaction between molecules and screening drugs by taking biological materials as targets comprises the following steps:
1) preparing TCIDDC or TCIDDCM for studying intermolecular interactions and screening drugs, comprising:
pretreating the capillary tube; the capillary is washed by methanol, ultrapure water, 0.1M HCl, ultrapure water, 0.1M NaOH and ultrapure water in sequence; adopting a TCIDDC or TCIDDCM mode with a thick front part and a thin back part to take the biological material as an interaction phase of the HPCE; wherein, the biological material is injected from the end part of the capillary with larger inner diameter and is intercepted in the capillary by the capillary with smaller inner diameter and provided with a detection window; the TCIDDC or TCIDDCM with the front coarse part and the back fine part are connected through a connector;
2) mounting the cartridge into a capillary electrophoresis apparatus;
3) and optimizing the type, concentration, PH, additive, sample introduction pressure, sample introduction time, separation voltage and separation pressure of the background buffer solution to determine the optimal conditions. Wherein the receptors in the biomaterial maintain natural conformation and biological activity, and have a real three-dimensional spatial structure and a three-dimensional microenvironment;
4) carrying out system applicability experimental study and interaction method study by using the negative control sample and the negative control sample; (ii) a
5) And carrying out electrophoretic screening on the sample. Detecting and analyzing the screened sample components, identifying the sample components which have interaction with the target in the biological material, and clarifying the structure-effect relationship.
Drawings
FIG. 1 is a schematic view of a detection system according to one embodiment of the present invention;
FIG. 2 is a schematic diagram of three different mounting positions of the connector in the detection system of FIG. 1, with the interface at the middle (A), front (B), or rear (C) of the cartridge, respectively;
FIG. 3 is a schematic diagram of TCIDDC or TCIDDCM;
FIG. 4 is a CR-EG, CR-EG noise plot and UV-EG noise plot for weak UV absorbing species, such as lysine (A, B, C), and for sodium chloride (D, E, F) for non-UV absorbing species using a detection system provided by embodiments of the present invention.
FIG. 5 shows CR-EG and UV-EG of negative compound LYS (A, B) and positive compound CWJZG (C, D) in empty TCIDDC (a), TCIDDC (b) in normal lung tissue, and TCIDDC (c) in tumor lung tissue, respectively, using the detection apparatus provided in the present invention.
Detailed Description
The invention is further described, by way of example, with reference to specific embodiments. It should be understood that these descriptions are not intended to limit the scope of the claims herein.
The basic structure of the detection system according to the invention is shown in fig. 1. In the figure, 1 is a front end sample plate; 2 is an electrode; 3 is background buffer solution; 4, a card box; 5 is a front end quartz capillary tube; 6 is a connector; 7 is a rear end capillary; 8 is a window; 9 is background buffer solution; 10 is an electrode; 11 is a rear end sample plate; 12 is a high-voltage power supply; 13 is a current detector and a signal amplifying device; and 14 is other detector.
The position of the connector according to the invention is shown in fig. 2. The connection mode of the front end capillary and the rear end capillary comprises a front end corrosion type, an outer sleeve type, a spiral interface type, a socket type and the like.
The interface structure according to the present invention is shown in fig. 3. In the figure 31 is a connector; 32 is a capillary tube with a smaller inner diameter; 33 is a capillary tube with a larger inner diameter; 34 is a column screen.
EXAMPLE I TCIDDC-CRD-UVD-HPCE for qualitative and quantitative analysis of substances with weak UV absorption and no UV absorption
In this example, Beckman P/ACE was usedTMThe MDQ HPCE system (Beckman Coulter, Fullerton, CA, USA) was equipped with a Diode Array Detector (DAD) and a 32 kart software (version 5.0, Beckman) workstation. FA1104N ten-thousandth electronic balance, Max 110g, d 0.1mg (precision scientific instruments ltd, national bridge, shanghai). KQ-250DE model digital control ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan). Dragonlab pipette 0.1-2, 2-20, 20-200, 100-. The polyimide coated fused silica capillary was purchased from Jian chromatographic devices, Yongnian (Hebei, China). And observing electrophoresis parameters in CR-EG at the running voltage of 1-9kV, the running pressure of 0.1-2psi and the air pressure of 0-10.0psi for 1-10s, and determining the position of each peak. FIG. 4 shows lysine as a weak ultraviolet-absorbing compound, CR-EG and UV-EG as non-ultraviolet-absorbing compounds, sodium chloride, respectively.
EXAMPLE two New method for studying intermolecular interactions and drug screening by TCIDDC-CRD-UVD-HPCE targeting tumor tissues
Taking animal tumor tissue as an example, firstly, preparing the animal tumor tissue, subculturing A549 tumor cells according to a cell culture method, taking logarithmic phase tumor cells for digestion, centrifugation and counting, resuspending the cells by using a culture medium containing 20% FBS, and adjusting the density to be 106-107one/mL, the cell suspension was injected subcutaneously into nude mice. Culturing nude mice in sterile environment, when the tumor volume is about 100cm3At that time, nude mice were sacrificed and tumor tissue and control tissue were removed. Immediately thereafter, tumor and control tissues were fixed in 4% paraformaldehyde for 24h and then removed and stored at 4 °.
Then a capillary tube of 15.0cm, 200 μm i.d., 365 μm o.d. was inserted 2mm into the tumor tissue at one end. A15.0 cm, 100 μm I.D.365 μm O.D. capillary was then used as the detection window with the coating removed at 5.0cm of one end. Then, a 365 mu m I.D. hollow sleeve is used for tightly connecting the 200 mu m I.D. capillary at one end with the tumor tissue with the 100 mu m I.D. capillary, and the tumor tissue in the front large inner diameter is blocked by the rear small inner diameter opening so as to be fixed in the capillary connected in series and not move along with the background electrolyte.
In this example, the HPCE used was Beckman P/ACETMMDQ systems (Beckman-Coulter, Fullerton, Calif., USA) were equipped with a two-stage array detector, 32KaratSoftware workstation (vision 5.0, Beckman), and TCIDDC. DMSO was used as a negative control and folic acid was used as a positive control in the experiment, and the chromatographic behavior in a capillary column with tumor/normal lung tissue as the interaction phase was studied and CR-EG and UV-EG were compared. Both CR-EG and UV-EG showed a symmetrical peak in TCIDDC-CRD-UVD-HPCE of tumor/normal lung tissues, with no major difference in peak width, peak height, and peak emergence time between the two. However, when the same concentration of folic acid was injected into TCIDDC-CRD-UVD-HPCE of tumor/normal lung tissues, the peak shape of UV-EG was changed. In TCIDDC-CRD-UVD-HPCE of normal lung tissue, the CR-EG/UV-EG of folic acid shows a symmetrical peak, while in TCIDDC-CRD-UVD-HPCE of tumor lung tissue, the CR-EG/UV-EG shows that the peak time is slowed down, the peak height is obviously reduced, the peak width is widened, and the tailing is serious. Since folate receptors are highly expressed on the surface of tumor tissue compared to normal lung tissue, folate specifically interacts with folate receptors when passing through tumor tissue, thereby showing different electrophoretic behaviors and finally showing a peak pattern with broadened tails in UV-EG. The CR-EG results were consistent with the UV-EG phenomenon, and kinetic parameters (K) were calculated for the bound active compoundsa、r、ka、kd) A novel method of TCIDDC-CRD-UVD-HPCE targeting tumor tissue was created.
On the basis, the method is further used for screening the monomer compounds in the ADI injection of the traditional Chinese medicine, and comprises 17 monomer compound samples such as RSZG, MRYHT, LYS, ZDXG, CWJZG and the like. As a result, the electrophoretic patterns of some compounds in TCIDDC-CRD-UVD-HPCE of tumor lung tissue/normal lung tissue are basically kept unchanged, and are consistent with the peak shape change of DMSO, and the compounds are negativeA compound (I) is provided. While other compounds showed similar peak shape change in TCIDDC-CRD-UVD-HPCE of tumor lung tissue/normal lung tissue as folic acid, exhibited delayed retention time and reduced peak height, severe tailing, and thus were positive compounds, and kinetic parameters (K) of bound active compound were calculateda、r、ka、kd) And further verifying the effectiveness and reliability of the created new method by adopting a cell level and animal level anti-tumor efficacy experiment, a computer-aided molecular simulation docking experiment and an HPCE competitive combination experiment. FIG. 5 shows that the negative compounds represent LYS and the positive compounds represent UV-EG and CR-EG of CWJZG.
Specific examples are described in detail herein, however, this is by way of illustration only and is not to be construed as limiting the scope of the invention. It should be understood that appropriate substitutions, alterations and modifications to the specific embodiments described herein are intended to fall within the scope of the invention as claimed.

Claims (10)

1. A high efficiency capillary electrophoresis detection system, comprising: the device comprises a separation buffer liquid bottle, two-end electrodes, a front-end capillary tube, a connector, a rear-end capillary tube, a high-voltage power supply, a signal amplification device and a detector; wherein:
the front end capillary tube and the rear end capillary tube are connected together through a connector; the inner diameter of the front end capillary is larger than that of the rear end capillary;
the High Performance Capillary Electrophoresis (HPCE) detection system comprises Tandem Capillary Interfaces (TCIDDC) with no screen and different inner diameters or Tandem Capillary Interfaces (TCIDDCM) with screen and different inner diameters, wherein a column screen is placed in a front Capillary of the latter;
the connector has 3 kinds of positions of placing, are respectively in card box front end, card box top and card box rear end.
2. The detection system of claim 1, wherein the detector comprises: a Current Detector (CRD), and an ultraviolet-visible light Detector (UVD), a Diode Array Detector (DAD), or a Laser induced fluorescence Detector (LIF) in combination therewith.
3. The detection system of claim 2, wherein the detection system is configurable into 12 systems, the system comprises a TCIDDC-CRD-HPCE detection system, a TCIDDC-CRD-UVD-HPCE detection system, a TCIDDC-CRD-DAD-HPCE detection system, a TCIDDC-CRD-LIF-HPCE detection system, a TCIDDC-CRD-UVD-LIF-HPCE detection system, a TCIDDC-CRD-DAD-LIF-HPCE detection system, a TCIDDCM-CRD-UVD-HPCE detection system, a TCIDDCM-CRD-DAD-HPCE detection system, a TCIDDCM-CRD-LIF-HPCE detection system, a TCIDDCM-CRD-UVD-LIF-HPCE detection system and a TCIDDCM-CRD-DAD-LIF-HPCE detection system.
4. The detection system of claim 3, wherein when the CRD is used alone, a current-electropherogram is provided; when the detectors are used in combination, the CRD-UVD provides a current-electropherogram and a UV-electropherogram, the CRD-DAD provides a current-electropherogram, a UV-electropherogram and a full-wavelength scanning UV-spectrogram, the CRD-LIF provides a current-electropherogram and a fluorescence-electropherogram, the CRD-UVD-LIF provides a current-electropherogram, a UV-electropherogram and a fluorescence-electropherogram, and the CRD-DAD-LIF provides a current-electropherogram, a UV-electropherogram, a full-wavelength scanning UV-spectrogram and a fluorescence-electropherogram.
5. The detecting system according to claim 1, wherein the current of the whole system changes when the sample passes through the interface due to the different inner diameters of the front end capillary and the rear end capillary, and the current fluctuation related to the state of the sample has a good linear relationship with the change of the concentration of the sample, thereby being used as the detecting signal.
6. The detection system according to claim 1, wherein the front capillary and the rear capillary are connected in a manner selected from the group consisting of a front-end etched type, an outer sleeve type, a screw interface type, and a socket type.
7. The detection system according to claim 1, wherein detection is performed using CRD on HPCE, useful for detecting substances not suitable for use in uv-vis detectors.
8. Use of the test system according to any of claims 1-7 for drug screening, interaction of biologically active macromolecules with active ligands, drug component analysis, purification and identification of drug active components.
9. A method of testing using the test system of any one of claims 1-7, comprising:
preparing TCIDDC which is resistant to acid, alkali and organic solvent, and optimizing the inner diameter ratio of capillaries with different inner diameters and the lengths of the capillaries at the front end and the rear end; connecting the capillaries with different inner diameters through a connector; pretreating the capillary tubes connected in series;
mounting the cartridge into a capillary electrophoresis apparatus;
optimizing the type, concentration, PH, additive, sample introduction pressure, sample introduction time, separation voltage and separation pressure of a background buffer solution, and determining the optimal conditions;
the performance index test experiment of the detector is carried out on the sample, which comprises the steps of detecting the noise, the sensitivity, the precision, the detection limit and the linear range of the detector, comparing the response values of the sample in a current-electrophoresis chart and an ultraviolet-electrophoresis chart, then carrying out the system applicability experiment, which comprises the precision, the separation degree, the column efficiency, the theoretical plate number and the symmetry factor, and comparing the complementary advantages of the two detectors used simultaneously, thereby carrying out the qualitative analysis and the quantitative analysis of the sample.
10. An HPCE for studying molecular interactions and screening drugs targeting biomaterials using the detection method of claim 9, wherein the detection method comprises:
preparing TCIDDC or TCIDDCM for researching intermolecular interaction and screening drugs, and pretreating capillary;
adopting TCIDDC or TCIDDCM with thick front part and thin back part to take biological materials as an interaction phase of HPCE, wherein the biological materials comprise biological tissues, cells, cell membranes, cell nuclei, endoplasmic reticulum, mitochondria, Golgi bodies, ribosomes, mesenchymal cells and solids; the biological material is injected from the end part of the capillary with larger inner diameter and is intercepted in the capillary by the capillary or the column screen with smaller inner diameter and a detection window, so that the detection of the detector is not influenced; the biological material can be updated at any time by punching in the opposite direction, so that the situation that the target is occupied by a compound with strong interaction to inactivate the biological material to cause false positive or false positive results is avoided;
installing the cartridge into an HPCE instrument;
optimizing the type, concentration, PH, additive, sample introduction pressure, sample introduction time, separation voltage and separation pressure of a background buffer solution, and determining the optimal experimental conditions; wherein, a buffer system close to physiological environment condition is used as a mobile phase, so that the target in the biomaterial keeps natural conformation and biological activity, and has a real three-dimensional space structure and a three-dimensional microenvironment;
performing a system suitability experiment of the new system using a negative compound with a biological material as an interaction phase, the biological material including biological tissue, cells, cell membranes, cell nuclei, endoplasmic reticulum, mitochondria, golgi apparatus, ribosomes, mesenchymal cells, solids;
screening the compound using a detection system, detecting and analyzing the screened sample constituents, identifying sample constituents that interact with a target in the biological material, and calculating a kinetic parameter (K)a、r、ka、kd) The structure-effect relationship is given.
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