CN116764704A - Preparation method of multichannel paper-based microfluidic chip and application of multichannel paper-based microfluidic chip in detection of food hazard - Google Patents
Preparation method of multichannel paper-based microfluidic chip and application of multichannel paper-based microfluidic chip in detection of food hazard Download PDFInfo
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Abstract
The invention belongs to the field of food safety detection, and particularly relates to a preparation method of a multichannel paper-based microfluidic chip and application of the multichannel paper-based microfluidic chip in detecting food hazardous substances; the method comprises the following steps: step one, preparing an oil-soluble up-conversion nano material; step two, preparing a water-soluble up-conversion nano material; step three, preparing an up-conversion fluorescent probe; step four, preparing a multichannel paper-based microfluidic chip; fifthly, detecting the content of the harmful substances in the food; the multichannel paper-based microfluidic chip is prepared on the paper substrate by combining laser printing with a thermal curing technology, has an independent detection area, and can realize simultaneous quantitative detection of various dangerous substances, such as enrofloxacin, ciprofloxacin, diarrhea escherichia coli and staphylococcus aureus. Therefore, the multi-channel paper-based microfluidic chip and the portable detection device based on the design have the capability of detecting various hazardous substances on site simultaneously, are rapid and accurate in detection, and have good practical prospects.
Description
Technical Field
The invention belongs to the field of food safety detection, and particularly relates to a preparation method of a multichannel paper-based microfluidic chip and application of the multichannel paper-based microfluidic chip in detecting food harmful substances.
Background
Food-borne diseases are often infectious or toxic, and result from food or water contaminated with food hazards such as food-borne pathogenic bacteria, viruses, parasites, or agricultural and veterinary drugs entering the human body.
Long-term accumulation of food hazards in the human body may lead to a range of health hazards, including damage to the reproductive system, nervous system, or even induction of cancer. Therefore, the development of a rapid and sensitive detection method of food dangerous substances has very important significance for food safety, human health and the like.
At present, conventional food hazard detection methods, such as high performance liquid chromatography, enzyme-linked immunosorbent assay and the like, often face the difficulties of expensive detection instruments and equipment, high cost, complicated steps and the like, and are difficult to realize rapid quantitative detection.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method of a multichannel paper-based microfluidic chip and application thereof in detecting food hazardous substances, so as to solve the technical problems of high detection cost, complex detection steps and inapplicability to on-site detection in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
step one, preparing an oil-soluble up-conversion nano material: adding yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate into a certain volume of methanol A for ultrasonic dissolution, then adding oleic acid and 1-octadecene, fully mixing, heating and stirring for the first time under a closed condition, and cooling to room temperature after stirring; removing the closed condition, then dropwise adding methanol B containing sodium hydroxide and ammonium fluoride, heating and stirring for the second time under the closed condition, cooling to room temperature after stirring, centrifugally separating to obtain precipitate, cleaning with a mixed solution of ultrapure water and ethanol, and drying to obtain an oleic acid coated up-conversion nanomaterial for later use;
preferably, the amount of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, methanol A, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride and methanol B in the first step is 236.6mg:77.6mg:7.6mg:10mL:4.0mL:9.0mL:50.0mg:74.1mg:10mL; the cleaning is carried out by using a mixed solution of ultrapure water and ethanol, wherein the volume ratio of the ultrapure water to the ethanol is 1:3, a step of; wherein methanol a and methanol B are both methanol, and the different letters are merely distinguished by name.
The first heating and stirring time is 20-30min, and the heating temperature is 160 ℃; the second heating and stirring temperature is 295-305 ℃, and the stirring time is 1.0-1.5h; the conditions of the centrifugal separation are as follows: the rotation speed is 6000-8000rpm/min, and the time is 5-8min.
Step two, preparing a water-soluble up-conversion nano material: the oleic acid coated up-conversion nano particles prepared in the first step are weighed and dispersed in hydrochloric acid solution for ultrasonic treatment; after the treatment is finished, adding ethanol, uniformly dispersing by ultrasonic, then adding ultrapure water and ammonia water, uniformly stirring at a certain temperature, adding tetraethyl orthosilicate after stirring for carrying out a first reaction, and then adding 3-aminopropyl triethoxysilane for carrying out a second reaction; after the reaction is finished, centrifugally separating, cleaning the obtained product with ultrapure water, and dispersing the cleaned product in the ultrapure water to obtain a water-soluble up-conversion nano material solution;
preferably, in the second step, the ratio of the up-conversion nanomaterial to the hydrochloric acid solution to the ethanol to the ultrapure water to the ammonia water to the tetraethyl orthosilicate to the 3-aminopropyl triethoxysilane is 20mg:0.5mL:60mL:10mL:2.5mL:20uL:50uL; the first reaction time is 4-6h, and the second reaction time is 1-2h; the concentration of the hydrochloric acid solution is 0.1mol/L; the certain temperature condition is 65 ℃; the conditions of the centrifugal separation are as follows: the rotating speed is 6000-8000rpm/min, and the time is 5-8min; the concentration of the water-soluble up-conversion nano material solution is 2mg/mL.
Step three, preparing an up-conversion fluorescent probe:
(1) Mixing the up-conversion nano material solution prepared in the second step with glutaraldehyde water solution for reaction to activate amino; separating out the activated up-conversion nano material, dispersing the activated up-conversion nano material in a phosphate buffer, adding a food hazard substance A aptamer solution, incubating at a first constant temperature, centrifuging after incubation, washing the obtained precipitate with ultrapure water, centrifuging again, drying to obtain an up-conversion nano material modified with the food hazard substance A aptamer, and finally adding the up-conversion nano material into the phosphate buffer containing graphene oxide, incubating at a second constant temperature to obtain a mixed solution, and marking the mixed solution as an up-conversion fluorescent probe solution A;
(2) The same operation as the step (1) is distinguished by only replacing the food hazard A aptamer solution with the food hazard B aptamer solution, and the rest operations are the same, so as to finally obtain an up-conversion fluorescent probe solution B;
preferably, in the step (1) of the third step, the usage amount relationship of the water-soluble up-conversion nanomaterial solution, glutaraldehyde aqueous solution, phosphate buffer solution, food hazard a aptamer solution, up-conversion nanomaterial and phosphate buffer solution containing graphene oxide is 1.0mL:1.0mL:5.0mL:50uL:1.0mg:2.0mL; the concentration of the phosphate buffer solution containing graphene oxide is 0.2mg/mL; the concentration of the target A aptamer solution is 0.1mmol/L; the concentration of the up-conversion fluorescent probe solution A is 0.5mg/mL; the concentration of the up-conversion fluorescent probe solution B is 0.5mg/mL.
The reaction time of the mixing reaction for activating the amino group is 2 hours, the first incubation is carried out on a shaking table, the temperature is 37 ℃, the time is 2 hours, and the rotating speed of the shaking table is 180-200rpm/min; the second incubation is carried out on a shaking table, the temperature is 37 ℃, the time is 0.5h, and the rotating speed of the shaking table is 180-200rpm/min; the conditions for centrifugal separation were: the rotating speed is 6000-8000rpm/min, and the time is 5-10min;
the aptamer solution is not limited to a sequence of a certain food hazard, and the corresponding aptamer sequence is matched according to the detected target food hazard type; wherein the food hazard comprises enrofloxacin, ciprofloxacin, diarrhea causing escherichia coli, staphylococcus aureus.
Step four, preparing a multichannel paper-based microfluidic chip:
cutting filter paper, performing pattern printing on the filter paper by using a laser printer, performing heat curing treatment on the filter paper after printing, and curing printer ink into fiber pores of the chromatographic paper to form a hydrophobic barrier zone in which solution cannot flow; in the area of the hydrophobic barrier, the ink encloses a microfluidic detection area; the microfluidic detection area consists of a detection area I, a detection area II, a sample adding area and a flow channel, wherein the detection area I and the detection area II are respectively arranged at two sides of the sample adding area and are communicated through the flow channel; the microfluidic detection area is arranged inside the hydrophobic barrier area and is a hydrophilic flow area; the two components form a multichannel paper-based microfluidic chip;
Preferably, in the fourth step, the filter paper is Whatman No. 1 chromatographic filter paper, and the size is 20×30mm;
the first detection area, the second detection area and the sample adding area are all round areas with the diameter of 3 mm; the flow channel is divided into a first flow channel and a second flow channel, the lengths of the first flow channel and the second flow channel are 3mm, and the widths of the first flow channel and the second flow channel are 1mm;
the temperature of the heat curing treatment is 200 ℃, and the curing time is 3 hours.
Fifthly, the application of the multichannel paper-based microfluidic chip for detecting food hazards is provided;
(1) Firstly, respectively dripping the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B prepared in the step three to a first detection area and a second detection area of the microfluidic paper substrate;
the dosage relation of the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B is 5uL:5uL.
(2) Establishment of a standard curve:
firstly, preparing a mixed solution containing a certain concentration of food dangers A and B, dripping the mixed solution into a sample adding area of the multichannel paper-based microfluidic chip obtained in the step four, respectively reaching a first detection area and a second detection area through a flow channel under the action of capillary driving force, incubating at room temperature to enable the food dangers A and B to react with corresponding up-conversion fluorescent probe solutions, and then detecting the fluorescent intensity of the first detection area and the second detection area;
Preferably, in the step (2.1), the amount of the mixed standard solution of the food hazard A and the food hazard B is 20uL; the incubation time at room temperature is 15min;
(2.2) the operation of the step (2.1) is different in that the concentration of the mixed solution containing the food dangers A and B is subjected to gradient change, and the rest operations are the same, so that the corresponding fluorescence intensities under different concentration conditions are finally obtained; performing linear fitting on the concentration of the food hazard A and the fluorescence intensity of the first detection area, and establishing a standard curve for detecting the content of the food hazard A; performing linear fitting on the concentration of the food hazard B and the fluorescence intensity of the second detection area, and establishing a standard curve for detecting the content of the food hazard B;
(3) Detection of actual samples: pretreating an actual sample to obtain a solution to be tested containing food hazardous substances; dropwise adding a solution to be detected into a sample adding area of the multichannel paper-based microfluidic chip, and incubating at room temperature; measuring the fluorescence intensity of the first detection area, carrying the fluorescence intensity into the food hazard A content detection standard curve obtained in the step five, and calculating the content of the food hazard A of the actual sample; and (3) measuring the fluorescence intensity of the second detection area, carrying the second detection area into the food hazard B content detection standard curve obtained in the step (V), and calculating the content of the food hazard B of the actual sample.
Preferably, in the step (3), the drop adding amount of the solution to be detected is 20uL; the incubation time was 15min.
Preferably, the detection of fluorescence intensity in steps (2) - (3) is performed by a portable food hazard detection device based on a multichannel paper-based microfluidic chip;
the detection device is formed by combining a base module, a shaft displacement fixing module, a clamping module of a laser transmitter and a paper-based bearing platform module;
the base module is horizontally installed; the two ends of the base module are A, B ends, and the A, B ends are respectively provided with an axle displacement fixing module; a clamping module is arranged above the shaft displacement fixing module at the end A, and a paper-based bearing platform module is arranged above the shaft displacement fixing module at the end B;
the shaft displacement fixing module comprises a base clamping knob, a quick-mounting plate clamping knob and a quick-mounting plate; the base clamping knob is arranged above the base module, and a quick-mounting plate clamping knob is arranged above the base clamping knob and clamps the quick-mounting plate;
the clamping module comprises a course axis damping adjusting knob, a pitching axis damping adjusting knob and a crab claw clamp holder; the steering shaft damping adjusting knob is arranged above the shaft displacement fixing module, and the pitching shaft damping adjusting knob is arranged above the steering shaft damping adjusting knob and is connected with the crab claw clamp holder;
The paper-based bearing platform module comprises a fixed base and a precise moving platform; wherein, the precise moving platform is provided with a Y-axis stepping knob, an X-axis stepping knob and a paper-based chip fixing clamp.
Parameter definition:
the dimensions of the base module are 230mm long by 38mm wide; the X axial direction of the base module is the long side of the base module, and the Y axial direction is the wide side of the base module; the X-axis displacement travel range of the base module is 0-200mm;
the course axis damping adjusting knob can adjust a course angle of 0-360 degrees; the pitching axis damping adjusting knob can adjust a pitching angle of 0-180 degrees; the clamping range of the crab claw clamp holder is 5-57mm;
the precision moving platform carries out Y-axis displacement and X-axis displacement through a Y-axis stepping knob and an X-axis stepping knob, the displacement precision is 1mm, and the displacement stroke is 40mm; the Y-axis direction of the precision moving platform is the Y-axis direction of the base module; the X-axis direction of the precision moving platform is the X-axis direction of the base module;
the operation method for detecting the fluorescence intensity by the portable detection device comprises the following steps:
placing a multichannel paper-based microfluidic chip to be detected on a precise moving platform of a detection device, fixing the multichannel paper-based microfluidic chip by using a paper-based chip fixing clamp, adjusting the X-axis distance of two shaft displacement fixing modules on a base module to be 100mm, and adjusting the pitching angle of a clamping module to be 45 degrees, so that laser of a laser transmitter is aligned with the center of a first detection area and the fluorescence intensity is measured; then, the X-axis stepping knob of the precise moving platform is adjusted to enable the paper-based chip to carry out X-axis displacement to be 12mm, so that laser of the laser transmitter is aligned to the center of a second detection area and fluorescence intensity is measured.
The invention discloses the following technical effects:
1. the invention discloses a preparation method of a multichannel paper-based microfluidic chip and application thereof in detecting food dangers, wherein up-conversion nano particles are adopted as luminescent materials, and the prepared up-conversion fluorescent probe is deposited in a microfluidic paper-based detection area, so that the change of characteristic fluorescent signals is caused based on the specific identification of an aptamer to the target, the accurate quantitative detection of the target is realized, and the high-sensitivity real-time detection of the food dangers can be realized only by using trace sample liquid (20 mu L).
2. The invention discloses a detection method of food hazard substances, which can realize simultaneous detection of various food hazard substances based on a prepared multichannel paper-based microfluidic chip, and particularly prepares two independent detection areas on a paper base by combining laser printing with a thermal curing technology, wherein up-conversion nano materials connected with an aptamer through graphene oxide in each detection area are respectively used as a fluorescence acceptor and a fluorescence donor, so that simultaneous quantitative detection of various hazard substances is realized, the defects of the traditional method are overcome, and the health and safety of the general diet are ensured.
3. When the modularized food hazard detection device constructed by the invention is assembled, the shaft displacement fixing module, the clamping module of the laser transmitter and the paper-based bearing platform module are axially aligned on the base module so as to ensure that the light path and the double detection areas are on the same axis. The immobilized stroke of the detection area of the multichannel paper-based microfluidic chip is mobilized through the precise mobile platform, so that errors caused by manual adjustment are effectively overcome, and the detection accuracy is improved.
4. The paper-based sensing detection method of the food hazard established by the invention has the linear concentration range of the fluorescent intensity signal characteristic value of enrofloxacin 1.0 multiplied by 10ng/mL-1 multiplied by 10 5 ng/mL, ciprofloxacin 1.0X10 ng/mL-1X 10 5 ng/mL, escherichia coli 1.0X10 CFU/mL-1.0X10) 7 CFU/mL, staphylococcus aureus 4.5X10 CFU/mL-4.5X10 7 CFU/mL, detection limits are enrofloxacin 1.84ng/mL, ciprofloxacin 2.22ng/mL, diarrhea E.coli 3.80CFU/mL, staphylococcus aureus 4.90CFU/mL. The designed paper-based sensing method can meet the requirement of high-sensitivity simultaneous detection of the harmful substances in the food, has good universality, and can provide a theoretical basis for real-time monitoring of the harmful substances in the food in the actual detection process.
Drawings
Fig. 1 is a schematic diagram of the construction of a multichannel paper-based microfluidic chip for food hazard detection.
FIG. 2 is a graph showing the characterization of the nanomaterial prepared in example 1; wherein A is a transmission electron microscope image of the oil-soluble up-conversion nano particles; b is a transmission electron microscope image of the water-soluble up-conversion nano particles.
FIG. 3 is characterization data of nanomaterial-modified aptamer sequences of example 1; wherein A is Zeta potential change before and after the nano material modifies the aptamer sequence; and B is an ultraviolet absorption spectrum before and after the sequence of the nano material modified aptamer.
Fig. 4 is a schematic structural diagram of a multichannel paper-based microfluidic chip; wherein A is the structure diagram of the chip; b is a size distribution diagram of the chip; reference numerals: the device comprises a hydrophobic barrier zone-4.1, a detection zone-4.2, a flow channel-4.3, a sample adding zone-4.4, a flow channel-4.5 and a detection zone-4.6.
FIG. 5 is a block diagram of a portable food hazard detection device based on a multi-channel paper-based microfluidic chip; wherein A is the structure diagram of the device; b is a front view of the device; c is the left side view of the device; d is a top view of the device; reference numerals: the device comprises a base module-5.1, an axle displacement fixing module-5.2, a clamp base clamping knob-5.2.1, a quick-mounting plate clamping knob-5.2.2, a laser emitter clamping module-5.3, a heading axis damping adjusting knob-5.3.1, a pitching axis damping adjusting knob-5.3.2, a crab clamp holder-5.3.3, a paper-based bearing platform module-5.4, a fixing base-5.4.1, a precision moving platform-5.4.2, a Y-axis stepping knob-5.4.2.1, an X-axis stepping knob-5.4.2.2 and a paper-based chip fixing clamp-5.4.2.3.
FIG. 6 is a fluorescence standard curve established for the detection of enrofloxacin and ciprofloxacin at different concentrations of example 1; wherein A is a sensor fluorescence signal diagram for detecting enrofloxacin with different concentrations; b is a standard curve established by enrofloxacin concentration and a fluorescence intensity signal characteristic value of a sensor at 654 nm; c is a sensor fluorescence signal diagram for detecting ciprofloxacin with different concentrations; d is a standard curve established with ciprofloxacin concentration and the characteristic value of the fluorescence intensity signal of the sensor at 654 nm.
FIG. 7 is a fluorescence standard curve established for detecting different concentrations of diarrhea causing Escherichia coli and Staphylococcus aureus in example 2; wherein A is a sensor fluorescence signal diagram for detecting the escherichia coli causing diarrhea with different concentrations; b is a standard curve established by the concentration of Escherichia coli and the characteristic value of the fluorescence intensity signal of the sensor at 654 nm; c is a sensor fluorescence signal diagram for detecting staphylococcus aureus with different concentrations; d is a standard curve established with staphylococcus aureus concentration and the characteristic value of the fluorescence intensity signal of the sensor at 654 nm.
Fig. 8 is a specific analysis of a multichannel paper-based microfluidic chip.
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 application 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 application. 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 application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
FIG. 1 is a schematic diagram of the construction of a multichannel paper-based microfluidic chip for food hazard detection; fig. 5 shows a portable detection device for food hazard based on a multichannel paper-based microfluidic chip, wherein the detection device is formed by combining a base module 5.1, a shaft displacement fixing module 5.2, a clamping module 5.3 of a laser transmitter and a paper-based bearing platform module 5.4;
The base module 5.1 is horizontally installed; the two ends of the base module 5.1 are A, B ends, and the A, B ends are respectively provided with an axle displacement fixing module 5.2; a clamping module 5.3 is arranged above the shaft displacement fixing module 5.2 at the end A, and a bearing platform module 5.4 is arranged above the shaft displacement fixing module 5.2 at the end B;
the shaft displacement fixing module 5.2 comprises a base clamping knob 5.2.1, a quick-mounting plate clamping knob 5.2.2 and a quick-mounting plate 5.2.3; wherein, the base clamping knob 5.2.1 is arranged above the base module 5.1, the quick-mounting plate clamping knob 5.2.2 is arranged above the base clamping knob 5.2.1, and the quick-mounting plate clamping knob 5.2.2 clamps the quick-mounting plate 5.2.3;
the clamping module 5.3 comprises a course axis damping adjusting knob 5.3.1, a pitching axis damping adjusting knob 5.3.2 and a crab clamp holder 5.3.3; the heading shaft damping adjusting knob 5.3.1 is arranged above the shaft displacement fixing module 5.2, the pitching shaft damping adjusting knob 5.3.2 is arranged above the heading shaft damping adjusting knob 5.3.1, and the pitching shaft damping adjusting knob 5.3.2 is connected with the crab clamp holder 5.3.3;
the paper-based bearing platform module 5.4 comprises a fixed base 5.4.1 and a precise moving platform 5.4.2; the precise moving platform comprises a Y-axis stepping knob 5.4.2.1, an X-axis stepping knob 5.4.2.2 and a paper-based chip fixing clamp 5.4.2.3;
The dimensions of the base module 5.1 are 230mm long by 38mm wide; the X axial direction of the base module 5.1 is the long side of the base module 5.1, and the Y axial direction is the wide side of the base module 5.1; the X axial displacement travel range of the base module 5.1 is 0-200mm;
the course axis damping adjusting knob 5.3.1 can adjust the course angle of 0-360 degrees; the pitching axis damping adjusting knob 5.3.2 can adjust the pitching angle by 0-180 degrees; the clamping range of the crab claw clamp holder 5.3.3 is 5-57mm;
the precise moving platform 5.4.2 can carry out Y-axis displacement and X-axis displacement through the Y-axis stepping knob 5.4.2.1 and the X-axis stepping knob 5.4.2.2, the displacement precision is 1mm, and the displacement stroke is 40mm; the Y-axis direction of the precision moving platform is the Y-axis direction of the base module; the X-axis direction of the precision moving platform is the X-axis direction of the base module;
the aptamer solutions used in the invention are all commercially available and the invention is purchased from the division of bioengineering (Shanghai); specific steps are shown in the examples;
example 1:
step one, preparing an oil-soluble up-conversion nano material: accurately weighing 236.6mg yttrium chloride hexahydrate, 77.6mg ytterbium chloride hexahydrate and 7.6mg erbium chloride hexahydrate, dispersing in 10mL methanol solvent by ultrasonic, adding 4.0mL oleic acid and 9.0mL 1-octadecene, and magnetically stirring and reacting for 30min at 160 ℃ in an argon environment to obtain a transparent solution; after cooling to room temperature, a mixed solution containing 50mg of sodium hydroxide and 74.1mg of ammonium fluoride dissolved in 10mL of methanol was added dropwise; then magnetically stirring for 1h at 300 ℃, cooling to room temperature, centrifuging at 6000rpm for 5min, centrifugally separating to obtain up-conversion nano particle precipitate, washing with mixed solution of ultrapure water and ethanol with volume ratio of 1:3 for three times, and drying to obtain pure oleic acid coated up-conversion nano particles;
Fig. 2A is a transmission electron microscope image of the oil-soluble up-conversion nanoparticle, and it can be seen that the prepared oil-soluble up-conversion nanoparticle has good crystal form and dispersibility.
Step two, preparing a water-soluble up-conversion nano material: the 20mg oleic acid coated up-conversion nano particles are weighed and dispersed in 0.5mL hydrochloric acid solution (the concentration is 0.1 mol/L) for ultrasonic treatment; after the treatment is finished, 60mL of ethanol is added and is dispersed evenly by ultrasonic, and then 10mL of ultrapure water and 2.5mL of ammonia water are added and are stirred evenly at 65 ℃; after stirring uniformly, adding 20uL of tetraethyl orthosilicate for reaction for 4 hours, and adding 50uL of 3-aminopropyl triethoxysilane for reaction for 1 hour; after the reaction is finished, the obtained solution is centrifuged for 5min at 6000rpm/min, the obtained product is washed by ultrapure water, and the washed product is dispersed in the ultrapure water to obtain the water-soluble up-conversion nano material solution with the concentration of 2mg/mL.
Fig. 2B is a transmission electron microscope image of the water-soluble up-conversion nanoparticle, and it can be seen that the prepared water-soluble up-conversion nanoparticle has a silica shell modified with amino groups, which indicates that the prepared nanoparticle has successfully undergone surface water-solubility modification.
Step three, preparing an up-conversion fluorescent probe:
(1) Mixing the up-conversion nano material solution (volume 1.0 mL) prepared in the second step with glutaraldehyde water solution (volume 1.0 mL) for reaction to activate amino groups; separating out activated up-conversion nano material, dispersing the activated up-conversion nano material in 5.0mL of phosphate buffer solution, adding 50uL of enrofloxacin aptamer solution (the concentration is 0.1mmol/L, the sequence is 5'-CCCATCAGGGGGCTAGGCTAACACGGTTCGGCTCTCTGAGCCCGGGTTATTTCAGGGG GA-3'), incubating for 2h at 37 ℃, wherein the incubation is performed on a shaking table, the rotating speed of the shaking table is 180rpm/min, centrifuging at 6000rpm for 5min after incubation, washing obtained precipitate with ultrapure water, centrifuging again (the rotating speed is 6000rpm, the time is 5 min), drying to obtain 1.0mg of up-conversion nano material modified with enrofloxacin aptamer, finally adding the up-conversion nano material into the phosphate buffer solution containing graphene oxide (the volume is 5mL, the concentration is 0.2 mg/mL), incubating for 0.5h at 37 ℃, the incubation is performed on the shaking table, the rotating speed of the shaking table is 180rpm/min, and the concentration is 0.5mg/mL, so as to obtain up-conversion fluorescent probe A solution;
(2) The operation in the step (1) is different from that the enrofloxacin aptamer solution is replaced by ciprofloxacin aptamer solution (the concentration is 0.1mmol/L, the sequence is 5'-ATACCAGCTTATTCAATTGCAGGGTATCTGAGGCTTGATCTAC TAAATGTCGTGGGGCATTGCTATTGGCGTTGATACGTACAATCGTAATCAGTTAG-3'), and the rest operations are the same, so as to finally obtain up-conversion fluorescent probe solution B;
fig. 3A shows Zeta potential changes before and after modification of an aptamer sequence with a nanomaterial, and it can be seen that after the aptamer sequence with negative potential is connected, the Zeta potential of the up-conversion nanoparticle with positive potential is converted into negative potential, and a substantial reduction occurs, which indicates successful connection of the aptamer sequence.
FIG. 3B is a graph of UV absorption before and after modification of an aptamer sequence by a nanomaterial, showing that after the aptamer sequence is attached, the up-conversion nanoparticle shows a strong UV absorption peak at 270nm, corresponding to the UV absorption of the aptamer sequence, further indicating successful attachment of the aptamer sequence.
Step four, preparing a multichannel paper-based microfluidic chip:
cutting Whatman No. 1 chromatographic filter paper (20 mm multiplied by 30 mm), performing pattern printing on the filter paper by using a laser printer, placing the filter paper in a blast drying oven for heat curing treatment at 200 ℃ for 3 hours after printing, and curing printer ink into the pores of chromatographic paper fibers to form a hydrophobic barrier zone 4.1 in which solution cannot flow; in the area of the hydrophobic barrier area 4.1, the micro-fluidic detection area is surrounded by ink; the microfluidic detection area consists of a detection area I4.2, a detection area II 4.6, a sample adding area 4.4, a flow channel I4.3 and a flow channel II 4.5, wherein the detection area I4.2 and the detection area II 4.6 are respectively arranged at two sides of the sample adding area 4.4 and are respectively communicated with the flow channel II 4.5 through the flow channel I4.3; the microfluidic detection area is in the hydrophobic barrier area 4.1 and is a hydrophilic flow area; the two components form a multichannel paper-based microfluidic chip;
Fig. 4 is a schematic structural diagram of a multichannel paper-based microfluidic chip, wherein a is a structural diagram of the chip; b is a size distribution diagram of the chip; reference numerals: the device comprises a hydrophobic barrier zone 4.1, a detection zone I4.2, a flow channel I4.3, a sample adding zone 4.4, a flow channel II 4.5 and a detection zone II 4.6.
Fifthly, detecting the content of enrofloxacin and ciprofloxacin in the food:
(1) Firstly, respectively dripping the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B prepared in the step three to a first detection area 4.2 and a second detection area 4.6 of the multichannel paper-based microfluidic chip; wherein the dosage of the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B is 5uL;
(2.1) firstly, preparing a mixed solution containing enrofloxacin and ciprofloxacin, wherein the concentrations of enrofloxacin and ciprofloxacin are respectively 1.0X10 ng/mL and 1.0X10 ng/mL; dripping the mixed solution into a sample adding area 4.4 of the multichannel paper-based microfluidic chip obtained in the step four, enabling the mixed solution to reach a detection area I4.2 and a detection area II 4.6 through a flow channel I4.3 and a flow channel II 4.5 under the action of capillary driving force, and incubating for 15min at room temperature to enable enrofloxacin and ciprofloxacin to react with an up-conversion fluorescent probe so as to obtain the multichannel paper-based microfluidic chip with the concentration I to be detected;
(2.2) the operation of step (2.1) is performed except that the concentration of enrofloxacin and ciprofloxacin is adjusted to 1.0X10 2 ng/mL、1.0×10 2 ng/mL, and the rest operations are the same, so as to finally obtain the multichannel paper-based microfluidic chip with the concentration II to be detected;
(2.3) the operation of step (2.1) is performed except that the concentration of enrofloxacin and ciprofloxacin is adjusted to 1.0X10 3 ng/mL、1.0×10 3 ng/mL, and the rest operations are the same, so as to finally obtain the multichannel paper-based microfluidic chip with the concentration of three to be detected;
(2.4) the operation of step (2.1) is performed except that the concentration of enrofloxacin and ciprofloxacin is adjusted to 1.0×10 4 ng/mL、1.0×10 4 ng/mL, and the rest operations are the same, so that a multichannel paper-based microfluidic chip with the concentration to be detected of four is finally obtained;
(2.5) the operation of step (2.1) is performed except that the concentration of enrofloxacin and ciprofloxacin is adjusted to 1.0X10 5 ng/mL、1.0×10 5 ng/mL, and the rest operations are the same, so that the multichannel paper-based microfluidic chip with the concentration of five to be detected is finally obtained;
(2.6) the operation of step (2.1) is performed except that the concentration of enrofloxacin and ciprofloxacin is adjusted to 1.0X10 6 ng/mL、1.0×10 6 ng/mL, and the rest operations are the same, so that the multichannel paper-based microfluidic chip with six to-be-detected concentration is finally obtained;
(2.7) the operation of step (2.1) is performed except that the concentration of enrofloxacin and ciprofloxacin is adjusted to 1.0X10 7 ng/mL、1.0×10 7 ng/mL, and the rest operations are the same, so that a multi-channel paper-based micro-fluidic chip with the concentration of seven to be detected is finally obtained;
(2.8) linearly fitting by using the enrofloxacin concentration and the fluorescent intensity of the first 4.2 detection area to establish a standard curve for detecting the enrofloxacin content; performing linear fitting by using the concentration of ciprofloxacin and the fluorescence intensity of the second detection zone 4.6, and establishing a standard curve for detecting the ciprofloxacin content;
wherein, the step of measuring the characteristic value of the fluorescence intensity signal of the detection solution comprises the following steps: the fluorescence intensity value at 654nm under the excitation of 980nm excitation light is the characteristic value of the fluorescence intensity signal of the detection solution; the measurement method is as follows:
placing a multichannel paper-based microfluidic chip to be detected on a precise moving platform 5.4.2 of a detection device, fixing the multichannel paper-based microfluidic chip by using a paper-based chip fixing clamp 5.4.2.3, adjusting the X axial distance of two shaft displacement fixing modules 5.2 on a base module 5.1 to be 100mm, and adjusting the pitching angle of a clamping module 5.3 to be 45 degrees, so that laser of a laser transmitter is aligned with the center of a circle of a detection area I4.2 and measuring the fluorescence intensity; then, the X-axis stepping knob 5.4.2.2 of the precise moving platform 5.4.2 is adjusted to enable the paper-based chip to perform X-axis displacement to be 12mm, so that laser of the laser transmitter is aligned to the center of a second detection area 4.6, and fluorescence intensity is measured.
FIG. 6 is a standard curve established for the fluorescent detection of enrofloxacin and ciprofloxacin at different concentrations; as can be seen from fig. 6A and 6B, as the enrofloxacin concentration increases, the fluorescence intensity at 654nm gradually increases, which shows a positive correlation between the enrofloxacin concentration and the increased fluorescence intensity; a good linear regression equation is obtained through linear fitting, y=1128.30x+1127.78, and the correlation coefficient R 2 0.9930, in the range of 1.0X10 ng/mL-1.0X10 5 The detection limit is 1.84ng/mL, and the requirements of enrofloxacin detection can be met. Where y represents the upconversion fluorescence intensity at 654nm and x represents the logarithmic concentration of enrofloxacin. As can be seen from fig. 6C and 6D, as the concentration of ciprofloxacin increases, the fluorescence intensity at 654nm gradually increases, which shows a positive correlation between the ciprofloxacin concentration and the increased fluorescence intensity; a good linear regression equation, y=1089.64x+964.66, is obtained by linear fitting, the correlation coefficient R 2 0.9955, in the range of 1.0X10 ng/mL-1.0X10 5 The detection limit is 2.22ng/mL, and the requirements of ciprofloxacin detection can be met. Where y represents the up-conversion fluorescence intensity at 654nm and x represents the logarithmic concentration of ciprofloxacin.
(3) Detection of actual samples:
firstly, preprocessing prawns: weighing 2g of prawn sample, and respectively concentrating 2mL to 5×10 and 1×10 2 And 1X 10 3 ng/mL enrofloxacin and ciprofloxacin standard solutions were added to the shrimp samples. After homogenization, 6mL of acetonitrile-acetone mixed solution is added, and the mixture is centrifuged for 10min at 4000 rmp; 3mL of the supernatant was heated in a rotary evaporator at 80℃to evaporate the acetonitrile. Finally, the dried residue was dissolved in 2mL of phosphate buffer to prepare a solution to be tested. And (3) dripping 20uL of the solution to be detected into a sample adding area 4.4, incubating for 15min at room temperature, detecting fluorescent intensity signal values of a detection area I4.2 and a detection area 4.6 on the multichannel paper-based microfluidic chip, taking the fluorescent intensity signal values into the standard curve obtained in the step (2), and calculating a labeling recovery result. Specifically as shown in table 1:
table 1: labeling detection result of enrofloxacin and cyclopropane Sha Xingwei harmful substances in prawn sample
RSD: relative standard deviation
Detection amount of harmful substances in prawns: x= (c×v×1000)/(m×1000)
Wherein:
x: the detection amount of the harmful substances in the prawns is expressed in micrograms per kilogram (mug/kg)
c: the concentration of the hazardous substances is measured in nanograms per milliliter (ng/mL)
V: the volume of the sample is determined in milliliters (mL)
m: the weight of the weighed prawn is gram (g)
Example 2:
step one, preparing an oil-soluble up-conversion nano material: accurately weighing 236.6mg yttrium chloride hexahydrate, 77.6mg ytterbium chloride hexahydrate and 7.6mg erbium chloride hexahydrate, dispersing in 10mL methanol solvent by ultrasonic, adding 4.0mL oleic acid and 9.0mL 1-octadecene, and magnetically stirring under argon environment at 160 ℃ for reaction for 25min to obtain transparent solution; after cooling to room temperature, a mixed solution containing 50mg of sodium hydroxide and 74.1mg of ammonium fluoride dissolved in 10mL of methanol was added dropwise; then magnetically stirring for 1.5h at 300 ℃, cooling to room temperature, centrifuging at 8000rpm for 8min to obtain up-conversion nanoparticle precipitate, washing with mixed solution of ultrapure water and ethanol with volume ratio of 1:3 for three times, and drying to obtain pure oleic acid coated up-conversion nanoparticle;
step two, preparing a water-soluble up-conversion nano material: the 20mg oleic acid coated up-conversion nano particles are weighed and dispersed in 0.5mL hydrochloric acid solution (the concentration is 0.1 mol/L) for ultrasonic treatment; after the treatment is finished, 60mL of ethanol is added and is dispersed evenly by ultrasonic, and then 10mL of ultrapure water and 2.5mL of ammonia water are added and are stirred evenly at 65 ℃; after stirring uniformly, adding 20uL of tetraethyl orthosilicate for reaction for 4 hours, and adding 50uL of 3-aminopropyl triethoxysilane for reaction for 1 hour; after the reaction is finished, the obtained solution is centrifuged for 8min at 8000rpm/min, the obtained product is washed by ultrapure water, and the washed product is dispersed in the ultrapure water to obtain the water-soluble up-conversion nano material solution with the concentration of 2mg/mL.
Step three, preparing an up-conversion fluorescent probe:
(1) Mixing the up-conversion nano material solution (volume 1.0 mL) prepared in the second step with glutaraldehyde water solution (volume 1.0 mL) for reaction to activate amino groups; separating out activated up-conversion nano material, dispersing the activated up-conversion nano material in 5.0mL of phosphate buffer solution, adding 50uL of diarrhea-causing escherichia coli aptamer solution (the concentration is 0.1mmol/L, the sequence is 5'-CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGG-3'), incubating for 2 hours at 37 ℃ on a shaking table, wherein the rotation speed of the shaking table is 180rpm/min, centrifuging for 5 minutes at 6000rpm after incubation, washing obtained precipitate with ultrapure water, centrifuging again (the rotation speed is 6000rpm, the time is 5 minutes), drying to obtain 1.0mg of up-conversion nano material modified with enrofloxacin aptamer, finally adding the up-conversion nano material into phosphate buffer solution containing graphene oxide (the volume is 5mL, the concentration is 0.2 mg/mL), incubating for 0.5 hours at 37 ℃, incubating on the shaking table, and obtaining up-conversion fluorescent probe A solution with the concentration of 0.5mg/mL at 180 rpm;
(2) The same operation as the step (1) is carried out, the difference is that the diarrhea causing escherichia coli is replaced by staphylococcus aureus aptamer solution (the concentration is 0.1mmol/L, the sequence is 5'-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGC GCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-3'), and the rest operations are the same, so as to finally obtain up-conversion fluorescent probe solution B;
Step four, preparing a multichannel paper-based microfluidic chip: cutting Whatman No. 1 chromatographic filter paper (20 mm multiplied by 30 mm), performing pattern printing on the filter paper by using a laser printer, placing the filter paper in a blast drying oven for heat curing treatment at 200 ℃ for 3 hours after printing, and curing printer ink into the pores of chromatographic paper fibers to form a hydrophobic barrier zone 4.1 in which solution cannot flow; in the area of the hydrophobic barrier area 4.1, the micro-fluidic detection area is surrounded by ink; the microfluidic detection area consists of a detection area I4.2, a detection area II 4.6, a sample adding area 4.4, a flow channel I4.3 and a flow channel II 4.5; the first detection area 4.2 and the second detection area 4.6 are respectively arranged at two sides of the sample adding area 4.4 and are respectively communicated with the second detection area 4.5 through the first flow channel 4.3; the microfluidic detection area is in the hydrophobic barrier area 4.1 and is a hydrophilic flow area; the two components form a multichannel paper-based microfluidic chip;
step five, detecting the content of diarrhea causing escherichia coli and staphylococcus aureus in food:
(1) Firstly, respectively dripping the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B prepared in the step three to a first detection area and a second detection area of the microfluidic paper substrate; wherein the amount of the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B is 5uL.
(2) Establishment of a standard curve:
(2.1) firstly preparing a mixed solution containing the diarrhea-causing escherichia coli and staphylococcus aureus, wherein the concentration of the diarrhea-causing escherichia coli and the concentration of the staphylococcus aureus are respectively 1.0x10CFU/mL and 4.5x10CFU/mL, dripping the mixed solution into a sample adding area 4.4 of the multichannel paper-based microfluidic chip obtained in the step four, enabling the mixed solution to reach a detection area I4.2 and a detection area II 4.6 through a flow channel I4.3 and a flow channel II 4.5 under the action of capillary driving force, and incubating the mixed solution at room temperature for 15 minutes to enable the diarrhea-causing escherichia coli and the staphylococcus aureus to react with an up-conversion fluorescent probe fully to obtain the multichannel paper-based microfluidic chip with the concentration I to be detected;
(2.2) the procedure of step (2.1) was followed except that the concentration of the diarrhea-causing Escherichia coli and Staphylococcus aureus was adjusted to 1.0X10% 2 CFU/mL、4.5×10 2 CFU/mL, the rest operations are the same, and finally the multichannel paper-based microfluidic chip with the concentration II to be detected is obtained;
(2.3) the procedure of step (2.1) was followed except that the concentration of the diarrhea-causing Escherichia coli and Staphylococcus aureus was adjusted to 1.0X10% 3 CFU/mL、4.5×10 3 CFU/mL, the rest operations are the same, and finally the multichannel paper-based microfluidic chip with the concentration to be detected III is obtained;
(2.4) the procedure of step (2.1) was followed except that the concentration of the diarrhea-causing Escherichia coli and Staphylococcus aureus was adjusted to 1.0X10% 4 CFU/mL、4.5×10 4 CFU/mL, all the other operations being identicalFinally, a multichannel paper-based microfluidic chip with the concentration to be detected IV is obtained;
(2.5) the procedure of step (2.1) was followed except that the concentration of the diarrhea-causing Escherichia coli and Staphylococcus aureus was adjusted to 1.0X10% 5 CFU/mL、4.5×10 5 CFU/mL, the rest operations are the same, and finally the multichannel paper-based microfluidic chip with the concentration of five to be detected is obtained;
(2.6) the procedure of step (2.1) was followed except that the concentration of the diarrhea-causing Escherichia coli and Staphylococcus aureus was adjusted to 1.0X10% 6 CFU/mL、4.5×10 6 CFU/mL, the rest operations are the same, finally the multi-channel paper-based micro-fluidic chip with six concentrations to be detected is obtained;
(2.7) the procedure of step (2.1) was followed except that the concentration of the diarrhea-causing Escherichia coli and Staphylococcus aureus was adjusted to 1.0X10% 7 CFU/mL、4.5×10 7 CFU/mL, the rest operations are the same, and finally the multi-channel paper-based micro-fluidic chip with the concentration to be detected of seven is obtained;
(2.8) performing linear fitting on the concentration of the diarrhea escherichia coli and the fluorescence intensity of the first detection zone 4.2, and establishing a standard curve for detecting the content of the diarrhea escherichia coli; performing linear fitting on the concentration of staphylococcus aureus and the fluorescence intensity of the second detection zone 4.6, and establishing a standard curve for detecting the content of staphylococcus aureus;
Wherein, the step of measuring the characteristic value of the fluorescence intensity signal of the detection solution comprises the following steps: the fluorescence intensity value at 654nm under the excitation of 980nm excitation light is the characteristic value of the fluorescence intensity signal of the detection solution; the measurement method is as follows:
placing a multichannel paper-based microfluidic chip to be detected on a precise moving platform 5.4.2 of a detection device, fixing the multichannel paper-based microfluidic chip by using a paper-based chip fixing clamp 5.4.2.3, adjusting the X axial distance of two shaft displacement fixing modules 5.2 on a base module 5.1 to be 100mm, and adjusting the pitching angle of a clamping module 5.3 to be 45 degrees, so that laser of a laser transmitter is aligned with the center of a circle of a detection area I4.2 and measuring the fluorescence intensity; then, the X-axis stepping knob 5.4.2.2 of the precise moving platform 5.4.2 is adjusted to enable the paper-based chip to perform X-axis displacement to be 12mm, so that laser of the laser transmitter is aligned to the center of a second detection area 4.6, and fluorescence intensity is measured.
FIG. 7 is a standard curve established for fluorescence detection of different concentrations of diarrhea-causing Escherichia coli and Staphylococcus aureus; as can be seen from fig. 7A and 7B, as the concentration of the diarrheal escherichia coli increases, the fluorescence intensity at 654nm gradually increases, which shows a positive correlation between the diarrheal escherichia coli concentration and the increased fluorescence intensity; a good linear regression equation is obtained through linear fitting, y=734.0x+1677.4, and the correlation coefficient R 2 0.9904, in the range of 1.0X10 CFU/mL-1.0X10 7 The detection limit of CFU/mL is 3.80CFU/mL, and the detection requirement of the diarrhea escherichia coli can be met. Wherein y represents the up-conversion fluorescence intensity at 654nm and x represents the logarithmic concentration of the diarrhea-causing Escherichia coli. As can be seen from fig. 7C and 7D, as the concentration of staphylococcus aureus increases, the fluorescence intensity at 654nm gradually increases, which shows a positive correlation between the concentration of staphylococcus aureus and the increased fluorescence intensity; a good linear regression equation is obtained through linear fitting, y=890.6x+572.8, and the correlation coefficient R 2 0.9894, in the range of 4.5X10 CFU/mL-4.5X10 7 The detection limit of CFU/mL is 4.90CFU/mL, and the requirements of staphylococcus aureus detection can be met. Where y represents the up-conversion fluorescence intensity at 654nm and x represents the logarithmic concentration of staphylococcus aureus.
(3) Detection of actual samples:
firstly, pretreatment of pork: 25g of fresh pork samples were washed three times with sterile physiological saline and then placed in a biosafety cabinet and sterilized using a 30W UV lamp irradiation for 15min to eliminate potential interference of the samples themselves with E.coli and Staphylococcus aureus. Then 1mL of the solution was concentrated to 1.25X10 respectively 4 、2.50×10 4 And 2.50X10 5 CFU/mL of a standard solution of E.coli and Staphylococcus aureus was added to the pork sample and incubated for 10min to simulate the natural growth of E.coli and Staphylococcus aureus in the sample. Then, the labeled pork sample is added into 225mL of sterile physiological saline and homogenized 3And (5) preparing the solution to be tested after min. And (3) dripping 20uL of the solution to be detected into a sample adding area 4.4, incubating for 15min at room temperature, detecting fluorescent intensity signal values of a first detection area 4.2 and a second detection area 4.6 on the multichannel paper-based microfluidic chip, taking the fluorescent intensity signal values into the standard curve obtained in the step (2), and calculating a labeling recovery result. As shown in table 2:
table 2: labeling detection result of diarrhea causing escherichia coli and staphylococcus aureus hazard in pork sample
RSD: relative standard deviation
Detection amount of hazardous substances in pork: x= (c×v)/m
Wherein:
x: the detection amount of the harmful substances in the pork is expressed in units of colony count per gram (CFU/g)
c: the concentration of the hazardous substances is measured in colony count per milliliter (CFU/mL)
V: the volume of the sample is determined in milliliters (mL)
m: the weight of the weighed pork is given in grams (g)
Specificity of the detection method:
In order to evaluate the specificity of the constructed multichannel paper-based microfluidic chip for detecting diarrhea escherichia coli and staphylococcus aureus, the invention selects some other common food-borne pathogenic bacteria including bacillus cereus, salmonella and pseudomonas aeruginosa as interfering pathogenic bacteria, and further researches the fluorescence characteristics of the multichannel paper-based microfluidic chip.
Comparative example 2:
the procedure is as in example 2; the difference from example 2 is that the sample solution to be tested in step (3) was replaced with a blank solution, bacillus cereus (1.0X10) 7 CFU/mL), salmonella (1.0X10) 7 CFU/mL), pseudomonas aeruginosa (1.0X10) 7 CFU/mL), escherichia coli (1.0X10) 7 CFU/mL), staphylococcus aureus (1.0X10) 7 CFU/mL), intoRows are detected separately. Wherein, the fluorescence response of the blank solution, bacillus cereus, salmonella and pseudomonas aeruginosa is the average value of the fluorescence intensities of the two detection areas, the fluorescence response of the diarrhea escherichia coli is the fluorescence intensity of the detection area I4.2, and the fluorescence response of staphylococcus aureus is the fluorescence intensity of the detection area II 4.6.
As shown in FIG. 8, the fluorescence response efficiency of the prepared sensor to diarrhea escherichia coli and staphylococcus aureus is obviously higher than that of other common food-borne pathogenic bacteria, and the result shows that the constructed multichannel paper-based microfluidic chip has better specificity.
Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.
Claims (10)
1. The preparation method of the multichannel paper-based microfluidic chip is characterized by comprising the following steps of:
step one, preparing an oil-soluble up-conversion nano material: adding yttrium chloride hexahydrate, ytterbium chloride hexahydrate and erbium chloride hexahydrate into a certain volume of methanol A for ultrasonic dissolution, then adding oleic acid and 1-octadecene, fully mixing, heating and stirring for the first time under a closed condition, and cooling to room temperature after stirring; removing the closed condition, then dropwise adding methanol B containing sodium hydroxide and ammonium fluoride, heating and stirring for the second time under the closed condition, cooling to room temperature after stirring, centrifugally separating to obtain precipitate, cleaning with a mixed solution of ultrapure water and ethanol, and drying to obtain an oleic acid coated up-conversion nanomaterial for later use;
Step two, preparing a water-soluble up-conversion nano material: weighing oleic acid coated up-conversion nano particles prepared in the first step, dispersing the oleic acid coated up-conversion nano particles in hydrochloric acid solution, and carrying out ultrasonic treatment; after the treatment is finished, adding ethanol, uniformly dispersing by ultrasonic waves, then adding ultrapure water and ammonia water, and uniformly stirring at a certain temperature; after stirring uniformly, adding tetraethyl orthosilicate to perform a first reaction, and then adding 3-aminopropyl triethoxysilane to perform a second reaction; after the reaction is finished, centrifugally separating, cleaning the obtained product with ultrapure water, and dispersing the cleaned product in the ultrapure water to obtain the water-soluble up-conversion nano material;
step three, preparing an up-conversion fluorescent probe:
(1) Mixing the up-conversion nano material solution prepared in the second step with glutaraldehyde water solution for reaction to activate amino; separating out the activated up-conversion nano material, dispersing the activated up-conversion nano material in a phosphate buffer, adding a food hazard substance A aptamer solution, incubating at a first constant temperature, centrifuging after incubation, washing the obtained precipitate with ultrapure water, centrifuging again, drying to obtain an up-conversion nano material modified with the food hazard substance A aptamer, and finally adding the up-conversion nano material into the phosphate buffer containing graphene oxide, incubating at a second constant temperature to obtain a mixed solution, and marking the mixed solution as an up-conversion fluorescent probe solution A;
(2) The same operation as the step (1) is distinguished by only replacing the food hazard A aptamer solution with the food hazard B aptamer solution, and the rest operations are the same, so as to finally obtain an up-conversion fluorescent probe solution B;
step four, preparing a multichannel paper-based microfluidic chip:
cutting filter paper, performing pattern printing on the filter paper by using a laser printer, performing heat curing treatment on the filter paper after printing, and curing printer ink into fiber pores of the chromatographic paper to form a hydrophobic barrier zone in which solution cannot flow; in the area of the hydrophobic barrier, the ink encloses a microfluidic detection area; the microfluidic detection area consists of a detection area I, a detection area II, a sample adding area and a flow channel; the first detection area and the second detection area are respectively arranged at two sides of the sample adding area and are communicated through a flow channel; the microfluidic detection area is arranged inside the hydrophobic barrier area and is a hydrophilic flow area; the two components form a multichannel paper-based microfluidic chip.
2. The method for preparing the multichannel paper-based microfluidic chip according to claim 1, wherein the dosage relationship of yttrium chloride hexahydrate, ytterbium chloride hexahydrate, erbium chloride hexahydrate, methanol a, oleic acid, 1-octadecene, sodium hydroxide, ammonium fluoride and methanol B in the step one is 236.6mg:77.6mg:7.6mg:10mL:4.0mL:9.0mL:50.0mg:74.1mg:10mL; the mixed solution of cyclohexane and ethanol is used for cleaning, wherein the volume ratio of the cyclohexane to the ethanol is 1:3, a step of;
The first heating and stirring time is 20-30min, and the heating temperature is 160 ℃; the second heating and stirring temperature is 295-305 ℃, and the stirring time is 1.0-1.5h; the conditions of the centrifugal separation are as follows: the rotation speed is 6000-8000rpm/min, and the time is 5-8min.
3. The method for preparing the multichannel paper-based microfluidic chip according to claim 1, wherein in the second step, the ratio of the up-conversion nanomaterial to the hydrochloric acid solution to the ethanol to the ultrapure water to the ammonia water to the tetraethyl orthosilicate to the 3-aminopropyl triethoxysilane is 20mg:0.5mL:60mL:10mL:2.5mL:20uL:50uL; the first reaction time is 4-6h, and the second reaction time is 1-2h; the concentration of the hydrochloric acid solution is 0.1mol/L; the certain temperature condition is 65 ℃; the conditions of the centrifugal separation are as follows: the rotating speed is 6000-8000rpm/min, and the time is 5-8min; the concentration of the water-soluble up-conversion nano material solution is 2mg/mL.
4. The method for preparing the multichannel paper-based microfluidic chip according to claim 1, wherein in the third step, the dosage relationship of the water-soluble up-conversion nanomaterial solution, glutaraldehyde aqueous solution, phosphate buffer, food hazard a aptamer solution, up-conversion nanomaterial, and phosphate buffer containing graphene oxide is 1.0mL:1.0mL:5.0mL:50uL:1.0mg:2.0mL; the concentration of the phosphate buffer solution containing graphene oxide is 0.2mg/mL; the concentration of the target A aptamer solution is 0.1mmol/L; the concentration of the up-conversion fluorescent probe solution A is 0.5mg/mL; the concentration of the up-conversion fluorescent probe solution B is 0.5mg/mL.
The reaction time of the mixing reaction for activating the amino group is 2 hours, the first incubation is carried out on a shaking table, the temperature is 37 ℃, the time is 2 hours, and the rotating speed of the shaking table is 180-200rpm/min; the second incubation is carried out on a shaking table, the temperature is 37 ℃, the time is 0.5h, and the rotating speed of the shaking table is 180-200rpm/min; the conditions for centrifugal separation were: the rotating speed is 6000-8000rpm/min, and the time is 5-10min;
the aptamer in the aptamer solution is not limited to a corresponding sequence of a certain food hazard, and the corresponding aptamer sequence is matched according to different detection target food hazards, wherein the food hazards comprise enrofloxacin, ciprofloxacin, diarrhea-causing escherichia coli and staphylococcus aureus.
5. The method for preparing the multichannel paper-based microfluidic chip according to claim 1, wherein in the fourth step, the filter paper is Whatman No. 1 chromatographic filter, and the size is 20×30mm; the first detection area, the second detection area and the sample adding area are all round areas with the diameter of 3 mm; the flow channel is divided into a first flow channel and a second flow channel, the lengths of the first flow channel and the second flow channel are 3mm, and the widths of the first flow channel and the second flow channel are 1mm;
the temperature of the heat curing treatment is 200 ℃, and the curing time is 3 hours.
6. Use of a multi-channel paper-based microfluidic chip prepared according to the method of any one of claims 1-5 for food hazard detection, characterized by the steps of:
(1) Firstly, respectively dripping the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B prepared in the step three to a first detection area and a second detection area of the multichannel paper-based microfluidic chip;
(2) Establishment of a standard curve:
firstly, preparing a mixed solution containing a certain concentration of food dangers A and B, dripping the mixed solution into a sample adding area of the multichannel paper-based microfluidic chip obtained in the step four, respectively reaching a first detection area and a second detection area through a flow channel under the action of capillary driving force, incubating at room temperature to enable the food dangers A and B to react with corresponding up-conversion fluorescent probe solutions, and then detecting the fluorescent intensity of the first detection area and the second detection area;
preferably, in the step (2.1), the amount of the mixed standard solution of the food hazard A and the food hazard B is 20uL; the incubation time at room temperature is 15min;
(2.2) the operation of the step (2.1) is different in that the concentration of the mixed solution containing the food dangers A and B is subjected to gradient change, and the rest operations are the same, so that the corresponding fluorescence intensities under different concentration conditions are finally obtained; performing linear fitting on the concentration of the food hazard A and the fluorescence intensity of the first detection area, and establishing a standard curve for detecting the content of the food hazard A; performing linear fitting on the concentration of the food hazard B and the fluorescence intensity of the second detection area, and establishing a standard curve for detecting the content of the food hazard B;
(3) Detection of actual samples: pretreating an actual sample to obtain a solution to be tested containing food hazardous substances; dropwise adding a solution to be detected into a sample adding area of the multichannel paper-based microfluidic chip, and incubating at room temperature; measuring the fluorescence intensity of the first detection area, carrying the fluorescence intensity into the food hazard A content detection standard curve obtained in the step five, and calculating the content of the food hazard A of the actual sample; and (3) measuring the fluorescence intensity of the second detection area, carrying the second detection area into the food hazard B content detection standard curve obtained in the step (V), and calculating the content of the food hazard B of the actual sample.
7. The use according to claim 6, wherein the up-conversion fluorescent probe solution A and the up-conversion fluorescent probe solution B in the step (1) are used in an amount of 5uL:5uL;
in the step (2.1), the dosage of the mixed standard solution of the food hazard A and the food hazard B is 20uL; the incubation time at room temperature is 15min; in the step (3), the dripping amount of the solution to be detected is 20uL; the incubation time was 15min.
8. The use according to claim 6, wherein the fluorescent intensity detection in steps (2) - (3) is performed by a portable food hazard detection device based on a multi-channel paper-based microfluidic chip, and the detection device is formed by combining a base module (5.1), an axis displacement fixing module (5.2), a clamping module (5.3) of a laser transmitter and a paper-based bearing platform module (5.4);
The base module (5.1) is horizontally installed; the two ends of the base module (5.1) are A, B ends, and the A, B ends are respectively provided with an axial displacement fixing module (5.2); a clamping module (5.3) is arranged above the shaft displacement fixing module (5.2) at the end A, and a bearing platform module (5.4) is arranged above the shaft displacement fixing module (5.2) at the end B;
the shaft displacement fixing module (5.2) comprises a base clamping knob (5.2.1), a quick-mounting plate clamping knob (5.2.2) and a quick-mounting plate (5.2.3); the base clamping knob (5.2.1) is arranged above the base module (5.1), the quick-mounting plate clamping knob (5.2.2) is arranged above the base clamping knob (5.2.1), and the quick-mounting plate clamping knob (5.2.2) clamps the quick-mounting plate (5.2.3);
the clamping module (5.3) comprises a course axis damping adjusting knob (5.3.1), a pitching axis damping adjusting knob (5.3.2) and a crab clamp holder (5.3.3); the device comprises a heading shaft damping adjusting knob (5.3.1), a shaft displacement fixing module (5.2), a pitching shaft damping adjusting knob (5.3.2) and a crab clamp holder (5.3.3), wherein the heading shaft damping adjusting knob (5.3.1) is arranged above the shaft displacement fixing module (5.2), and the pitching shaft damping adjusting knob (5.3.2) is connected with the crab clamp holder (5.3.3);
the paper-based bearing platform module (5.4) comprises a fixed base (5.4.1) and a precise moving platform (5.4.2); wherein, a Y-axis stepping knob (5.4.2.1), an X-axis stepping knob (5.4.2.2) and a paper-based chip fixing clamp (5.4.2.3) are arranged on the precise moving platform (5.4.2).
9. Use according to claim 8, characterized in that the dimensions of the base module (5.1) are 230mm long x 38mm wide; the X axial direction of the base module (5.1) is the long side of the base module (5.1), and the Y axial direction is the wide side of the base module (5.1); the X-axis displacement travel range of the base module (5.1) is 0-200mm;
the course axis damping adjusting knob (5.3.1) can adjust the course angle of 0-360 degrees; the pitching axis damping adjusting knob (5.3.2) can adjust the pitching angle by 0-180 degrees; the clamping range of the crab claw clamp holder (5.3.3) is 5-57mm;
the precise moving platform (5.4.2) can carry out Y-axis displacement and X-axis displacement through a Y-axis stepping knob (5.4.2.1) and an X-axis stepping knob (5.4.2.2), the displacement precision is 1mm, and the displacement stroke is 40mm; the Y-axis direction of the precision moving platform is the Y-axis direction of the base module; the X-axis direction of the precision moving platform is the X-axis direction of the base module.
10. The use according to claim 8, wherein the method for detecting fluorescence intensity of the portable food hazard detection device based on the multichannel paper-based microfluidic chip is as follows:
placing a multichannel paper-based microfluidic chip to be detected on a precise moving platform (5.4.2) of a detection device, fixing by using a paper-based chip fixing clamp (5.4.2.3), adjusting the X axial distance of two shaft displacement fixing modules (5.2) on a base module (5.1) to be 100mm, and adjusting the pitch angle of a clamping module (5.3) to be 45 degrees, so that the laser of a laser transmitter is aligned with the center of a first detection area and the fluorescence intensity is measured; then, an X-axis stepping knob (5.4.2.2) of the precise moving platform (5.4.2) is adjusted to enable the paper-based chip to be displaced to be 12mm in the X-axis direction, so that laser of the laser transmitter is aligned to the center of a second detection area and fluorescence intensity is measured.
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