CN105675592B - A kind of temperature control Capillary Electrophoresis interface of chemiluminescence reaction - Google Patents
A kind of temperature control Capillary Electrophoresis interface of chemiluminescence reaction Download PDFInfo
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- CN105675592B CN105675592B CN201610054382.5A CN201610054382A CN105675592B CN 105675592 B CN105675592 B CN 105675592B CN 201610054382 A CN201610054382 A CN 201610054382A CN 105675592 B CN105675592 B CN 105675592B
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 87
- 238000005251 capillar electrophoresis Methods 0.000 title claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 52
- 238000000926 separation method Methods 0.000 claims abstract description 41
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 238000003780 insertion Methods 0.000 claims abstract description 5
- 230000037431 insertion Effects 0.000 claims abstract description 5
- 239000003292 glue Substances 0.000 claims description 30
- 239000011521 glass Substances 0.000 claims description 14
- 238000002844 melting Methods 0.000 claims description 4
- 230000008018 melting Effects 0.000 claims description 4
- 239000012491 analyte Substances 0.000 claims description 2
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 claims 3
- 229920005372 Plexiglas® Polymers 0.000 claims 3
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000004020 luminiscence type Methods 0.000 description 32
- 239000008236 heating water Substances 0.000 description 23
- 238000004458 analytical method Methods 0.000 description 7
- 238000002038 chemiluminescence detection Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000004615 ingredient Substances 0.000 description 5
- 239000012472 biological sample Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000005684 electric field Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000003760 hair shine Effects 0.000 description 3
- 238000000520 microinjection Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 2
- 239000004568 cement Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000219784 Sophora Species 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000001499 laser induced fluorescence spectroscopy Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明公开了一种化学发光反应的控温毛细管电泳接口，包括套管模块和观测模块，套管模块包括分离毛细管、高压电极、发光试剂室、反应毛细管和水浴加热腔，分离毛细管用于待测物注入与分离，其在套管模块一侧插入，反应毛细管在套管模块另一侧插入，高压电极与分离毛细管的插入始端相邻；套管模块内的分离毛细管插置于反应毛细管内，两毛细管内外壁间留有间隙；发光试剂室设有发光试剂入口，反应毛细管始端与发光试剂室相通以导出发光试剂，中段穿过水浴加热腔，水浴加热腔内设有热水入口和热水出口；分离毛细管末端出口之后的反应毛细管部位是反应区，反应区固接于观测模块处。本发明易于装配制作，以套管结构实现对化学发光检测的温度控制。
The invention discloses a temperature-controlled capillary electrophoresis interface for chemiluminescence reaction, which includes a sleeve module and an observation module. Sample injection and separation, which is inserted on one side of the casing module, and the reaction capillary is inserted on the other side of the casing module, and the high-voltage electrode is adjacent to the insertion end of the separation capillary; the separation capillary in the casing module is inserted into the reaction capillary , there is a gap between the inner and outer walls of the two capillaries; the luminescent reagent chamber is provided with a luminescent reagent inlet, the beginning of the reaction capillary communicates with the luminescent reagent chamber to lead out the luminescent reagent, the middle part passes through the water bath heating chamber, and the water bath heating chamber is provided with a hot water inlet and a heating chamber. Water outlet; the part of the reaction capillary after the outlet of the separation capillary end is the reaction area, and the reaction area is fixedly connected to the observation module. The invention is easy to assemble and manufacture, and realizes the temperature control of the chemiluminescent detection with the casing structure.
Description
Technical field
The present invention relates to chemical reaction instrument, the temperature control Capillary Electrophoresis interfaces of especially a kind of chemiluminescence reaction.
Background technique
Chemiluminescence (chemiluminescence, CL) detection is not necessarily to additional light source, and ambient noise is small, has sensitive
Degree high (high sensitivity that available and laser induced fluorescence compares favourably), the range of linearity is wide, analysis speed is fast, instrument and equipment is simple
The advantages that single, is a kind of effective Analytical Methods of Trace, has been used for biological sample analysis.However, the complicated component of biological sample,
The shortcomings that chemoluminescence method intrinsic poor selectivity, limits the application of this method.The Capillary Electrophoresis of CL and high separating efficiency
(capillary electrophoresis, CE) is combined, and is directly used in the separation and measurement of trace constituent in complex sample, can
It solves the problems, such as that poor selectivity and sensitivity are low simultaneously, provides development opportunity to CL research and CE technology.CE-CL is combined skill
Art be applied to biological sample, Food and environment sample analysis in, analysis object have been directed to protein, amino acid, alkaloid,
Carbohydrate and metal ion etc..But what up to the present CL was used in the detection mode of CE is relatively fewer, is primarily due to two
Person's combination remains some difficulties, therefore the development of CE-CL detection interface is still the research hotspot of CE-CL analysis field.
In CE-CL joint technology, the Interface design of analyte and chemical illuminating reagent mixed mode is one and closes very much
The factor of key.Mode and detection position according to fluid mixing, interface arrangement, which is commonly divided into, merges flow pattern, coaxial in column from column
Flow pattern, four kinds from the coaxial flow pattern of column and styletable liquid pool type etc..In recent years, domestic and international researcher is also constantly designed some novel
CE-CL interface.Zhike He etc. develops one kind in column slit/past column reaction interface.The interface can introduce simultaneously two kinds with
Upper luminescence reagent solves the compatibility issue of CE separation and luminescent condition, and luminescence-producing reaction and electric field isolation, effectively prevents
The electrochemical decomposition and migration of luminescence reagent have the advantages that from column and in column interface.Jieke Cheng etc. is to styletable liquid pool
Type improves, and devises the CE-CL interface of rotation pond type, by revolving detection cell, separates the chemistry hair at capillary outlet end
Light reagent is timely updated, and solves the problems, such as to interrupt electrophoresis because of bubble formation in conventional coaxial stream interface.Dumke
Deng by the way that luminol and horseradish peroxidase are fixed in capillary, widely used commercialization CE instrument is changed
It makes for CL on-line checking, although improving the operability of CE-CL detection, the most of CE-CL detections of remolding sensitivity are low
The 2-3 order of magnitude, and fix that CL reagent is also more difficult, reproducibility is also not good enough in capillary.
Although researcher has developed a variety of different CE-CL connecting interfaces, these interface arrangements are still in laboratory
There are respective advantage and disadvantage in the voluntarily process segment, all cannot solve the introducing due to CL reagent simultaneously and cause separative efficiency, sensitive
The problems such as degree and stability reduce；Hf etching postcapillary is fragile, not easy to operate；Designed interface does not all consider temperature
The influence reacted CL to be spent, CL is reacted without temperature regulating device substantially and carries out temperature control, the reproducibility for causing CL to detect is poor,
Also the sensitivity of detection can not be improved by improving CL reaction temperature.Therefore in very mature CE-CL combined instrument not yet
Before device occurs, exploitation temperature control, highly sensitive, high stable and convenient CE-CL detect interface, expand CE-CL technology in biological sample
Application in product analysis, is of great significance to life science and biomedical development.
Summary of the invention
The present invention proposes a kind of temperature control Capillary Electrophoresis interface of chemiluminescence reaction, production is easily assembled, with casing knot
Structure realizes the temperature control to chemiluminescence detection.
The present invention uses following technical scheme.
A kind of temperature control Capillary Electrophoresis interface of chemiluminescence reaction, the Capillary Electrophoresis interface include set tube module and
Module is observed, described sleeve pipe module includes separation capillary, high-field electrode, luminescence reagent room, reaction capillary and heating water bath
Chamber, it is described separation capillary for determinand injection with separate, set tube module side be inserted into, reaction capillary in casing
The insertion of the module other side, the high-field electrode are adjacent with the insertion beginning of capillary is separated；Cover the separation capillary in tube module
Be inserted in reaction capillary, separation capillary outer wall with react between capillary tube inner wall that there are the gaps flowed for luminescence reagent；
The luminescence reagent room is equipped with luminescence reagent entrance, and the reaction capillary beginning mutually passes to the luminous examination of export with luminescence reagent room
Agent, middle section pass through heating water bath chamber, and the heating water bath is intracavitary to be equipped with hot water inlet and hot water outlet；In the reaction capillary
On internal luminescence reagent flow direction, the reaction capillary position after the outlet of separation capillary end is reaction zone,
The reaction zone is fixed at observation module.
The normal melting point capillary that the reaction capillary is diameter about 1mm.
The observation module is adjacent with set tube module.
The production of the interface is divided into following steps:
A1, I is drilled on organic glass (polymerthyl acrylate) first, the organic glass for being drilled with hole I is set tube module
Shell, I diameter of hole is about 3-5mm；Then II, III, IV and V is drilled respectively in casing module housing two sides；II is the examination that shines
Agent entrance, III is high-field electrode hole, and IV is heating water bath chamber hot water inlet, and V is heating water bath chamber hot water outlet (heating water bath
The length of chamber is about 1.5cm)；Then on the face vertical with hole I, drill a and b；Drilling VI is made on another piece of organic glass
To observe module, drill c on the face vertical with hole VI.
A2, make four round rubber mats with rubber stopper, diameter and hole I are suitable；Rubber mat centre drill aperture, is just passed through it instead
Answer capillary；It is inserted into hole I after rubber mat being all through on reaction capillary, the first rubber mat and the second rubber mat is made to be located just at hole a two
Side cooperates with I inner wall of hole to form the first glue chamber, and third rubber mat and the 4th rubber mat are located at the hole two sides b；With I inner wall of hole cooperate with
Form the second glue chamber；The AB glue mixed is poured into the first glue chamber and forms the first AB glue plug, is poured into and is mixed in the second glue chamber
AB glue form the second AB glue plug, two rubber plugs fixed reaction capillary jointly；At this time between the first AB glue plug and the second AB glue plug
I inner cavity of hole forms heating water bath chamber.
A3, the reaction zone for reacting capillary is inserted into hole VI, the AB glue mixed, fixed reaction capillary is poured into the c of hole
Pipe；Set tube module and observation module are fixed on one piece of glass slide with glass cement；Face reaction capillary is anti-on glass slide
The region of area's detection position is answered to bore a loophole.
A4, it takes diameter to be less than the separation capillary of caliber in reaction capillary, is placed through a conical plug center and cut out
With suitable capillary pipe length, conical plug is inserted into hole I, makes to separate capillary and be inserted in react in capillary, at this time conical plug
I inner cavity of hole between the first AB glue plug forms luminescence reagent room.
The step A4, concrete operations are to take a conical plug, are passed through in its center with a syringe needle, then separation
Capillary is passed through from syringe needle, after cutting out the separation capillary with appropriate length, extracts syringe needle, makes to separate capillary indwelling
In conical plug center；Then plug is filled in hole I and stoppered, separated capillary and hole I at this time and be coaxially disposed, automatic inserting apparatus in
In the reaction capillary that hole I is coaxially disposed.
The indoor luminescence reagent of luminescence reagent is injected by external medical two pass micro-injection pump.
The intracavitary water of the heating water bath is heat cycles water, is provided by external thermostat water bath.
The present invention use chemical illuminating reagent reach detection zone before first luminescence reagent is heated, then again with it is to be measured
After object mixing reaction generates chemiluminescent strategy, realizes the temperature to chemiluminescence detection with sleeve type structure interface arrangement
Control is easy to make and assemble, can effectively promote the reproducibility of CL detection, moreover it is possible in practical applications, by improving CL reaction
Temperature improves the sensitivity of detection.
It is about 1mm normal melting point capillary to react capillary that the present invention, which uses diameter, and translucency is good, and internal diameter is big, shines
Face is also big, improves luminous intensity, and easy to assembly, interface is durable, is carried out using coaxial manner with capillary is separated in assembly
Socket, it is easy to accomplish, and can effectively avoid reaction interface assembling cumbersome, capillary wall is frangible, is easily detained in pipe gap
The problems such as bubble.
The present invention heats luminescence reagent using water-bath mode, can carry out in this kind of microenvironment of capillary to reagent uniform
Heating, and it is easy to accurate temperature controlling.
It in the present invention, reacts capillary and is heated in heating water bath chamber, and separate capillary and be inserted in reaction capillary
In chamber, this, which to separate between capillary and heating water bath chamber, heat insulation structure, when chemical illuminating reagent is heated, separates capillary
Determinand electrophoretic separation process in pipe is not easy affected by heat, can promote the sensitivity of chemiluminescence reaction in this way,
So that electrophoretic separation is not easy to be influenced by temperature rise.
In the present invention, the indoor luminescence reagent of luminescence reagent is injected by external medical two pass micro-injection pump, the injection side
The injection rate of formula is adjustable and degree of regulation is high, and injection rate is not easy to be mutated, so that the luminescence reagent flow velocity in reaction capillary
It is easy to same electrophoretic velocity matching, promotes the effect of chemiluminescence reaction.
Detailed description of the invention
The present invention is described in more detail with reference to the accompanying drawings and detailed description:
Attached drawing 1 is a kind of structural schematic diagram of the temperature control Capillary Electrophoresis interface of chemiluminescence reaction of the present invention；
Attached drawing 2 is the schematic diagram in interface manufacturing process A1 step of the present invention；
Attached drawing 3 is interface manufacturing process A2 step inner sleeve of the present invention showing there are four the reaction capillary of round rubber mat
It is intended to；
Attached drawing 4 be interface of the present invention in specific application, the contrast schematic diagram of chemiluminescence intensity under different temperatures；
Attached drawing 5 is the Capillary Electrophoresis figure of sophora bud sample and standard sample in Fig. 4 environment；
In figure: the hole I of 1- set tube module；2- luminescence reagent entrance；3- high-field electrode hole, 4- heating water bath chamber hot water enter
Mouthful；The hot water outlet of 5- heating water bath chamber；6- reacts capillary；7- high-field electrode；8- observes module；9- separates capillary；10-
Luminescence reagent room；11- heating water bath chamber；12- reaction zone；13- covers tube module；The first rubber mat of 14-；The second rubber mat of 15-；16-
Three rubber mats；The 4th rubber mat of 17-；The hole 19- a；The hole 20- b；The hole 21- c；22- conical plug.
Specific embodiment
As shown in figure 1, figure 2, figure 3, figure 4 and figure 5, the temperature control Capillary Electrophoresis interface of a kind of chemiluminescence reaction, the hair
Cons electrophoresis interface includes set tube module 13 and observation module 8, and described sleeve pipe module 13 includes separation capillary 9, high-field electrode
7, luminescence reagent room 10, reaction capillary 6 and heating water bath chamber 4, the separation capillary 9 are used for the injection of determinand and divide
From, be inserted into set 13 side of tube module, reaction capillary 6 is inserted into set 13 other side of tube module, the high-field electrode 7 with point
Insertion beginning from capillary 9 is adjacent；Separation capillary 9 in set tube module 13 is inserted in reaction capillary 6, separates capillary
9 outer wall of pipe with react between 6 inner wall of capillary that there are the gaps flowed for luminescence reagent；The luminescence reagent room 10 is equipped with the examination that shines
Agent entrance 2,6 beginning of reaction capillary pass to export luminescence reagent with 10 phase of luminescence reagent room, and middle section passes through heating water bath
Chamber 11, the heating water bath chamber 11 is interior to be equipped with hot water inlet 4 and hot water outlet 5；Luminous examination inside the reaction capillary 6
On agent flow direction, the reaction capillary position after separation 9 end outlet of capillary is reaction zone 12, the reaction zone
12 are fixed at observation module.
The normal melting point capillary that the reaction capillary 6 is diameter about 1mm.
The observation module 8 is adjacent with set tube module 13.
The production of the interface is divided into following steps:
A1, drill on organic glass (polymerthyl acrylate) I (1) first, and the organic glass for being drilled with hole I (1) is casing
The shell of module, hole I (1) diameter is about 3-5mm；Then it drills respectively II (2), III (3), IV in casing module housing two sides
(4) and V (5)；II (2) are luminescence reagent entrance 2, and III (3) are high-field electrode hole 3, and IV (4) are heating water bath chamber hot water inlet
4, V (5) are that the length of heating water bath chamber hot water outlet 5(heating water bath chamber is about 1.5cm)；Then vertical with hole I (1)
On face, drill a(19) and b(20)；Drilling VI, which is used as, on another piece of organic glass observes module 8, on the face vertical with hole VI
Drill c21.
A2, make four round rubber mats with rubber stopper, diameter and hole I (1) are suitable；Rubber mat centre drill aperture, wears it just
Cross reaction capillary 6；It is inserted into hole I (1) after rubber mat being all through on reaction capillary 6, makes the first rubber mat and the second rubber mat just
Positioned at the hole two sides a19, cooperate with hole I (1) inner wall to form the first glue chamber, third rubber mat and the 4th rubber mat are located at the hole two sides b20；
Cooperate with hole I (1) inner wall to form the second glue chamber；The AB glue that mixes is poured into the first glue chamber and forms the first AB glue plug, the
The AB glue mixed is poured into two glue chambers and forms the second AB glue plug, and two rubber plugs are jointly fixed to react capillary 6；First AB glue at this time
Hole I (1) inner cavity between plug and the second AB glue plug forms heating water bath chamber 11.
A3, the reaction zone for reacting capillary 6 is inserted into hole VI, the AB glue mixed, fixed reaction hair is poured into the c21 of hole
Tubule 6；Set tube module 13 and observation module 8 are fixed on one piece of glass slide with glass cement；Face reacts hair on glass slide
Bore a loophole in the region of 6 reaction zone of tubule, 12 detection position.
A4, it takes diameter to be less than the separation capillary 9 of caliber in reaction capillary, is placed through 22 center of a conical plug simultaneously
Suitable capillary pipe length is cut out, conical plug 22 is inserted into hole I (1), makes to separate capillary 9 and be inserted in react in capillary 6, this
When conical plug 22 and the first AB glue plug between I inner cavity of hole formed luminescence reagent room 10.
The step A4, concrete operations are to take a conical plug 22, are passed through in its center with a syringe needle, then point
It is passed through from syringe needle from capillary 9, after cutting out the separation capillary with appropriate length, extracts syringe needle, make to separate capillary 9
Indwelling is in 22 center of conical plug；Then plug 22 is filled in hole I (1) and stoppered, separated capillary 9 at this time and hole I (1) is coaxial sets
It sets, automatic inserting apparatus reacts in capillary (6) in what hole I (1) was coaxially disposed.
Luminescence reagent in the luminescence reagent room 10 is injected by external medical two pass micro-injection pump.
Water in the heating water bath chamber 11 is heat cycles water, is provided by external thermostat water bath.
Embodiment:
Operator starts high-field electrode 7, injects separation capillary using the electrophoretic buffer containing ingredient to be measured as determinand
9, under the action of electrophoretic effect, ingredient to be measured from determinand separation and under electric field action towards separation capillary 9 end
Outlet movement, into the cannula structure part of capillary；Hot water is injected heating water bath chamber by operator, and luminescence reagent is injected and is sent out
Light reagent chamber 10, luminescence reagent enter reaction capillary 6 through luminescence reagent room 10, heating water bath chamber are continuously moved in lumen
11 are sufficiently heated；Determinand is not heated due to moving in separation capillary, operation is separated by electrophoresis not by shadow
It ringing, ingredient to be measured is sufficiently separated under electric field action, since separation 9 end outlet of capillary is located at reaction capillary lumen,
Therefore when ingredient to be measured is flowed out from separation 9 end outlet of capillary, chemical reaction directly is generated with the luminescence reagent being heated
And shine, operator is according to the light generated in chemical reaction, to test to ingredient to be measured.
Claims (6)
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