CN117427700A - Microfluidic synthesis device, method for preparing colloidal gold by using microfluidic synthesis device, product and application - Google Patents
Microfluidic synthesis device, method for preparing colloidal gold by using microfluidic synthesis device, product and application Download PDFInfo
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 48
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- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 38
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- 229910052737 gold Inorganic materials 0.000 claims abstract description 22
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- 239000002243 precursor Substances 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims abstract description 14
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Urology & Nephrology (AREA)
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- General Health & Medical Sciences (AREA)
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- Biotechnology (AREA)
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- Microbiology (AREA)
- Food Science & Technology (AREA)
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- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention provides a microfluidic synthesis device, a method for preparing colloidal gold by using the microfluidic synthesis device, a product and application of the microfluidic synthesis device, and belongs to the technical field of nano colloidal gold preparation. The microfluidic chip in the microfluidic synthesis device comprises a herringbone double-spiral mixed channel and a microchannel formed by a cylindrical convex serpentine channel, and glucose-pepsin solution and gold precursor solution (Au 3+ ) Mixing, pumping into a sample inlet of a microfluidic chip together with NaOH solution, rapidly generating pepsin doped colloidal gold (AuNPs@Pep) in a functional microchannel of the chip, and finally collecting through a sample outlet. The AuNPs@Pep preparation method is simple, quick, automatic, green, pollution-free, high in product stability, excellent in color development and photo-thermal signal indication function and capable of being used as'The paper-based microfluidic device is prepared by using a chromogenic and photo-thermal double-readout probe and is used for detecting the small molecular antibiotic florfenicol.
Description
Technical Field
The invention belongs to the technical field of nano-colloidal gold preparation, and particularly relates to a microfluidic synthesis device, a method for preparing colloidal gold by using the microfluidic synthesis device, a product and application.
Background
Gold nanoparticles (AuNPs) have high quality Surface Plasmon Resonance (SPR), biomolecules with strong binding affinity, high absorption coefficient of X-rays, and unique electronic characteristics, and thus, in the past few years, auNPs have attracted great attention in the fields of biosensing, drug delivery, imaging, photothermal therapy, etc., with the accompanying increase in demand for AuNPs, and thus, auNPs must be mass-produced. Compared with the common chemical and physical synthesis methods, the green synthesis of the AuNPs is an eco-friendly method, and can furthest reduce toxic byproducts and harmful chemical substances generated in the traditional AuNPs preparation process. At present, green material plants, fungi, microorganisms, enzymes, biopolymers and the like are reported to be used for preparing AuNPs, so that the AuNPs are endowed with various surface functions, and the application effect is improved. However, most of the green synthesis techniques currently used involve a large amount of gold ions, reducing agents and surfactants, and various manual processes, and have the disadvantages of low yield and long synthesis time. In addition, particle morphology, size distribution and batch stability of AuNPs are affected by preparation conditions, interaction of gold ions with a reducing agent, and adsorption process of a stabilizer with AuNPs, for example, glucose can be used as a green reducing agent in the prior art, which can convert Au within 26s at room temperature 3+ The stability of glucose-AuNPs was greatly affected by experimental pH and physiological saline ions, though it was converted into Au particles. Therefore, developing an eco-friendly AuNPs synthesis method capable of controlling the size, shape, stability and other characteristics has become a technical problem of great concern.
To address the limitations described above, microfluidic technology is increasingly being used to automatically synthesize AuNPs of size, shape, morphology and particle size distribution in a short period of time. The continuous flow microfluidic device is the most widely used microfluidic device in nanoparticle synthesis, and the flow pattern of the device has simplicity, uniformity and versatility in controlling process parameters. However, a continuous flow state means that the reagents are inter-diffused between them, resulting in a laminar flow of two or more reagents, since the mixing of the fluids is mainly dependent on diffusion, resulting in a reduced mixing efficiency. While there are established microfluidic techniques for producing AuNPs, most procedures are based on general chemical methods and are performed at specific temperatures, longer reaction times or longer channels.
Therefore, the invention combines the green synthesis method with the micro-fluidic technology, combines the green, automatic and controllable synthesis around AuNPs, and utilizes the new technical scheme of micro-fluidic synthesis of colloidal gold.
Disclosure of Invention
In order to solve the technical problems, the invention provides a microfluidic synthesis device, and a method, a product and application for preparing colloidal gold by using the microfluidic synthesis device.
In order to achieve the above purpose, the present invention provides the following technical solutions:
one of the technical schemes of the invention is as follows:
the microfluidic chip in the microfluidic synthesis device comprises a substrate, a sample inlet, a surge chamber, a micro-channel and a sample outlet are arranged on the substrate, the micro-channel comprises a double-spiral mixing channel and a serpentine channel, the double-spiral mixing channel is connected with the sample inlet through the surge chamber, and the other end of the double-spiral mixing channel is connected with the sample outlet through the serpentine channel.
Further, the double-spiral mixing channel is internally provided with herringbone bulges, the height of each herringbone bulge is 40 microns, the herringbone bulges are uniformly arranged along the liquid flow direction, and the tip parts of the herringbone bulges face the direction of the sample inlet, so that the liquid is transversely split in the cross-sectional area direction of the pipeline to form circulating flow; cylindrical protrusions are vertically arranged in the serpentine channel, the diameter of each cylindrical protrusion is 50 microns, the height of each cylindrical protrusion is 40 microns, and the heights of the double-spiral mixing channel and the serpentine channel are 90 microns.
Further, the number of the sample inlets is two, and the surge chambers are hexagonal surge chambers.
The second technical scheme of the invention is as follows:
the preparation method of pepsin doped colloidal gold (AuNPs@Pep) adopts the microfluidic synthesis device for synthesis, and comprises the following steps:
glucose-pepsin (Glu-Pep) solution and gold precursor solution (Au) 3+ ) Mixing to obtain Glu-Pep-Au 3+ Solution of Glu-Pep-Au 3+ The solution and NaOH solution are pumped into a surge chamber through a sample inlet of the microfluidic chip, then enter a micro-channel, and pepsin doped colloidal gold is synthesized in situ in the micro-channel and then collected through a sample outlet.
The raw material Glu-Pep used in the invention contains pepsin and is characterized in that the raw material Glu-Pep contains a large amount of tyrosine, tryptophan and cysteine, wherein the tyrosine and the tryptophan can reduce Au under the environment of alkaline pH value 3+ While cysteine can provide a thiol group to immobilize the reduced gold atoms when the nanoparticles are produced, greatly enhancing the stability and dispersibility of aunps@pep compared to colloidal gold prepared using glucose alone as a reducing agent.
Further, the gold precursor solution is HAuCl 4 The concentration of the solution was 2g/mL.
Further, the mass ratio of glucose to pepsin in the glucose-pepsin (Glu-Pep) solution is (0-100) to (0-100), more specifically m Glucose ∶m Pepsin The total concentration of glucose and pepsin in the glucose-pepsin (Glu-Pep) solution is 100mg/mL, which is 100:0, 98:2, 96:4, 95:5, 90:10, 60:40, 30:70, 0:100.
Further, the volume ratio of the gold precursor solution to the Glu-Pep solution is 2 μL:1 mL.
Further, the concentration of the NaOH solution is 3mg/mL, and the volume ratio of the NaOH solution to the gold precursor solution is 500 [ mu ] L to 2 [ mu ] L.
Further, the Glu-Pep-Au 3+ Flow of solutionThe speed is 20 mu L/min-300 mu L/min.
The flow rate and concentration of the reactants were optimized and the reactant solutions were mixed in a double spiral herringbone mixing channel and then AuNPs@Pep was formed in the serpentine channel to ensure particle uniformity.
The AuNPs@Pep prepared by the method of the invention mainly depends on a reactant Glu-Pep-Au 3+ The solution and NaOH solution rapidly move in the microfluidic environment formed by the microfluidic synthesis device to realize rapid mixing, so that nucleation is rapid and uniform. The main determinants of particle formation rate and particle uniformity are mass transfer of the reactive components, rapid mixing and rapid nucleation spacing to help increase particle uniformity: the double-spiral herringbone mixing channel can enable liquid to transversely split in the cross-sectional area direction of the pipeline to form circulating flow, the effect of the double-spiral herringbone mixing channel is similar to that of fluid stirring, mixing is promoted, the spiral structure is conducive to generating vortex for promoting liquid mixing, the herringbone structure in the flowing direction is conducive to breaking a flowing boundary layer, and disorder of flowing is promoted; the serpentine channel with the dispersed cylindrical structure is adopted to further promote the rapid mixing of reactant solutions and form colloidal gold particles in the mixing process. After passing through the double spiral herringbone mixing channel, the fluids with different properties can generate a speed difference, so that the mixing of the fluids is accelerated. When the combined fluid flows through the serpentine channel with the cylindrical lobes, mixing is actually completed, and the cylindrical lobe configuration further increases the degree of mixing while slowing the flow rate of the fluid and extending the residence time of the reaction components interactions.
The third technical scheme of the invention:
pepsin doped colloidal gold (AuNPs@Pep) is prepared according to the preparation method.
The technical scheme of the invention is as follows:
the pepsin doped colloidal gold (AuNPs@Pep) is applied to food safety detection.
Further, the pepsin doped colloidal gold (aunps@pep) is used for detecting the antibiotic florfenicol (FF) in foods.
Compared with the prior art, the invention has the following advantages and technical effects:
(1) According to the invention, pepsin is introduced into a glucose reduction system as a stabilizer, and a novel pepsin doped AuNPs (AuNPs@pep) green automatic synthesis strategy based on a microfluidic chip is provided, and the strategy has the advantages of high synthesis speed, environment friendliness and remarkable stability under the conditions of high salt ion concentration and pH value.
(2) The AuNPs@Pep prepared based on the microfluidic chip has excellent colorimetric and photothermal biosensing characteristics, can be used as a double-readout probe to prepare a paper-based microfluidic device, and is used for evaluating food safety and detecting small molecular antibiotics florfenicol (FF).
Drawings
The accompanying drawings, which are included to provide a further understanding of the application, illustrate and explain the application and are not to be construed as limiting the application. In the drawings:
FIG. 1 is a schematic structural diagram of a microfluidic chip in a microfluidic synthesis device according to the present invention, wherein the microfluidic chip comprises a 1-first sample inlet, a 2-second sample inlet, a 3-surge chamber, a 4-double spiral mixing channel, a 5-serpentine channel, and a 6-sample outlet;
FIG. 2 is a schematic diagram of the internal three-dimensional structure of a double-helix mixing channel of a microfluidic chip in a microfluidic synthesis device according to the present invention;
FIG. 3 is a schematic view of the internal three-dimensional structure of a serpentine channel of a microfluidic chip in a microfluidic synthesis device according to the present invention;
in FIG. 4, A is a process diagram of generating Glucose-AuNPs in 26s by taking Glucose as a reducing agent, B is a transmission electron microscope diagram of AuNPs@Pep, C is an AuNPs@Pep particle size distribution diagram prepared by Glucose-Pepsin solutions with different concentrations, D is an influence result of different pH values on the stability of AuNPs@Pep and Glucose-AuNPs, E is an influence result of NaCl with different concentrations on the stability of AuNPs@Pep and Glucose-AuNPs, F is an influence result of centrifugation time on the absorbance of AuNPs@Pep and Glucose-AuNPs;
FIG. 5 is an ultraviolet-visible spectrum of AuNPs@Pep prepared in examples 1-10 of the invention, wherein C is an ultraviolet-visible spectrum of AuNPs@Pep prepared in examples 2-6 of the invention, and D is an ultraviolet-visible spectrum of AuNPs@Pep prepared in examples 1, 7-10 of the invention;
fig. 6a is a transmission electron microscope image of aunps@peps prepared in example 1 of the present invention, and B is a distribution diagram of particle size of aunps@peps prepared in example 1 of the present invention; c is the single crystal XRD pattern of AuNPs@Pep prepared in the embodiment 1 of the invention; d is the result of measuring the digestive enzyme activity of the AuNPs@Pep and pepsin on casein, which are prepared in the embodiment 1 of the invention; e is the SDS-PAGE result of AuNPs@Pep prepared in the embodiment 1 of the invention, and F is the photothermal performance analysis result of AuNPs@Pep;
FIG. 7 is a detection curve of a color development mode;
FIG. 8 shows the color development result of the color development mode;
FIG. 9 is a graph of detection of photo-thermal mode;
FIG. 10 shows the color development result of the photo-thermal mode;
fig. 11 shows the detection line temperature and gray value measurement results of paper-based microfluidic devices prepared from aunps@pep for detection of florfenicol amine (a), chloramphenicol (b), flumequine (c), danofloxacin (d) and gatifloxacin (e), a-e mixture (f) and florfenicol (g) antibiotics.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The embodiment of the invention provides a microfluidic synthesis device, wherein a microfluidic chip in the microfluidic synthesis device comprises a substrate, a sample inlet, a hexagonal surge chamber, a micro-channel and a sample outlet are arranged on the substrate, the micro-channel comprises a double-spiral mixing channel and a serpentine channel, the double-spiral mixing channel is connected with the sample inlet through the surge chamber, and the other end of the double-spiral mixing channel is connected with the sample outlet through the serpentine channel. The micro-fluidic chip structure is schematically shown in fig. 1, wherein the micro-fluidic chip structure comprises a 1-first sample inlet, a 2-second sample inlet, a 3-surge chamber, a 4-double-spiral mixing channel, a 5-serpentine channel and a 6-sample outlet.
The inside setting of double helix mixed channel is the chevron, the bellied high 40 microns of chevron, the inside three-dimensional structure schematic diagram of double helix mixed channel is see fig. 2, the inside perpendicular cylindrical arch that is provided with of snake-shaped channel, the bellied diameter of cylindrical is 50 microns, and the height is 40 microns, the inside three-dimensional structure schematic diagram of snake-shaped channel is see fig. 3, the double helix mixed channel with the height of snake-shaped channel is 90 microns.
The preparation method of the microfluidic chip is a conventional technical means in the field, and the preparation method specifically comprises the following steps in the embodiment of the invention:
cleaning silicon wafer with isopropyl ultrasonic wave for 10min, transferring the silicon wafer into ultrapure water, ultrasonic cleaning for 10min, plasma treating for 1min, adsorbing a first layer SU-8 3050 on the pretreated silicon wafer by a rotary film machine, heating for 15min at 95deg.C on an electric furnace, placing the silicon wafer carrying SU-8 3050 and photomask A in a contact aligner, and treating at 365nm wavelength of 200mj/cm 2 Exposing, baking at 95deg.C for 3min, and coating a second SU-8 3050 film on the silicon wafer with a photomask B according to the above process to form the above structure. Next, the wafer was baked at 95℃for 3min, and then the wafer was immersed in SU-8 developing solution together with the exposed film, with gentle stirring for 5min. The silicon wafer was then rinsed with isopropyl alcohol and blow-dried with nitrogen. After drying at 150℃for 5min, the wafers were subjected to release treatment with TMCS solution. Then, PDMS (polydimethylsiloxane) was injected into the mold and solidified at 85 ℃ for 1 hour. After separating PDMS from the mold, the PDMS chips were washed with isopropyl alcohol and deionized water. Finally, the chip was attached to a glass substrate and dried at 80℃for 30min.
The embodiment of the invention also provides a preparation method of pepsin doped colloidal gold (AuNPs@Pep), which is synthesized by adopting the microfluidic synthesis device and comprises the following steps of:
glucose-pepsin (Glu-Pep) solution and gold precursor solution (Au) 3+ ) Mixing to obtain Glu-Pep-Au 3+ Solution of Glu-Pep-Au 3+ The solution and NaOH solution are pumped into the surge chamber 3 through the first sample inlet 1 and the second sample inlet 2 of the microfluidic chip respectively, and then the mixed reaction solution sequentially enters the double-spiral mixing channel 4 (double-spiral herringbone mixing channel) and the serpentine channel 5, in the double-spiral mixing channel 4, the mixed reaction solution generates transverse flow distribution in the cross-sectional area direction of the pipeline to form circulating flow, the effect is similar to fluid stirring, the mixing is promoted, the spiral structure is helpful to generate vortex for promoting the liquid mixing, and herringbone junctions in the flowing direction are formedThe structure is favorable for breaking a flowing boundary layer, promoting disorder of flowing and generating a speed difference so as to accelerate fluid mixing, then the mixed reaction liquid enters the serpentine channel 5, the dispersion cylindrical structure of the mixed reaction liquid further promotes rapid mixing of reactant solution, improves the mixing degree, simultaneously slows down the flow velocity of the fluid, and prolongs glucose pepsin and Au 3+ The residence time of the interaction was determined by in situ synthesis in the microchannel to form aunps@pep, which was then collected through the sample outlet.
In a preferred embodiment of the present invention, the gold precursor solution (Au 3+ ) Is HAuCl 4 The concentration of the solution is 2g/mL, and the gold precursor solution is prepared by mixing 1g of HAuCl 4 ·3H 2 O was dissolved in 500. Mu.L of ultrapure water.
In a preferred embodiment of the invention, the mass ratio of glucose to pepsin in the glucose pepsin solution is 100:0, 98:2, 96:4, 95:5, 90:10, 60:40, 30:70, 0:100, more preferably the mass ratio of glucose to pepsin is 98:2, and the total concentration of glucose to pepsin in the glucose pepsin solution is 100mg/mL.
In a preferred embodiment of the invention, the volume ratio of the gold precursor solution to the glucose pepsin solution is 2. Mu.L:1 mL.
In a preferred embodiment of the invention, the concentration of the NaOH solution is 3mg/mL, and the volume ratio of the NaOH solution to the gold precursor solution is 500 μL to 2 μL.
In a preferred embodiment of the present invention, the Glu-Pep-Au 3+ The flow rate of the solution is 20-300 mu L/min.
The raw materials used in the examples of the present invention are all commercially available.
The microfluidic synthesis device can be connected with common commercial devices by arranging the sample inlet and the sample outlet.
The method for preparing AuNPs@Pep by using the preparation microfluidic synthesis device is carried out at room temperature (25+/-2 ℃), and the process does not need to provide energy, does not need additional chemical reactants toxic and harmful to the environment, and is green and pollution-free.
The technical scheme of the invention is further described by the following examples.
Example 1
1g of HAuCl 4 ·3H 2 O was dissolved in 500. Mu.L of ultrapure water to obtain a gold precursor solution having a concentration of 2 g/mL; mixing 1mL glucose-pepsin solution (Glu-Pep, mass ratio of glucose to pepsin is 98:2, total mass is 100 mg) with 2 μl of the above gold precursor solution to obtain Glu-Pep-Au 3+ Solution of Glu-Pep-Au obtained 3+ The solution and 500 mu L of NaOH solution with the concentration of 3mg/mL are pumped into the surge chamber 3 through the first sample inlet 1 and the second sample inlet 2 of the microfluidic chip respectively, glu-Pep-Au 3+ The flow rate of the solution is 100 mu L/min, then the mixed reaction solution sequentially enters the double-spiral mixing channel 4 and the serpentine channel 5, the mixed reaction solution is synthesized into AuNPs@Pep in situ in the double-spiral mixing channel 4 and the serpentine channel 5, and then the mixed reaction solution is collected through the sample outlet 6.
Example 2
The procedure is as in example 1, except that the glucose-pepsin solution is diluted with water to a concentration of 50mg/mL.
Example 3
The procedure is as in example 1, except that the glucose-pepsin solution is diluted with water to a concentration of 25mg/mL.
Example 4
The procedure is as in example 1, except that the glucose-pepsin solution is diluted with water to a concentration of 12.5mg/mL.
Example 5
The procedure is as in example 1, except that the glucose-pepsin solution is diluted with water to a concentration of 6.25mg/mL.
Example 6
The procedure is as in example 1, except that the glucose-pepsin solution is diluted with water to a concentration of 3.13mg/mL.
Example 7
As in example 1, the only difference is Glu-Pep-Au 3+ The flow rate of the solution was 300. Mu.L/min.
Example 8
As in example 1, the only difference is Glu-Pep-Au 3+ The flow rate of the solution was 200. Mu.L/min.
Example 9
As in example 1, the only difference is Glu-Pep-Au 3+ The flow rate of the solution was 50. Mu.L/min.
Example 10
As in example 1, the only difference is Glu-Pep-Au 3+ The flow rate of the solution was 20. Mu.L/min.
Performance testing
1. Performance comparison of Glu-Pep with glucose as reducing agent
In order to verify that Glu-Pep has higher stability as a reducing agent than glucose, auNPs@pep is prepared in a laboratory by adopting a direct mixing mode, and parameters such as the consumption, concentration and the like of each raw material are the same as those of example 1, and the specific preparation method is as follows:
1g of HAuCl 4 ·3H 2 O was dissolved in 500. Mu.L of ultrapure water to prepare a gold precursor (Au 3+ ) A solution; then, 2. Mu.L of the gold precursor solution (Au 3+ ) Adding into 1mL of Glucose Pepsin (Glucose-Pepsin) solution with different concentrations to obtain Glu-Pep-Au 3+ After mixing the solution with 500. Mu.L of NaOH solution (3 mg/mL), a red AuNPs@Pep solution was rapidly formed, and after washing by centrifugation (20000 rpm,30 min), auNPs@Pep was obtained and dissolved in ultrapure water for storage.
Glucose-AuNPs (Gluconoes-AuNPs) were prepared as described above, except that the mass ratio of the two in the Glucose-Pepsin (Glucose-Pepsin) solution used was m Glucose :m Pepsin =100:0. As can be seen from FIG. 4A, glucose produced Gluconoes-AuNPs in 500. Mu.L NaOH solution (3 mg/mL) for 26 s.
With Glucose-Pepsin (m) at a concentration of 25mg/mL (diluted to a specific concentration by addition of water, the same applies hereinafter) Glucose :m Pepsin Transmission electron microscopy of aunps@pep prepared as described above see B in fig. 4, showing that aunps@pep is predominantly in the form of a single dispersed sphere.
As can be seen from the graph C in FIG. 4, when the concentration of Glucose-Pepsin is reduced from 50mg/mL to 3.13mg/mL, the particle size increases with concentration to 8, 16, 28, 40 and 62nm, respectively, indicating that the size of the concentration of Glucose-Pepsin affects the particle size of AuNPs@pep, and the larger the concentration, the smaller the particle size.
Will use 25mg/mL of Glucose-Pepsin (m Glucose :m Pepsin Aurpin@pep prepared from a Glucose-Pepsin solution =98:2) and the Glucose-AuNPs prepared as described above were placed in centrifuge tubes, pH was adjusted to 2, 4, 6, 8 and 10, and when pH < 6, the color of the Glucose-AuNPs changed from tarry red to purple because Glucose was fully protonated at lower pH, resulting in a sharp decrease in surface negative charge, leading to aggregation, and reduced stability of AuNPs. At pH > 7, the Gluconoes-AuNPs solution remained reddish in color and no aggregation occurred because deprotonated glucose retained the negative surface charge and a repulsive effect occurred between adjacent AuNPs. The stability test results are shown at D in FIG. 4, and it can be seen that the effect of pH on the stability of AuNPs@Pep is significantly less than that of Glucous-AuNPs when the pH is changed from 2 to 10. When different concentrations of NaCl were added to the AuNPs@pep and Gluconoes-AuNPs described above, respectively, the stability test results are shown as E in FIG. 4, and it was seen that Gluconoes-AuNPs began to aggregate. This is because when the concentration of NaCl increases, the surface charge of the nanoparticles is significantly reduced, thereby decreasing the electrostatic repulsive force thereof, resulting in aggregation thereof. However, naCl has little effect on the stability of AuNPs@Pep when the NaCl concentration is increased from 0mM to 100 mM. The AuNPs@Pep and Gluconoes-AuNPs described above were centrifuged at 5000rmp, and the absorbance change with time was shown as F in FIG. 4.
The above results show that the incorporation of pepsin prepared according to the present invention onto AuNPs can greatly enhance its stability or dispersibility even under extreme pH conditions and high concentration NaCl conditions without the addition of any surfactant or stabilizer, which provides an opportunity for aunps@pep to serve as a stable probe in biological applications. And when the AuNPs@Pep solution is treated by centrifugal force, the AuNPs@Pep can also be kept stable under extreme conditions and in a high concentration NaCl solution. AuNPs@pep also showed better stability than Glusoes-AuNPs, because pepsin has better hydrophobic protection.
In summary, the colloidal gold prepared by using Glu-Pep as a reducing agent has higher stability than glucose.
2. Glu-Pep-Au 3+ Effects of concentration and flow Rate of (A) on AuNPs@Pep
The ultraviolet visible spectrum of the Glucose-Pepsin solution (Glucose-Pepsin) prepared in examples 2-6 of the present invention is shown as C in FIG. 5, and it can be seen from C in FIG. 5 that when the concentration of Glucose-Pepsin is changed from 50mg/mL to 3.13mg/mL, the peak position of the ultraviolet visible spectrum of AuNPs@pep is red shifted, indicating that the particle size of AuNPs@pep increases with increasing injection concentration of Glucose-Pepsin.
The ultraviolet and visible spectrum of AuNPs@Pep prepared in examples 1 and 7-10 of the invention is shown as D in FIG. 5, and as can be seen from D in FIG. 5, when Glu-Pep-Au 3+ The peak position of the UV-visible spectrum at 521nm remained unchanged at a flow rate of 20. Mu.L/min to 300. Mu.L/min, but the maximum absorbance at 521nm increased from 6.9a.u. To 7.6a.u. And then decreased to 3.9a.u., indicating that the particle size of AuNPs@pep was hardly affected by the flow rate, and the density of the produced AuNPs@peps decreased with decreasing flow rate. At a lower Glu-Pep-Au 3+ The AuNPs@peps in the microfluidic device is generated at a slower speed under the conditions of concentration and injection flow rate, and Glu-Pep-Au is higher 3+ At a concentration of most Au 3+ Will be converted into larger particles.
3. Investigation of photo-thermal Properties of AuNPs@peps
The transmission electron microscope image and the particle size distribution diagram of AuNPs@peps prepared in the embodiment 1 of the invention are shown as A and B in fig. 6, so that the functional mixed structure of the micro-channel of the invention can lead the generated particles to be spherical and have the diameter of about 20nm.
The single crystal XRD pattern of aunps@pep prepared in example 1 of the present invention is shown as C in fig. 6, from which it can be seen that the face-centered cubic crystal (fcc) structure of gold of the metal, the bragg diffraction peaks of aunps@pep are located at 38.73, 44.11, 64.87, 77.86 and 81.62 ° respectively, corresponding to (111), (200), (220), (311) and (222) planes respectively.
With casein of different concentrations as a substrate, auNPs@Pep prepared in example 1 and commercially available Pepsin (Pepsin) were added, respectively, and the results of measurement of the digestive enzyme activity of AuNPs@Pep and Pepsin on casein are shown as D in FIG. 6, so that it can be seen that AuNPs@Pep inhibits the digestive enzyme activity of Pepsin, resulting in that casein is not digested. Pepsin is also a nonspecific endopeptidase commonly used to cleave antibodies of the IgG type, forming a bivalent F (ab') by cleavage of the heavy chain around the hinge region 2 Short peptides of fragments and Fc fragments. When aunps@pep is used as immunosensor platform, pepsin binding to the surface of AuNPs may result in antibody digestion. Therefore, the endopeptidase activity of AuNPs@Pep was studied in this case.
SDS-PAGE results of AuNPs@pep prepared in example 1 of the present invention are shown in E (1-3: igG in the figure, pepsin-treated IgG, reduced SDS-PAGE electrophoresis of AuNPs@pep treated IgG; M: marker;4-6: igG, pepsin-treated IgG, non-reduced SDS-PAGE electrophoresis of AuNPs@pep treated IgG) in FIG. 6, whereby a large number of mouse IgG antibodies were seen to have only one 150kDa band under non-reducing conditions, and two 50kDa (heavy chain) and 25kDa (light chain) bands were seen under reducing conditions. After pepsin addition, the IgG antibodies were cleaved into Fc fragments and bivalent F (ab') 2 The short peptide of the fragment was 100kDa (F (ab') 2 ) And the corresponding band appears at 25kDa (Fab) after reduction. However, when running a mixture of AuNPs@Pep and IgG, F (ab') was not found, either under non-reducing or reducing conditions 2 Fragment bands, which indicate that aunps@pep has no endopeptidase activity on IgG antibodies.
Preparation of paper-based microfluidic: the sample pad, the nitrocellulose membrane and the absorbent pad form a test strip. BSA-FF (0.5 mg/mL) was sprayed onto nitrocellulose membrane as a detection line. After drying the nitrocellulose membrane overnight at 37 ℃, the sample pad, nitrocellulose membrane and absorbent pad were adhered to a PVC plate. For the detection of FF, sample buffer containing anti-FFIgG covered aunps@pep was added directly to the sample pad. The analyte and test line (BSA-FF conjugate) were then competitively bound to the anti-FF IgG coated aunps@pep, and the excess conjugate migrated to the tail of the nitrocellulose membrane. The test strips were then photographed with a charge-coupled device (CCD) camera and either gray value analysis by imageJ software or photothermal analysis directly using a thermal imager (FOTRIC, shanghai) under laser radiation (520 nm,1.5W/cm 2). The analysis results are shown as F in FIG. 6.
F in FIG. 6 shows that the AuNPs@Pep was dropped onto the nitrocellulose membrane, the temperature of the AuNPs@Pep spot was rapidly raised to 65℃within 5 seconds under continuous irradiation of 520nm laser, and the temperature of the AuNPs@Pep-IgG spot was raised to 60℃throughout the irradiation period. This directly demonstrates the unique thermal properties of aunps@pep as a thermal probe.
4. Application of AuNPs@Pep in detection of antibiotics florfenicol (FF) in foods
For FF analysis, 1.5ng/mL, 20ng/mL and 50ng/mL of FF were added to commercial milk samples without FF, respectively. Subsequently, 10mL of milk sample was centrifuged (30 min,10000 rpm) to remove fat, and the skimmed milk was diluted 10-fold with PBS buffer to take 50. Mu.L of sample for PAD test (test procedure as above).
The FF standard with the concentration is added into the milk extract solution without FF, and the detection in the extract solution has good linear relation (y=8.95-10.24 lgx, R 2 =0.986), the range of the logarithm of gray-scale to FF concentration (lgx) is 0.05-6.25ng/mL, the detection limit is 0.05ng/mL (the detection curve result of the color development mode is shown in fig. 7), and the color development detection mode result is shown in fig. 8. The result of the photo-thermal mode detection curve is shown in fig. 9, and the result of the photo-thermal mode detection curve is shown in fig. 10.
Compared with a chromogenic detection mode, the analysis sensitivity of the photothermal detection mode of AuNPs@Pep prepared by the method disclosed by the embodiment 1 is improved by 2.5 times, the detection limit is 0.02ng/mL, and the detection range is obviously enlarged and is 0.02-100 ng/mL.
To evaluate the specificity of the PAD assay prepared by aunps@pep of the present invention, five potentially harmful antibiotics in milk were selected. As shown in FIG. 11, when 4ng/mL of FF was added to PAD, the temperature of the detection line (g) was 54.9℃which was significantly lower than the temperature and gray scale values of the detection lines reacting with the 40ng/mL antibiotics (a) florfenicol amine, (b) chloramphenicol, (c) flumequine, (d) danofloxacin, and a mixture of (e) gatifloxacin and (f) a-e, as shown in FIG. 11. A similar phenomenon was also found in the chromogenic detection mode, which indicated that PAD had a higher selectivity for FF analysis.
In addition, to evaluate the precision and accuracy of the developed dual read PAD, milk samples with three FF concentrations of 1.5, 20 and 50ng/mL, respectively, were also tested. Table 1 shows recovery rates for the PoT and colorimetric detection FFs of 87.05% -96.06% and 93.38% -102.39%, respectively. The result is consistent with the result of a commercial ELISA kit, and shows that the FF PAD developed by AuNPs@Pep prepared by the method is suitable for FF quantitative analysis.
Table 1 measurement results
The foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily conceivable by those skilled in the art within the technical scope of the present application should be covered in the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (10)
1. The microfluidic synthesis device is characterized in that a microfluidic chip in the microfluidic synthesis device comprises a substrate, a sample inlet, a surge chamber, a micro-channel and a sample outlet are arranged on the substrate, the micro-channel comprises a double-spiral mixing channel and a serpentine channel, the double-spiral mixing channel is connected with the sample inlet through the surge chamber, and the other end of the double-spiral mixing channel is connected with the sample outlet through the serpentine channel.
2. The microfluidic synthesis device according to claim 1, wherein a herringbone protrusion is arranged inside the double-spiral mixing channel, the herringbone protrusion has a height of 40 microns, a cylindrical protrusion is vertically arranged inside the serpentine channel, the diameter of the cylindrical protrusion is 50 microns, the height of the cylindrical protrusion is 40 microns, and the heights of the double-spiral mixing channel and the serpentine channel are 90 microns.
3. A method for preparing pepsin doped colloidal gold, which is characterized by adopting the microfluidic synthesis device according to any one of claims 1-2 for synthesis, and comprising the following steps:
mixing glucose-pepsin solution with gold precursor solution to obtain Glu-Pep-Au 3+ Solution of Glu-Pep-Au 3+ The solution and NaOH solution are pumped into a surge chamber through a sample inlet of the microfluidic chip, then enter a micro-channel, and pepsin doped colloidal gold is synthesized in situ in the micro-channel and then collected through a sample outlet.
4. The method for preparing pepsin doped colloidal gold according to claim 3, wherein the gold precursor solution is HAuCl 4 The concentration of the solution was 2g/mL.
5. The method for preparing pepsin doped colloidal gold according to claim 3, wherein the mass ratio of glucose to pepsin in the glucose-pepsin solution is (0-100) to (0-100), and the total concentration of glucose and pepsin in the glucose-pepsin solution is 100mg/mL.
6. The method for preparing pepsin doped colloidal gold according to claim 3, wherein the volume ratio of the gold precursor solution to the glucose pepsin solution is 2 μΙ_, 1mL.
7. The method for preparing pepsin doped colloidal gold according to claim 3, wherein the concentration of the NaOH solution is 3mg/mL, and the volume ratio of the NaOH solution to the gold precursor solution is 250:1.
8. The method for preparing pepsin doped colloidal gold according to claim 3, wherein the Glu-Pep-Au 3+ The flow rate of the solution is 20-300 mu L/min.
9. Pepsin doped colloidal gold, characterized in that it is prepared according to the preparation method of any one of claims 3-8.
10. Use of pepsin doped colloidal gold according to claim 9 for food safety detection, wherein the pepsin doped colloidal gold is used for detecting the antibiotic florfenicol in food.
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| US20090117045A1 (en) * | 2007-10-01 | 2009-05-07 | The Curators Of The University Of Missouri | Soy or lentil stabilized gold nanoparticles and method for making same |
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