CN108722506B

CN108722506B - Method for controlling hydrophilization modification effect inside micro-fluidic chip

Info

Publication number: CN108722506B
Application number: CN201810535581.7A
Authority: CN
Inventors: 吕超; 田锐; 段雪
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2018-05-29
Filing date: 2018-05-29
Publication date: 2020-05-19
Anticipated expiration: 2038-05-29
Also published as: CN108722506A

Abstract

The invention discloses a method for controlling hydrophilization modification effect in a microfluidic chip, belonging to the technical field of hydrophilization control of microfluidic chips. The method realizes the in-situ visual control of the micro-channel in the micro-fluidic chip, can solve the problem that the in-situ detection of the interior of the chip cannot be implemented in the traditional measurement, is a safe, nondestructive, rapid and effective visual identification method, and can be widely applied to the rapid screening of the hydrophilization treatment effect in materials.

Description

Method for controlling hydrophilization modification effect inside micro-fluidic chip

Technical Field

The invention belongs to the technical field of hydrophilic treatment of microfluidic chips, and particularly relates to a method for performing specific identification dyeing and fluorescence imaging control on an elastic PDMS chip after hydrophilic modification.

Background

The micro-fluidic chip is concerned about the realization of sample preparation, separation and detection in a micron scale environment. The Polydimethylsiloxane (PDMS) is a main manufacturing material of the microfluidic chip due to the characteristics of good optical transparency, simple preparation, excellent elasticity and the like. In order to play an important role of the PDMS microfluidic chip in a sample reaction system, the PDMS microfluidic chip has important significance for surface hydrophilization treatment.

Commonly used hydrophilization treatment methods include polymer adsorption, acid treatment, plasma or ultraviolet irradiation, and the like. Because the pore of the microfluidic chip is in the micron level, the evaluation of the hydrophilization effect is mostly based on the simulation treatment of the surface of an external material at present, and the direct characterization of the interior of the pore of the microfluidic chip is difficult to realize. The stress and the structure of the inner and the outer surfaces of the pore channel are different, and the external simulation test is difficult to represent the real level of the hydrophilization modification effect in the pore channel. Therefore, an in-situ and nondestructive specific identification method is urgently needed to realize real and effective control of the hydrophilization effect in the microfluidic channel.

The method is based on the selective bonding identification of aggregation-induced emission molecules (DB-TPE) with two boric acid groups to hydroxyl on polyvinyl alcohol (PVA), the hydrophilic treatment effect inside the micro-fluidic chip modified by the PVA is visually evaluated, and the method can be expanded to the monitoring of the surface hydroxyl content after other hydrophilic treatment. The method is safe, quick and effective, can be used for in-situ detection and visual control of hydrophilic micro-channels in the micro-fluidic chip, and lays a foundation for the effect of the micro-fluidic chip in the subsequent adsorbent reaction.

Disclosure of Invention

Based on the important role of hydrophilicity in the microfluidic chip, the invention provides an in-situ hydrophilic effect visual control method in order to overcome the problems of the prior art.

The technical scheme of the invention is as follows: preparing DB-TPE solution, and adjusting the pH value of the solution to 10 to ensure ionization. Adding 3% of polyvinyl alcohol (PVA) in different amounts into the DB-TPE solution, and fully oscillating to obtain the composite material with enhanced fluorescence property. The control experiment and the infrared characterization verify that the PVA and the DB-TPE form specific and stable combination in a covalent bond form. Based on the method, PVA is used for modifying the interior of a micro-fluidic chip pore channel constructed by PDMS, DB-TPE is used for dyeing after washing, and the redundant DB-TPE is washed by deionized water again after dyeing. The fluorescence distribution condition inside the pore channel of the microfluidic chip is qualitatively and quantitatively analyzed through a laser scanning confocal microscope, and the visual screening of the hydrophilization degree inside the pore channel is realized. In addition, the method is also suitable for visual evaluation of other hydrophilization treatment modes, and has the advantages of convenience and reliability in operation.

The method can visualize the hydrophilicity of the pore canal in the chip in situ, effectively solves the limitation of the traditional method in a microscopic region, and has important significance for chip modification and subsequent application due to the rapid and lossless imaging method.

The hydrophilization control method for the inner pore passage of the PDMS microfluidic chip is characterized by comprising the following steps:

(1) preparing an alkaline solution of a fluorescent substance having a boric acid modification;

(2) preparation of PDMS microfluidic chip

Preferably: designing and photoetching a metal-based template as a mold of the microfluidic chip; mixing a curing agent and PDMS according to a volume ratio of 1:10, pouring the mixture into a mold groove, and vacuumizing for 30min to discharge air bubbles; placing the mould in an oven at 80 ℃ for curing; finally stripping the cured PDMS from the mould for standby;

(3) hydrophilic treatment of PDMS micro-fluidic chip

Ultrasonically cleaning the prepared PDMS chip for 5-20min by using deionized water; injecting the prepared hydrophilization treatment reagent solution into the PDMS microfluidic channel, and sucking out the solution after reserving for different time periods; rinsing the pore channel with deionized water for 3-4 times;

(4) fluorescent mark of PDMS micro-fluidic chip

Injecting the aqueous solution of the boric acid modified fluorescent substance prepared in the step (1) into the PDMS chip subjected to hydrophilization treatment in the step (3), keeping for 2-10min, sucking out the solution, and rinsing the pore channel with deionized water for 3-4 times for later use;

(5) confocal imaging process

Blowing the chip processed in the step (4) with nitrogen, directly placing the chip on a glass slide, and inversely placing the chip on a laser scanning confocal microscope for imaging observation; performing fluorescence intensity analysis on the obtained imaging picture, and intercepting a specific area to measure the fluorescence distribution condition;

the hydrophilization agent solution is an inorganic or organic solution of hydroxyl-containing material, such as polyvinyl alcohol with different alcoholysis degree, different polymerization degree, different viscosity and different molecular weight, other hydroxyl-containing material includes polyethylene glycol, glycerol and other small molecules or polymers with different structures and hydroxyl groups, and the acid-base treatment includes treatment with concentrated H₂SO₄-H₂O₂、HCl-H₂O₂Strong acid and strong alkali solutions such as NaOH.

According to the result, the type, concentration and retention time of the solution of the different hydrophilizing agents in the step (3) are further adjusted to further adjust the hydrophilizing effect.

The fluorescent substance being modified by one or more boronic acidsAggregation-induced emission materials, fluorescent substances with boric acid groups, preferably tetraboric acid-modified tetraphenylethylene molecules (DB-TPE); more preferably, the bis-boronic acid modified tetraphenylethylene molecule (DB-TPE) is dissolved in dimethyl sulfoxide to obtain a stock solution with a concentration of 15-25mM, and K is added₂CO₃And KHCO₃A buffer solution with the pH value of 10 is formed; the volume fraction of the final dimethyl sulfoxide is 1-5%, and the concentration of DB-TPE is 50-1000 mu mol/L.

And (3) observing the change condition of the fluorescence intensity and the average pixel distribution of the micro-fluidic chip pore channel marked by the fluorescent substance by using a fluorescence microscope or a laser scanning confocal microscope.

The invention is based on the fact that a hydroxyl-rich material and a boric acid group can form a covalent B-O bond, so that the boric acid modified aggregation-induced emission molecule is used for specific recognition of the hydroxyl material, and is applied to the visual evaluation of the hydrophilicity in a micro-channel. The method does not need to carry out destructive treatment such as cutting and the like on the micro-fluidic chip, and carries out in-situ, safe and nondestructive fluorescent marking on the micron-level pore canal. The used boric acid group fluorescence has small molecular weight, short marking time, quick response, stable result and reliable and accurate identification information. The invention realizes the in-situ nondestructive visual identification research on the hydrophilicity of the microfluidic chip and provides a basis for further screening the microfluidic chip with excellent efficacy.

Drawings

FIG. 1 is a fluorescence emission spectrum of a boric acid group modified aggregation-induced emission molecule (DB-TPE) in an alkaline solution, wherein the excitation wavelength of the fluorescence emission spectrum is 330nm, and an inset is a fluorescence photograph of the solution under an ultraviolet lamp.

FIG. 2 is a diagram of the combined action of polyvinyl alcohol (PVA) and DB-TPE, wherein A is a fluorescence spectrum obtained by adding different amounts of 3% polyvinyl alcohol (PVA) solution into DB-TPE solution, B is a diagram of the change of fluorescence intensity of the composite material at 465nm after adding different amounts of PVA, and C is a diagram of the change of quantum yield of the composite material after adding different amounts of PVA.

FIG. 3 shows the addition of different polymers to the DB-TPE solution: the fluorescence intensity change value of the compound at 465nm after polyvinylidene fluoride (PVDF), poly (diallyl dimethyl ammonium chloride) (PDDA) and sodium polystyrene sulfonate (PSS);

FIG. 4 is a graph of the infrared transmission spectrum of the composite material after different amounts of PVA were added.

FIG. 5 is an image of a confocal microscope; a and B are confocal microscope imaging pictures of the upper surface of a PDMS chip under a bright field and a dark field after TPEDB is injected into the PDMS chip and cleaned respectively; c and D are confocal microscope imaging images of the lower surface of the PDMS chip under a bright field and a dark field after the PDMS chip is injected with TPEDB and cleaned.

FIG. 6 is an image of a clear field confocal microscope after PDMS chips were treated with PVA for different periods of time, TPEDB was injected and cleaned, and the image size was 1200X 1200 μm²；

FIG. 7 is an image of a dark field confocal microscope after PDMS chips were treated with PVA for different times, TPEDB was injected and cleaned, and the image size was 1200X 1200 μm²。

FIG. 8 shows the average pixel point analysis of PDMS chip imaged by a dark field confocal microscope after being treated with PVA for different time periods, injected with TPEDB and cleaned.

FIG. 9 shows the contact angles obtained by simulating hydrophilicity measurements in an external environment after PDMS modules were treated with PVA for various times.

FIG. 10 is an image of a dark field confocal microscope image after injecting TPEDB and cleaning after hydrophilizing PDMS by different methods: wherein A is acid solution (V)_H2O:V_HCl:V_H2O21:1), B is 3% polyethylene glycol, C is 3% glycerol, D is 3% PVA solution, the fluorescence intensity profile is taken at the arrow with the image size of 800 × 800 μm²。

Detailed Description

The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.

Example 1:

(1) preparation of DB-TPE solution

0.042g of the tetraphenylethylene molecule (DB-TPE) modified by the diboronic acid is weighed and dissolved in 5.0mL of dimethyl sulfoxide, and ultrasonic dissolution is carried out for 15min to obtain a stock solution with the concentration of 20.0 mM. Preparing a buffer solution with pH 10: 1.12g of K are weighed₂CO₃0.19g of KHCO₃Dissolved in 96.0mL of deionized water. To this buffer solution was added 3.5mL of dimethyl sulfoxide, and 0.5mL of the prepared 10.0mM TPEDB stock solution was added with constant stirring to give a solution with a concentration of 100. mu.M. The solution was subjected to fluorescence spectroscopy, and as shown in fig. 1, the emission spectrum of DB-TPE obtained by excitation at 330nm had a fluorescence emission peak at 415nm, with low intensity.

(2) Construction of PVA and DB-TPE composite material

3.0g of PVA solid was weighed out and dissolved in 100mL of hot water at 90 ℃ and stirred for 2 hours to give a transparent and homogeneous solution. The solution was cooled to room temperature for use. And (2) dropwise adding the prepared PVA solution into the DB-TPE solution prepared in the step (1) in different amounts, continuously oscillating to obtain the final PVA contents of 0.00%, 0.15%, 0.30%, 0.60%, 0.90%, 1.35% and 1.80%, and continuously oscillating the suspension for 5 minutes. Drying the film in an oven at 60 ℃ for 2h to obtain the transparent film. The obtained composite material was subjected to a fluorescence spectrum test to obtain a fluorescence spectrum shown in FIG. 2A, and as the PVA content increased, the fluorescence of the composite material first increased and then decreased, and the change in fluorescence intensity at 460nm was shown in FIG. 2B. Through the measurement of the quantum yield of the composite material, the quantum yield of the composite material also shows the trend of increasing and then decreasing as shown in fig. 2C.

(3) Interaction research of PVA and DB-TPE composite material

Other non-hydroxyl containing polymers such as PVDF, PSS and PDDA, in a mass fraction of 3%, were also suitable, mixed with DB-TPE, the final concentration of polymer in the compound being 0.6%. The fluorescence intensity at 460nm was characterized (FIG. 3), and it was found that none of these three polymers was able to bind effectively to DB-TPE, and the fluorescence intensity of the compound was not significantly changed from that of the original DB-TPE. Performing infrared characterization on the composite film obtained in the step (2), as shown in FIG. 4, wherein the composite film is 1310-1430cm^-1The vibrational peak at which the B-O bond formed, demonstrates the effective binding of DB-TPE to PVA.

(4) Preparation of PDMS microfluidic chip

And designing and photoetching a metal-based template with the width of 500 mu m and the depth of 500 mu m to be used as a mold of the microfluidic chip. 20mL of PDMS and 2mL of curing agent are uniformly mixed, and the mixture is vacuumized to remove air bubbles, poured into a mold and vacuumized again to discharge the generated air bubbles. The mold was placed in an oven at 80 ℃ for 2h to cure. Finally, stripping the cured PDMS from the mould for standby.

(5) Hydrophilic treatment of PDMS micro-fluidic chip

And ultrasonically cleaning the prepared PDMS chip for 10min by using deionized water, and blowing the chip dry by using nitrogen. Injecting the prepared PVA solution with the mass fraction of 3% into the PDMS microfluidic channel, and sucking out the PVA solution after respectively retaining for 0min, 1 min, 2 min, 3 min, 5min, 10min, 15min and 20 min. And cleaning the pore channel for 3 times by using deionized water, and removing redundant PVA for later use.

(6) Fluorescent mark of PDMS micro-fluidic chip

And continuously injecting the prepared DB-TPE solution into the PDMS chip modified by the PVA, and keeping for 5 min. And then sucking the DB-TPE solution out, and washing the pore channel for 3 times by using deionized water to remove the redundant DB-TPE. The cleaned PDMS chip is dried by nitrogen purging, placed on a glass slide, covered and sealed by a cover glass, and the surface is guaranteed to be flat, smooth and transparent.

(7) Confocal imaging process

A laser scanning confocal microscope with 20-time magnification is selected, ultraviolet light with the wavelength of 405nm is used as a light source, and emitted light with the wavelength of 430-520nm is subjected to image acquisition. As for the PDMS chip which is not modified by PVA, an imaging graph after the DB-TPE dyeing is shown in the figure, the surface is smooth and regular, and no fluorescence color block can be observed on the upper surface and the lower surface of the PDMS chip. The surface condition of the PVA-modified PDMS chip is shown in FIG. 6, and the surface condition is smooth and flat in a bright field without causing large influence on the appearance of the PDMS chip. As the PVA modification time increases, it is obvious from FIG. 7 that the fluorescence distribution of the chip after DB-TPE staining is more uniform, and the fluorescence intensity is gradually increased. The average pixel value of the fluorescence in the pore canal is analyzed, and the average pixel is obtained to increase along with the increase of the modification time (as shown in figure 8), which proves that the hydrophilicity of the fluorescence is continuously enhanced. While the increase in hydrophilicity was consistent with the contact angle results of the conventional method in the outer surface simulation test (see fig. 9).

To is coming toThe universality of the method is proved, and other hydrophilic treatment methods are adopted to carry out hydrophilic modification on the PDMS microfluidic chip. And the modification results of the four methods were compared: a is acid solution (V)_H2O:V_HCl:V_H2O21:1), B is 3% polyethylene glycol, C is 3% glycerol, D is 3% PVA solution. As can be seen from FIG. 10, all four methods produced hydroxyl groups after surface modification, and showed fluorescence after staining with DB-TPE. The fluorescence intensity distribution diagram intercepted by the arrow shows that the fluorescence color lumps obtained by the processing of the first three methods have weak intensity and uneven dispersion. The three methods are shown to have uneven hydrophilization treatment effect and weak hydrophilicity, and PVA has better hydrophilization performance after treatment.

Claims

The hydrophilization control method for the inner pore passage of the PDMS microfluidic chip is characterized by comprising the following steps:

(1) preparing an alkaline solution of a fluorescent substance having a boric acid modification; the fluorescent substance is one or more boric acid modified aggregation-induced emission materials or a fluorescent substance with boric acid groups;

(2) preparation of PDMS microfluidic chip

(3) Hydrophilic treatment of PDMS micro-fluidic chip

Ultrasonically cleaning the prepared PDMS chip for 5-20min by using deionized water; injecting the prepared hydrophilization treatment reagent solution into the PDMS microfluidic channel, and sucking out the solution after reserving for different time periods; rinsing the pore channel with deionized water for 3-4 times; the hydrophilization treatment reagent solution is a solution of inorganic or organic substances of hydroxyl-containing materials;

(4) fluorescent mark of PDMS micro-fluidic chip

Injecting the aqueous solution of the boric acid modified fluorescent substance prepared in the step (1) into the PDMS chip subjected to hydrophilization treatment in the step (3), keeping for 2-10min, sucking out the solution, and rinsing the pore channel with deionized water for 3-4 times for later use;

(5) confocal imaging process

Blowing the chip processed in the step (4) with nitrogen, directly placing the chip on a glass slide, and inversely placing the chip on a laser scanning confocal microscope for imaging observation; performing fluorescence intensity analysis on the obtained imaging picture, and intercepting a specific area to measure the fluorescence distribution condition;

according to the result, the type, concentration and retention time of the solution of the different hydrophilizing agents in the step (3) are further adjusted to further adjust the hydrophilizing effect.
2. The method for controlling hydrophilization of an internal channel of a PDMS microfluidic chip according to claim 1, wherein the step (2) of preparing the PDMS microfluidic chip comprises: designing and photoetching a metal-based template as a mold of the microfluidic chip; mixing a curing agent and PDMS according to a volume ratio of 1:10, pouring the mixture into a mold groove, and vacuumizing for 30min to discharge air bubbles; placing the mould in an oven at 80 ℃ for curing; finally, stripping the cured PDMS from the mould for standby.
3. The method for controlling hydrophilization of an internal channel of a PDMS microfluidic chip according to claim 1, wherein the fluorescent substance is tetraphenylethylene (DB-TPE) modified with diboronic acid.
4. The method for controlling hydrophilization of an internal channel of a PDMS microfluidic chip according to claim 3, wherein the step (1) is: dissolving tetraphenylethylene molecule (DB-TPE) modified by diboronic acid in dimethyl sulfoxide to obtain stock solution with the concentration of 15-25mM, and adding K₂CO₃And KHCO₃A buffer solution with the pH value of 10 is formed; the volume fraction of the final dimethyl sulfoxide is 1-5%, and the concentration of DB-TPE is 50-1000 mu mol/L.
5. The method for controlling hydrophilization of inner channels of a PDMS microfluidic chip according to claim 1, wherein the fluorescent material-labeled microfluidic chip channels are observed for fluorescence intensity variation and average pixel distribution by a fluorescence microscope or a laser scanning confocal microscope.
6. Hydrophilization control of inner channels of PDMS microfluidic chips according to claim 1The method is characterized in that the hydrophilization reagent is polyvinyl alcohol, polyethylene glycol, glycerol or acid-base treatment solution with different alcoholysis degrees, different polymerization degrees, different viscosities and different molecular weights, and the acid-base treatment solution is selected from concentrated H₂SO₄-H₂O₂、HCl-H₂O₂Or a strong acid and strong base solution of NaOH.