CN109142754A - A kind of protein detecting chip and preparation method thereof based on micro-nano fluid - Google Patents
A kind of protein detecting chip and preparation method thereof based on micro-nano fluid Download PDFInfo
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- CN109142754A CN109142754A CN201811148599.8A CN201811148599A CN109142754A CN 109142754 A CN109142754 A CN 109142754A CN 201811148599 A CN201811148599 A CN 201811148599A CN 109142754 A CN109142754 A CN 109142754A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a kind of protein detecting chips and preparation method thereof based on micro-nano fluid for belonging to biotechnology, sample introduction, reaction and detection are integrated on one piece of micro-fluidic chip by protein detecting chip, and high detection sensitivity is realized by the enhancement effect of fluorescence of metal Nano structure.It is compared with the traditional method, the present invention realizes the fast high-sensitive degree detection of protein, automatically completes detection overall process without complex operations.Meanwhile the present invention is based on micro-nano technology techniques to propose its large-scale producing method, reduces chip cost, is suitble to large-scale production and application.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of protein detecting chip and its system based on micro-nano fluid
Preparation Method.
Background technique
Currently, fluorescence immunoassay detection has great significance in the early diagnosis of disease.Fluorescence immunoassay passes through fluorescence mark
Specific antigen binding in the antibody and sample to be tested of note detects corresponding fluorescence signal by equipment such as microscopes to mark
Antigen in this is identified and is positioned.However traditional fluorescence detection needs personnel and the instrument of profession, step is complicated, detection
Time is longer.Interfered its detection sensitivity lower by biomolecule background fluorescence etc. simultaneously, so that there are biggish diagnosis to prolong
When, it is unfavorable for making a definite diagnosis and treating early for disease.
The micro-nano fluid chip technology occurred in recent years passes through the micro-nano technology technology of microelectronics, during medical analysis
The operations such as sample pretreatment, reaction, separation, detection be integrated on the chip of one piece of micro-meter scale, be automatically performed the full mistake of analysis
Journey, has that sample and reactant dosage are few, detection time is short, at low cost, flux is high, the advantages such as at low cost.
Summary of the invention
It is low for the sensitivity during general protein detection, it is complicated for operation and detection speed it is slow the disadvantages of, propose one
Protein detecting chip and preparation method thereof of the kind based on micro-nano fluid.The chip is mainly used for quick, high sensitivity quantitation inspection
Survey protein molecule.
The present invention proposes a kind of protein detecting chip based on micro-nano fluid, each component part include sample introduction region,
Deposition region, conversion zone and capillary tube pump region (such as Fig. 1).The detection entrance of sample introduction region offer biological sample.Deposition
Region embeds the detection antibody (dAb) of fluorescent marker in advance, provides fluorescence signal.Conversion zone bonding capture antibody (cAb), mentions
For the high specific capture of target antigen.Power needed for capillary tube pump region provides sample introduction and reaction delay.
Further, it is identical respectively to form partial depth for above-mentioned protein detecting chip, and range is 10-300 μm.
Further, sample introduction region is connected to form by injection port and lateral main channel.It is heavy that deposition region is connected by vertical passage
Product region injection port and sample introduction region main channel.Intersect with deposition region vertical passage, there is into the array channel of periodic arrangement,
Total volume determines the amount of capacity of deposition region.Conversion zone main channel passes through flow resistance one end and sample introduction region main channel phase
Even, the other end connects capillary tube pump region.Capillary tube pump regional channel is the array channel at periodic arrangement, and the present invention has " tree "
Shape arrangement, " mesh " like arrangement and " honeycomb " shape arrange 3 kinds of structures.At the same time, the tail portion in capillary tube pump area array channel connects
Port is picked out, the air in channel is excluded during the automatic sampling of sample.
Further, there are two types of preparation methods for the chip.The first preparation method (such as Fig. 2): preparation has above 4 kinds of regions
Band device dimethyl silicone polymer (PDMS) in channel, and be bonded to have obtained with sheet glass by plasma reaction (plasma)
Whole chip.Second of preparation method (such as Fig. 3): the band device glass piece with above 4 kinds of regional channels is prepared, and is passed through
Plasma is bonded to obtain complete chip with smooth PDMS substrate.
Further, in the conversion zone of chip, the present invention utilizes glancing angle deposition (oblique angle
Deposition, OAD) method prepare nano-array.OAD is a kind of physical gas-phase deposite method, and principle is to pass through shade
Effect and the diffusion of adatom grow controllable nano array in substrate.The nanostructure prepared in the present invention is average straight
Diameter is 50nm-150nm, and with a thickness of 200nm-300nm, spacing is gold/silver nanoparticle array of 100nm-200nm, is increased for fluorescence
By force, the high detection sensitivity (such as Fig. 4) of chip is realized.
Further, to the single conversion zone chip (single channel chip) being made of above 4 regions, conversion zone can
To obtain the chip (multi-channel chip) (such as Fig. 5) of multiple conversion zones at periodicity arrangement.Multi-channel chip is each anti-
It answers region to share a sample introduction region and identical sample is provided, while connecting identical capillary tube pump region and identical power is provided
It is delayed with reacting, provides identical detection environment for each conversion zone.
Further, for the protein detecting chip, capillary tube pump region shares 3 kinds of structures (such as Fig. 6).The first knot
Structure: being formed an angle " tree " shape structure for form of intersection with two channels of same widths, wherein length be interconnection for by
The array channel of some cycles arrangement provides uniform power and sufficient delay to react.The intersecting channels to form an angle
Connect each interconnection and tail portion connection gas outlet.Second of structure: with one fixed width channel at tangent to each other group of regular hexagon
At " honeycomb " shape structure, entire length is determined with width reacts delay.The third structure: with one fixed width channel at square
Shape, the long side center of each rectangle is by connecting the " mesh " like structure formed with rectangle short side.Entire length is determined with width reacts
Delay.Three kinds of structures are mutually advantageous.Wherein, " tree " shape structure is the simplest, and fluid flow direction determines, thus most stable." honeycomb "
Shape structure efficiently uses the reaction delay longest that area is maximum, provides under same size.Each channel Cheng Zhi of " mesh " like structural fluid
Angle intersection, the power provided is minimum, thus fluid flow rate is minimum, facilitates the more uniform capture biomarker of conversion zone.
Further, capture antibody (cAb) is bonded for conversion zone, it is of the invention the preparation method comprises the following steps: the first technique stream
Journey is to be bonded cAb (such as Fig. 7 a) on glass, first beats plasma to sheet glass, certain density 3- aminopropyl is immediately added
Trimethoxy siloxane (APTMS) is reacted at room temperature in the short time, after reaction using 80 DEG C in cleaning, air-dried and long-time
Lower reaction, crosslinking agent is (such as double sulfo group suberate (BS between certain density amino is added after cooling3)) in the short time at room temperature
Reaction cleans after reaction and certain density cAb is added and reacts for a long time, certain using cleaning and being added after reaction
The bovine serum albumin(BSA) (BSA) of concentration reacts for a long time, obtains being bonded in glass using cleaning, air-dried or freeze-drying after reaction
The cAb of on piece.Second of preparation method is to be bonded cAb (such as Fig. 7 b) on gold nanorods, and it is thio to be first added certain density two
Two succinimides (DSP) or dithio dipropyl acid esters (DSU) react for a long time at room temperature, clean after reaction and are added one
The cAb for determining concentration reacts for a long time at room temperature, cleans after reaction and that certain density BSA is added is anti-for a long time at room temperature
It answers, obtains being bonded in the cAb on gold nanorods using cleaning, air-dried or freeze-drying after reaction.
Further, the detection antibody (dAb) of fluorescent marker, preparation method of the invention are embedded in advance for deposition region
Are as follows: micro a certain concentration dAb is instilled in deposition region and air-dries or is lyophilized, and some sugar and amino acid can be added in dAb solution
For protecting the activity of dAb for a long time.
10-20 μ L sample solution to be detected need to only be instilled injection port by operator, what solution will be provided in capillary tube pump
Automatic sampling cocurrent expires all microchannels under power, and sample injection time and speed can pass through capillary tube pump regional structure and size control
System, generally 1-30min.
The utility model has the advantages that a kind of protein detecting chip based on micro-nano fluid proposed by the present invention is by sample introduction, reaction and inspection
Survey is integrated on one piece of micro-fluidic chip, and high detection sensitivity is realized by the enhancement effect of fluorescence of metal Nano structure,
Realize the fast high-sensitive degree detection of protein.It is compared with the traditional method, which automatically completes inspection without complex operations
Overall process is surveyed, 10-20 μ L sample solution to be detected need to only be instilled injection port by operator, what solution will be provided in capillary tube pump
Automatic sampling cocurrent expires all microchannels under power, and sample injection time and speed can pass through capillary tube pump regional structure and size control
System, generally 1-30min.The present invention is based on micro-nano technology techniques to propose its large-scale producing method simultaneously, reduces chip cost,
It is suitble to large-scale production and application.
Detailed description of the invention
Fig. 1 is that protein detecting chip respectively forms partial schematic diagram.
Fig. 2 is protein detecting chip figure of the device channel on PDMS.
Fig. 3 is the protein detecting chip figure of device channel on the glass sheet.
Fig. 4 is the protein detecting chip figure that conversion zone integrates gold nanorods.
Fig. 5 is multichannel protein detecting chip figure.
Fig. 6 is 3 kinds of capillary pump configuration figures of protein detecting chip.
Fig. 7 is that protein detecting chip conversion zone is bonded cAb schematic diagram.
Fig. 8 is to be schemed using the protein detecting chip detection sheep IgG of integrated gold nanorods.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but implementation of the invention is not limited only to this.
Embodiment 1
Prepare protein detecting chip of the device channel on PDMS
First by positive photoetching SU8 photoresist, the SU8 silicon wafer template with a thickness of 20-200 μm is prepared on silicon wafer.It will
The 30g PDMS solution of A/B=1/10 vacuumizes bubble removing after being mixed evenly.Limpid PDMS mixed liquor is added in SU8 again
Silicon wafer template surface simultaneously solidifies 1h at 80 DEG C.The PDMS and PDMS comprising individual devices cuts with a knife of gently tearing is cut point
From.Each band device PDMS is cleaned, process is first to be cleaned by ultrasonic 5min with acetone, then be cleaned by ultrasonic 5min with isopropanol, then use DI
Water ultrasonic cleaning 5min simultaneously uses N2Air-blowing is dry.60s is beaten simultaneously at 60W plasma to clean band device PDMS and sheet glass
Bonding together, obtains protein detecting chip of the device channel on PDMS at once.
Embodiment 2
Prepare the protein detecting chip of device channel on the glass sheet
Photoresist is prepared on the glass sheet with a thickness of 1-10 μm of sheet glass template by front lighting quarter first.Again by the mould
Plate is immersed in 3-6h in BOE (6:1) solution wet etching, or uses dry etching to the template, obtains each logical on sheet glass
Road is with a thickness of 20-200 μm.Device each on sheet glass is cut and separates and cleans up.To clean band device glass piece and put down
Whole PDMS substrate beats 60s at 60W plasma and bonding together, obtains the albumen of device channel on the glass sheet at once
Quality detection chip.
Embodiment 3
Prepare the protein detecting chip that conversion zone integrates gold nanorods
Gold nanorods are grown on the glass sheet by the method for glancing angle deposition (OAD).Glass substrate normal direction and steaming
Vapour direction is (vapor deposition angle) into θ angle.Device lower section gun filament is under high pressure in launching electronics beam (electron beam) evaporator crucible
Raw material source (gold, silver, silica etc.).
Steam first substrate surface be nucleated, due to limited surface atom diffusion and shadow effect, these surfaces at
Core center will continue to be grown to nanostructure.Can by the rotation speed of substrate when control vapor deposition angle θ, deposition, deposition thickness,
Deposition rate, power etc. control the structural parameters of nanostructure, and reach maximum fluorescence enhancement effect by optimization.This implementation
In example, during metal nano-rod evaporation process, the titanium (Ti) of angle deposition 10nm thickness is deposited, then with 0 ° first with 86 ° of vapor deposition angles
The silver (Ag) of 1.5 μ m-thicks is deposited, the gold (Au) that angle deposition 10nm thickness finally is deposited with 10 °, which obtains surface growth, there are gold nanorods
Sheet glass.Gold nanorods zone length is 1-5mm, and width is 500 μm of -2mm, with a thickness of 100-300nm.Further in accordance with embodiment 1
Method the sheet glass of clean PDMS and integrated gold nanorods with device are bonded together, wherein gold nanorods are integrated in instead
Region is answered, the protein detecting chip that conversion zone integrates gold nanorods is obtained.
Embodiment 4
The automatic sampling of PBS buffer, cow's serum and human blood in protein detecting chip
To above-mentioned each protein detecting chip, including embodiment 1,2,3 protein detecting chip and its advanced optimize
Obtained multi-channel chip.To the protein detecting chip that PDMS is bonded with sheet glass, 10-20 is instilled in injection port respectively
3 kinds of μ L PBS buffer, cow's serum and human blood sample solutions, experimental verification energy automatic sampling stream expire all microchannels.Its
In, it is PBS buffer for sample, the automatic sampling time is 1-5min;It is cow's serum for sample, the automatic sampling time is
5-10min;It is human blood for sample, the automatic sampling time is 15-30min.
Embodiment 5
Sheep IgG is detected using protein detecting chip
For protein detecting chip of the device channel in embodiment 1 on PDMS.60W first is beaten to sheet glass
Plasma 90s is immediately added the 5%APTMS that solvent is alcohol and reacts 10min, cleaned after reaction using absolute alcohol
And natural air drying, 2h is reacted at 80 DEG C, and the 5mg/ml BS that solvent is PBS buffer is added after cooling3It reacts at room temperature
25min is cleaned using PBS buffer after reaction, and adding solvent is the sheep IgG that PBS buffer concentration is 20 μ g/ml
2h is reacted, is cleaned after reaction using PBS buffer, the 5%BSA that solvent is PBS buffer is added and reacts 1h, reaction
After using PBS buffer cleaning and natural air drying obtain being bonded cAb on the glass sheet.Again in sheet glass deposition region
The rabbit-anti sheep IgG for the fluorescent marker that 0.2 μ L concentration is 20 μ g/ml and natural air drying or freeze-drying are instilled, solvent is PBS buffer,
The wherein L-phenylalanine (L-Phenylalanine) of the trehalose containing 25mM (Trehalose) and 70 μ g/ml, acts on and is
Protect the activity of dAb.By protein treated sheet glass together with each regional alignment bonding of the PDMS with device, obtain
To the protein detecting chip of detectable sheep IgG.10-20 μ L PBS buffer is added to injection port, as PBS buffer flows
Full entire device channel, the rabbit-anti sheep IgG of fluorescent marker, which is first dissolved in deposition region, then flows through conversion zone, and is bonded in
The sheep IgG generation antigen-antibody reaction of conversion zone is captured, and realizes and detects sheep IgG using protein detecting chip, entirely certainly
Simultaneously detection process continues 1-5min (such as Fig. 8) to dynamic sample introduction.
Embodiment 6
Sheep IgG is detected using the protein detecting chip of integrated gold nanorods
The protein detecting chip of gold nanorods is integrated for the conversion zone in embodiment 3, first in the Jenner of sheet glass
Sheep IgG is bonded on rice stick.Process is that addition solvent is that the DSP that dimethyl sulfoxide (DMSO) concentration is 4mg/ml reacts 2h, reaction
After cleaned using DMSO and deionized water (DI water), it is 20 μ g/ml that add solvent, which be PBS buffer concentration,
Sheep IgG reacts 2h, is cleaned after reaction using PBS buffer and DI water, and adding solvent is PBS buffer's
5%BSA reacts 1h, is bonded in after reaction using PBS buffer and DI water cleaning and natural air drying or freeze-drying
Sheep IgG on gold nanorods.The rabbit-anti sheep for the fluorescent marker that 0.2 μ L concentration is 20 μ g/ml is instilled in sheet glass deposition region again
IgG and natural air drying, solvent are the L-Phenylalanine that PBS buffer contains 25mM Trehalose and 70 μ g/ml.It will
Protein processing after and be integrated with gold nanorods sheet glass together with each regional alignment bonding of the PDMS with device, obtain
To the integrated gold nanorods protein detecting chip of detectable sheep IgG.10-20 μ L PBS buffer is added to injection port, with
PBS buffer flows full entire device channel, and the rabbit-anti sheep IgG of fluorescent marker, which is first dissolved in deposition region, then flows through reaction
Region occurs antigen-antibody reaction with the sheep IgG for being bonded in conversion zone and is captured, realizes the albumen using integrated gold nanorods
Quality detection chip detects sheep IgG, and simultaneously detection process continues 1-5min to entire automatic sampling.Comparative example 5, this detection fluorescence letter
It number is significantly increased by the effect of the surface plasmon resonances of gold nanorods, there is high detection sensitivity.
Claims (10)
1. a kind of protein detecting chip based on micro-nano fluid, which is characterized in that including sample introduction region, deposition region, reaction
Region and capillary tube pump region, the sample introduction region is for providing the detection entrance of biological sample, and the deposition region is for pre-
The detection antibody for first embedding fluorescent marker, provides fluorescence signal, and it is anti-to provide target for being bonded capture antibody for the conversion zone
Former high specific capture, capillary tube pump region is for power needed for providing sample introduction and reaction delay.
2. a kind of protein detecting chip based on micro-nano fluid according to claim 1, which is characterized in that the sample introduction
Region, deposition region, the depth in conversion zone and capillary tube pump region are identical, and range is 10-300 μm.
3. a kind of protein detecting chip based on micro-nano fluid according to claim 1, which is characterized in that the sample introduction
Region is connected to form by injection port and lateral main channel, and the deposition region is by vertical passage connection deposition region injection port and sinks
Product region main channel, the conversion zone main channel are connected by flow resistance one end with sample introduction region main channel, and the other end connects hair
Tubule pumps region, and the capillary tube pump regional channel is the array channel at periodic arrangement, and the tail portion of array channel connects outlet
Mouthful.
4. a kind of protein detecting chip based on micro-nano fluid according to claim 1, which is characterized in that the reaction
Region prepares nano-array, nanostructure diameter 50nm-150nm, with a thickness of 200nm- using the method for glancing angle deposition
300nm, spacing are the gold or silver nanoparticle array of 100nm-200nm, are used for fluorescence enhancement, realize that the high detection of chip is sensitive
Degree.
5. a kind of protein detecting chip based on micro-nano fluid according to claim 1, which is characterized in that described 4
The single conversion zone chip of region composition, conversion zone can obtain having multiple conversion zones at periodicity arrangement
Chip, each conversion zone of chip with multiple conversion zones shares sample introduction region and provides identical sample,
Identical capillary tube pump region is connected simultaneously identical power is provided and be delayed with reacting, provide identical inspection for each conversion zone
Survey environment.
6. a kind of protein detecting chip based on micro-nano fluid according to claim 3, which is characterized in that the capillary
The logical array channel in tube pump region can be tree-shaped, honeycomb or reticular structure;The tree is with the two of same widths
The structure of a channel intersection composition, wherein interconnection is the array channel by periodic arrangement, provides uniform power for reaction
With sufficient delay, intersecting channels connect each interconnection and tail portion connects gas outlet;The honeycomb is into regular hexagon
The structure of composition tangent to each other, entire length is determined with width reacts delay;The reticular structure is rectangle reticular structure,
Entire length is determined with width reacts delay.
7. a kind of preparation method of protein detecting chip based on micro-nano fluid described in claim 1, which is characterized in that system
The standby band device dimethyl silicone polymer with sample introduction region, deposition region, conversion zone and capillary tube pump region, and pass through
Ion precursor reactant is bonded to obtain complete chip with sheet glass.
8. a kind of preparation method of protein detecting chip based on micro-nano fluid described in claim 1, which is characterized in that system
The band device glass piece in standby sample introduction region, deposition region, conversion zone and capillary tube pump region, and by plasma with it is smooth
PDMS substrate is bonded to obtain complete chip.
9. a kind of preparation method of protein detecting chip based on micro-nano fluid according to claim 7 or 8, feature
It is, the preparation method of the conversion zone bonding capture antibody, first method is the bonding capture antibody on glass, first right
Sheet glass beats plasma, and 3- aminopropyl trimethoxy siloxane is immediately added, reacts at room temperature, after reaction using clear
It washes, air-dry and reacted at 80 DEG C in long-time, crosslinking agent between amino is added after cooling, reacts, cleans after reaction at room temperature
And capture antibody response is added, clean after reaction and bovine serum albumin(BSA) reaction is added, after reaction using cleaning, wind
Dry or freeze-drying obtains being bonded capture antibody on the glass sheet;Second of preparation method is that bonding capture is anti-on gold nanorods
Body is first added two thio two succinimides or dithio dipropyl acid esters reacts at room temperature, cleans and be added after reaction and catch
It obtains and is reacted under antibody at room temperature, clean and be added bovine serum albumin(BSA) after reaction, react at room temperature, after reaction using clear
Wash, air-dry or be lyophilized the capture antibody for obtaining being bonded on gold nanorods.
10. a kind of preparation method of protein detecting chip based on micro-nano fluid according to claim 7 or 8, special
Sign is that the deposition region need to embed the detection antibody of fluorescent marker, preparation method in advance are as follows: instills in deposition region micro-
The detection antibody-solutions of amount are simultaneously air-dried or are lyophilized, and sugar is added in the detection antibody-solutions and amino acid is used to protect detection antibody
Activity.
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CN111771126B (en) * | 2019-01-30 | 2022-05-06 | 苏州宇测生物科技有限公司 | Single-molecule quantitative detection method and detection system |
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