CN105112293A - Microfluidic double-water-phase cocurrent laminar flow enzymatic reaction technique - Google Patents

Microfluidic double-water-phase cocurrent laminar flow enzymatic reaction technique Download PDF

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CN105112293A
CN105112293A CN201510568228.5A CN201510568228A CN105112293A CN 105112293 A CN105112293 A CN 105112293A CN 201510568228 A CN201510568228 A CN 201510568228A CN 105112293 A CN105112293 A CN 105112293A
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phase
enzymatic reaction
laminar flow
double
water
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孟涛
孟世昕
郭婷
童志平
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Southwest Jiaotong University
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Southwest Jiaotong University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00783—Laminate assemblies, i.e. the reactor comprising a stack of plates
    • B01J2219/00786—Geometry of the plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00801—Means to assemble
    • B01J2219/00804—Plurality of plates
    • B01J2219/00806—Frames
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00819—Materials of construction
    • B01J2219/00831—Glass
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00781—Aspects relating to microreactors
    • B01J2219/00891—Feeding or evacuation
    • B01J2219/00894—More than two inlets

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  • Health & Medical Sciences (AREA)
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  • Physical Or Chemical Processes And Apparatus (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The invention discloses a microfluidic double-water-phase cocurrent laminar flow enzymatic reaction technique. By using the double-water-phase solution as the carrier, enzymatic reaction separation is performed in a double-Y-shaped microfluidic device, an injection pump is used for providing flow powder for the fluid, and an injector needle and a glass sheet are used for preparing a minitype device, thereby constructing a double-water-phase cocurrent laminar flow mass transfer interface; and thus, the enzymatic reaction can be performed quickly and continuously, and the product can be separated and purified. The device has the advantages of simple structure and the like, and is convenient to manufacture. The technique has better separating effect than the traditional batch-type double-water-phase biological reaction, has the advantages of continuous operational process, low energy consumption and flexible control on the laminar flow interface, and can continuously and circularly use the enzyme solution to save the cost. The microscale double-water-phase laminar flow interface is utilized to reinforce the interphase mass transfer, enzymatic reaction and separation, and thus, the technique has wide application prospects in the field of biological reaction separation with expensive raw materials and high product added value.

Description

Micro-fluidic double water-phase parallel laminar flow enzymatic reaction technology
Technical field
The present invention relates to biochemical technology, especially microminiaturized biological respinse engineering and apparatus field thereof.
Background technology
Increasing enzymic catalytic reaction is little by little applied in the middle of industrial production in the world, the development of huge output to human society creates immeasurable prograding, such as at food, drink, medicine, makeup, the fields such as healthcare products have and relate to enzymatic reaction, also relate to the every aspect of human lives simultaneously, bring huge economic interests.Enzymatic reaction is one and relates to face extremely extensively, the biochemical industry reaction that using value is very high.With regard to the actual production of enzymatic reaction, how to realize efficiently the rapid reaction of substrate and enzyme, sharp separation, realize enzyme repeat recycle, be urgent need to solve the problem in enzyme industrial production always.Double-aqueous phase system, as a kind of Reaction Separation extraction system of gentleness, is very suitable for biological respinse and is separated.But double-aqueous phase system becomes that phase material price is high, the phase-splitting time is long, can not operate continuously, leave standstill again to separation time consumption and energy consumption from being mixed into, greatly limit its practical application.Compared with traditional biological Chemical Engineering Technology, micro-Chemical Engineering Technology has that volume is little, transfer efficiency is high, security is high, be easy to the advantage such as integrated, developed into the prominent example of biochemical industry process intensification nearly ten years.
By Two-phase system and macro work technology set " micro-fluidic double water-phase parallel laminar flow enzymatic reaction technology " together, provide a new way for solving double water-phase enzymatic reaction problems.It utilizes simple miniaturization structure to form, and the sample introduction of double water-phase enzymatic reaction, reaction, separation concentrated on one piece of miniature reactor.Utilize microscale parallel layers stream interface to increase two-phase mass transfer area, multiplication enzyme ' s reaction speeding, idetified separation effect, improves the handiness of interface manipulation, and can realize the recirculation utilization etc. of enzyme.Because it carries out under micro-meter scale, so be especially applicable to the biological respinse that cost of material is high and added value of product is high.This technology has broad application prospects at biological respinse and separation field.
Summary of the invention
In view of the bottleneck of existing Two-phase system and the advantage of microflow control technique, the present invention is intended to design micro-fluidic double water-phase parallel laminar flow enzymatic reaction method and device thereof, enable to overcome the shortcoming of double water-phase biological respinse in batches, effectively improve the efficiency of enzymatic reaction.
Object of the present invention is realized by following means.
A kind of micro-fluidic double water-phase parallel laminar flow enzymatic reaction device, with slide glass and cover glass structure microchannel.Slide glass 1. for substrate slide be placed in device orlop, pair of end cover glass (2. with 4.) and a pair side cover slide (3. with 5.) are relative between two is jointly combined into double-Y shaped parallel layers flow structure; 6. cover glass is covered in the device the superiors; Entrance 1 is connected miniature volume pump respectively with entrance 2, entrance 1 place, is responsible for conveying enzyme liquid phase; Entrance 2 place, is responsible for conveying substrate phase; Outlet 1 ' place, is responsible for collecting enzyme liquid phase, and outlet 2 ' place is responsible for collecting product phase; The enzyme liquid phase circulation that outlet 1 ' is collected injects laminar flow apparatus by entrance 1 place, carries out repeatedly enzymic catalytic reaction, to save enzyme liquid; Microchannel height should be less than or equal to 0.2mm, and width should be less than or equal to 0.6mm, and Y shape feeder connection place's angle and exit angle all should be less than or equal to 90 degree.
The present invention also aims to use device as above to realize micro-fluidic double water-phase parallel laminar flow enzymatic reaction method, the reaction adopting micro-fluidic double water-phase laminar flow interface to realize biologically active substance be separated a step and complete, in micro-fluidic parallel double aqueous phase laminar flow apparatus, utilize parallel layers stream interface as the mass transfer interface of two phase reaction thing, carry out enzymatic successive reaction and be continuously separated.
Adopt technology of the present invention, control two-phase fluid with the contact of parallel layers stream mode, mass transfer area is large, and Reaction Separation one step flexibly, can complete by interface manipulation in micro fluidic device.Provide one method fast and efficiently for biological enzymatic reaction is separated, be especially suitable for some enzymatic reactions and be worth high, expensive starting materials, is not suitable for the biological respinse completed in main equipment.
Accompanying drawing is described as follows:
Fig. 1 is micro-fluidic double water-phase parallel laminar flow enzymatic reaction know-why and apparatus structure schematic diagram.
Fig. 2 is the A-A ' sectional view of Fig. 1.
Fig. 3 is the B-B ' sectional view of Fig. 1.
Embodiment
Below in conjunction with accompanying drawing, enforcement of the present invention is further described.But it is emphasized that embodiment is exemplary below, instead of in order to limit the scope of the invention and application.
Embodiment one
Micro-fluidic double water-phase parallel laminar flow enzymatic reaction technology utilizes fluidised bed stream interface as double water-phase mass transfer interface, carries out the separation and purification of micro-enzymatic reaction process and enzyme product.In flat shape laminar flow reaction unit, utilize syringe pump to provide power, in the channel form layers stream interface, double water-phase two-phase Rapid contact carries out mass transfer enzymatic reaction and is then separated.Compared with traditional double water-phase enzymatic reaction, the method has significantly increases two-phase mass transfer area, strengthen separating effect, improve the advantages such as interface manipulation handiness.From accompanying drawing, one is entered by entrance 1, and another is entered by entrance 2.In the parallel channels of centre, realize double water-phase two-phase contacting laminar flow, complete enzymatic reaction.Two-phase is flowed out by outlet 1 ' corresponding on the right of device and outlet 2 ', to realize being separated of product and enzyme liquid.The center-aisle of this device provides parallel laminar flow contact surface, and Y type has exported the parallel laminar flow two-phase laminated flow of double water-phase, regulates two-phase flow velocity and device parameter can control the height of two-phase rate of mass transfer.
Implement the parallel laminar flow urase reaction of micro-fluidic double water-phase and product monitoring
(1) preparation of experiment double water-phase
The dextran (Dex) of the polyoxyethylene glycol (PEG) and 3.2g that utilize electronic weigher to take 3.2g is respectively placed in 2 clean beakers, then adds the deionized water of 16.8mL respectively to two beakers.Be mixed with PEG and the Dex solution that massfraction is all 16%.After treating that solute dissolves all completely, take out 10mL solution respectively, be fully uniformly mixed, be mixed with the double water-phase solution 20mL that two-phase high molecule mass mark is 8%.Leave standstill to the complete phase-splitting of two-phase.
(2) the parallel laminar flow urase reaction of micro-fluidic double water-phase
Draw each 5mL of double water-phase solution upper and lower phase of above-mentioned standing phase-splitting respectively, put into two clean beakers.0.3g urea powder is joined in the beaker that upper phase is housed, is uniformly mixed rear stand for standby use, be substrate phase; 5mg urase powder is joined in the beaker that lower phase is housed, is uniformly mixed rear stand for standby use, be enzyme liquid phase.
Enzyme liquid phase and substrate are sucked in two disposable syringes mutually respectively, puts on syringe pump operator's console fixing, with silicone tube communication apparatus entrance 1 and entrance 2.Opening syringe pump adjustment substrate phase flow velocity is 5mLh -1, adjustment enzyme liquid phase flow rate is 0.6mLh -1.Start logical liquid, and place two beakers collections in outlet 1 ' and outlet 2 ' place.Collect product sal volatile at outlet 2 ' place, and utilize conductivitimeter to test its conductivity variations.Calculate micro-fluidic enzyme ' s reaction speeding according to the data obtained, and contrast with the enzyme ' s reaction speeding data in beaker.
(3) experimental result:
Enzymatic reaction can be realized and product separation one step completes in the parallel laminar flow apparatus of micro-fluidic double water-phase, in enzymatic reaction efficiency ratio beaker, improve nearly 50 times.

Claims (3)

1. a micro-fluidic double water-phase parallel laminar flow enzymatic reaction device, with slide glass and cover glass structure microchannel, it is characterized in that: slide glass 1. for substrate slide be placed in device orlop, pair of end cover glass (2. with 4.) and a pair side cover slide (3. with 5.) are relative between two is jointly combined into double-Y shaped parallel layers flow structure; 6. cover glass is covered in the device the superiors; Entrance 1 is connected miniature volume pump respectively with entrance 2, entrance 1 place, is responsible for conveying enzyme liquid phase; Entrance 2 place, is responsible for conveying substrate phase; Outlet 1 ' place is responsible for collecting enzyme liquid phase, outlet 2 ' place, is responsible for collecting product phase; The enzyme liquid phase circulation that outlet 1 ' is collected injects laminar flow apparatus by entrance 1 place, carries out repeatedly enzymic catalytic reaction, to save enzyme liquid; Microchannel height should be less than or equal to 0.2mm, and width should be less than or equal to 0.6mm, and Y shape feeder connection place's angle and exit angle all should be less than or equal to 90 degree.
2. micro-fluidic double water-phase parallel laminar flow enzymatic reaction method, the step adopting the two Y shape parallel layers stream interface of micro fluidic device according to claim 1 structure to realize enzymatic reaction and product separation completes, it is characterized in that, in two Y shape device, utilize double water-phase parallel layers stream interface as interphase mass transfer interface, flow interface is carried out enzymatic reaction be coupled with product separation, realize successive reaction.
3. micro-fluidic double water-phase according to claim 2 parallel laminar flow enzymatic reaction method, for ensureing the carrying out of micro-fluidic double water-phase enzymatic, the ratio of substrate phase flow velocity and enzyme liquid phase flow rate should be between 9:1 ~ 8:1, and enzyme liquid phase flow rate is more than or equal to 0.6mLh -1.
CN201510568228.5A 2015-09-08 2015-09-08 Microfluidic double-water-phase cocurrent laminar flow enzymatic reaction technique Pending CN105112293A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108613973A (en) * 2018-04-17 2018-10-02 西南交通大学 A kind of micro-fluidic aqueous two-phase liquid dripping method and its device of easy quickly detection urea
CN111304189A (en) * 2020-02-28 2020-06-19 西南交通大学 Enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on aqueous two-phase system

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102179064A (en) * 2011-03-24 2011-09-14 西南交通大学 Micro flow control aqueous two-phase annular space extraction technology and device

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102179064A (en) * 2011-03-24 2011-09-14 西南交通大学 Micro flow control aqueous two-phase annular space extraction technology and device

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YUSHI HUANG ET AL: "Aqueous two-phase extraction for bovine serum albumin(BSA) with co-laminar flow in a simple coaxial capillary microfluidic device", 《MICROFLUID NANOFLUID》 *
黄宇石: "微流控环隙流双水相蛋白质分离和酶促反应研究", 《中国优秀硕士学位论文全文数据库基础科学辑》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108613973A (en) * 2018-04-17 2018-10-02 西南交通大学 A kind of micro-fluidic aqueous two-phase liquid dripping method and its device of easy quickly detection urea
CN108613973B (en) * 2018-04-17 2020-11-03 西南交通大学 Microfluidic double-aqueous-phase droplet flow method and device for simply, conveniently and rapidly detecting urea
CN111304189A (en) * 2020-02-28 2020-06-19 西南交通大学 Enzyme-loaded calcium alginate microsphere enhanced cascade enzymatic reaction method based on aqueous two-phase system

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