CN116083222A - Long-period fiber grating biosensor coupling micro-fluidic chip and preparation method and application thereof - Google Patents
Long-period fiber grating biosensor coupling micro-fluidic chip and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of drug sensitivity testing of virulent strains, and discloses a long-period fiber grating biosensor coupling microfluidic chip and a preparation method and application thereof. And sequentially carrying out surface silanization treatment, polydopamine modification and phage modification on the long-period fiber grating, sealing to obtain a long-period fiber grating biosensor, and clamping the long-period fiber grating biosensor into a chip to obtain the long-period fiber grating biosensor coupling microfluidic chip. The long-period fiber grating biosensor coupling micro-fluidic chip provided by the invention can be used for carrying out drug sensitivity test on the virulent strain, shortens the detection time to within 4 hours, and improves the sensitivity of the drug sensitivity test on the virulent strain; the method has the advantages of high sensitivity, high accuracy, short test time and simple and convenient operation. The preparation method of the long-period fiber grating biosensor coupling microfluidic chip is simple, large-scale equipment is not needed, and the reaction condition is mild; has the advantages of low cost and high safety coefficient.
Description
Technical Field
The invention relates to the technical field of drug sensitivity testing of virulent strains, in particular to a long-period fiber grating biosensor coupling microfluidic chip and a preparation method and application thereof.
Background
Antibiotics are generally considered as good agents for treating virulent strains, however, with irregular use of antibiotics in clinic and in farmed animals, virulent strains are increasingly resistant to drugs, resistant strains are increasing, and even multiple resistant phenomena are generated. The drug-resistant strain, in particular to a multi-drug-resistant strain, enters the human body through a food chain, so that the conventional antibiotics are invalid, and a great challenge is presented to clinical treatment, thereby seriously threatening the life safety of the human. Thus, research on resistance of drug-resistant strains to antibiotics has become a hot problem in the fields of biomedicine and the like.
Currently, identification of virulent strains of bacteria is primarily dependent on traditional microbiological methods such as isolation culture, biochemical identification, broth dilution, paper diffusion, and the like. They often involve multiple time consuming steps: (1) isolating a pathogen from a sample; (2) enriching the isolated bacteria to a detectable level; (3) Identifying microbial pathogens using a bacterial specific biochemical method and incubating the cells with antibiotics in a multiwell plate; (4) Bacterial growth was determined using absorbance spectra or visual assessment. Typically, the entire process takes 3 to 4 days, resulting in a delay in testing. With the development of technology, new assay methods, especially techniques based on molecular biology and immunology, such as different kinds of Polymerase Chain Reaction (PCR) methods and enzyme-linked immunosorbent assay (ELISA), are expected to shorten the identification time. However, they still present some significant drawbacks in themselves, such as the need for appropriate primers for PCR techniques, cumbersome protocols, and the susceptibility to false positive results. Although the above-mentioned techniques provide standards for the identification of resistance to different species of pathogenic bacteria and antibacterial drugs in laboratories, they cannot meet the different requirements of various analyses such as clinical diagnosis and emergency treatment sites when dealing with public emergencies in the fields of foods and biomedicine. Therefore, there is a need to establish a rapid, accurate and repeatable method that can achieve drug resistance monitoring and identification of virulent strains.
For bacterial structures, the action mechanism of general antibiotics on bacteria mainly comprises the steps of preventing bacterial cell wall synthesis to enable bacteria to swell and rupture under a low osmotic pressure environment, enhancing permeability of bacterial cell membranes to enable internal substances of bacteria to leak out, preventing reproduction and transcription of bacterial DNA from being replicated, preventing nucleic acid synthesis from being hindered to inhibit reproduction due to influence on folic acid metabolism, and inhibiting protein synthesis through action with bacterial ribosomes or reaction substrates, so that bacteria are ruptured and atrophic or are inhibited from reproduction. Thus, resistance of a target bacterium to an antibiotic can be assessed by a change in the morphology and number of bacteria propagated after the bacterium has bound to the antibiotic.
Microfluidic technology is the forefront technology field of the hottest in instant detection technology. Compared with the traditional experimental platform, the method has the advantages of high integration level, low cost, high sensitivity, less reagent consumption, short detection time and the like. In addition, the advantages of flexible combination of various unit operations, overall controllability and the like of the micro-flow control enable the micro-flow control to be combined with the research thought of the system biology, and the micro-flow control has become an important technical platform in the field of biological research. Bacterial culture is a basic operation unit for biological research, and compared with the traditional bacterial culture method, bacterial culture performed in the microfluidic has unique advantages. The relatively closed environment formed by the microfluidic channels and the two-dimensional flow of fluid in the channels enable more complex physicochemical operations to be performed on bacteria. Therefore, researches on micro-fluidic technology targeting bacteria have attracted attention from various researchers at home and abroad, and have been rapidly developed, and are also used in drug resistance analysis of virulent strains. Whereas most of the existing studies rely on in vitro detection devices. Such as electrochemical workstations, enzyme-labeled instruments, surface-enhanced raman scattering, mass spectrometry, etc., cannot be portable and integrated. Therefore, how to realize integrated and portable detection by using the microfluidic chip technology is a current research trend.
In recent years, the combination of microfluidic chips and optical systems has been widely used because the optical field formed between the optical path and the microfluidic enhances the interaction between light and fluid. At the same time, manipulation of the light may be achieved by controlling the microfluidics. However, the conventional optical inspection apparatus requires complicated optical devices such as a microscope, a lens, etc., which is disadvantageous in terms of the development of miniaturization of the chip. In order to integrate light with a microfluidic chip better, sensing measurement is realized, and an optical fiber becomes a suitable optical transmission carrier. The combination of the optical fiber and the micro channel in the micro-fluidic chip is the key for realizing the detection of the target object. The sensitivity of the fiber optic sensor to external changes enables a sensing measurement of the sample in the microchannel. In addition, the micro-fluidic chip not only can provide stable sensing environment for some fragile and special optical fiber sensing structures, but also can protect the sensor from external environmental factors, and the stability of the sensor is obviously improved. More and more micro-fluidic chips and optical sensors manufactured by optical fiber coupling are widely applied to sensing measurement.
In summary, the integration of the optical fiber and the microfluidic chip has very important application value in the field of biochemical sensing. In the future, the main research direction of the optical fiber integrated micro-fluidic sensor chip is still integration, miniaturization, portability, high flux and multifunction, and automatic analysis and on-site real-time measurement are realized. The combination of the two can break through the bottlenecks of low sensitivity, time consumption, complicated steps, low accuracy and the like, develop towards the directions of low cost, easy operation and industrialization, are applied to the field drug sensitivity test of virulent strains, and have wide industrialization prospect. Therefore, developing an optical fiber integrated microfluidic sensor chip for drug sensitivity test of a virulent strain with high sensitivity, high accuracy, short test time, simple operation and low cost becomes a urgent need in the art.
Disclosure of Invention
The invention aims to provide a long-period fiber grating biosensor coupling micro-fluidic chip and a preparation method and application thereof, so as to solve the problems of low identification speed, low accuracy, low repeatability and high cost of the existing identification method for drug resistance of virulent strains, and solve the problem of blank research on the existing fiber integrated micro-fluidic sensor chip for drug sensitivity test of virulent strains.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a long-period fiber grating biosensor coupling micro-fluidic chip, which comprises the following steps:
sequentially carrying out surface silanization treatment, modification of polydopamine and modification of phage solution on the long-period fiber grating, and then sealing to obtain a long-period fiber grating biosensor; clamping the long-period fiber bragg grating biosensor into a chip to obtain a long-period fiber bragg grating biosensor coupling micro-fluidic chip;
the chip comprises a glass plate and a polydimethylsiloxane template;
the grating period of the long period fiber grating is 320-400 mu m, the total grating length is 15-20 mm, the grating diameter is 100-120 mu m, and the loss peak intensity is 25-35 dB.
Preferably, the specific steps of the surface silanization treatment are as follows: soaking the long-period fiber bragg grating in a hydrochloric acid methanol solution, a sodium hydroxide solution and a 3-aminopropyl trimethoxy silane acetonitrile solution in sequence; the volume ratio of hydrochloric acid to methanol in the hydrochloric acid methanol solution is 0.5-1.5: 0.5 to 1.5; the concentration of the sodium hydroxide solution is 0.5-1.2 mol/L; the volume concentration of the 3-aminopropyl trimethoxy silane acetonitrile solution is 1.5-2.5%.
Preferably, the time for soaking in the hydrochloric acid methanol solution is 1-5 hours; soaking in the sodium hydroxide solution for 5-12 h; the soaking time in the 3-aminopropyl trimethoxy silane acetonitrile solution is 12-20 h.
Preferably, the reagent used for modifying the polydopamine is a dopamine hydrochloride solution; the dopamine hydrochloride solution comprises dopamine hydrochloride and Tris-HCl buffer solution, and the mass volume ratio of the dopamine hydrochloride to the Tris-HCl buffer solution is 8-12 mg: 0.8-1.2 mL; the concentration of the Tris-HCl buffer solution is 8-12 mmol/L, and the pH value is 7.5-8.5; the time for modifying the polydopamine is 1-2 h.
Preferably, the phage solution has a concentration of 10 4 ～10 7 CFU/mL; the temperature of the modified phage solution is 15-35 ℃, and the modified phage solutionThe time of (2) is 20-50 min.
Preferably, the blocking is performed in a bovine serum albumin solution, and the mass concentration of the bovine serum albumin solution is 3-7%; the sealing temperature is 15-25 ℃, and the sealing time is 5-12 h.
Preferably, the polydimethylsiloxane-based template comprises 4 independent units, and each unit comprises a sample injection area, a microfluidic channel, a reaction area and a test area.
Preferably, the sensing area of the long-period fiber grating biosensor is clamped into a testing area in the chip.
The invention also provides the long-period fiber grating biosensor coupling microfluidic chip prepared by the preparation method of the long-period fiber grating biosensor coupling microfluidic chip.
The invention also provides application of the long-period fiber grating biosensor coupling microfluidic chip in preparing a virulent strain drug sensitivity test product.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention shortens the drug sensitivity test time of the virulent strain within 4 hours by combining micro-culture of the microfluidic chip with high sensitivity of the long-period fiber grating biosensor, is more suitable for actual emergency detection, and has the advantages of high sensitivity, high accuracy and strong specificity;
(2) The long-period fiber grating biosensor coupling microfluidic chip has the advantages of simple preparation process, no need of large-scale equipment, mild reaction conditions, low cost and high safety coefficient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic structural diagram of a unit in a polydimethylsiloxane-based template as described in example 1;
fig. 2 is a schematic structural diagram of a coupling microfluidic chip of a long-period fiber bragg grating biosensor obtained in embodiment 1;
FIG. 3 is a graph showing the shift of resonance wavelength of a long period fiber grating biosensor coupled microfluidic chip according to example 1 after two anti-biopharmaceuticals are used to culture a strain of Staphylococcus aureus, wherein Δλλ 1-1 、Δλ 1-2 、Δλ 1-3 、Δλ 1-4 Ampicillin, deltalambda, at various concentrations 1-1 、Δλ 1-2 、Δλ 1-3 、Δλ 1-4 Representative concentrations of ampicillin are 0 μg/mL, 0.005 μg/mL, 0.08 μg/mL, 2.5 μg/mL; Δλ (delta lambda) 2-1 、Δλ 2-2 、Δλ 2-3 、Δλ 2-4 For different concentrations of cefoxitin, deltalambda 2-1 、Δλ 2-2 、Δλ 2-3 、Δλ 2-4 Representative concentrations of cefoxitin are 0 μg/mL, 0.005 μg/mL, 0.08 μg/mL, 2.5 μg/mL.
Detailed Description
The invention provides a preparation method of a long-period fiber grating biosensor coupling micro-fluidic chip, which comprises the following steps:
sequentially carrying out surface silanization treatment, modification of polydopamine and modification of phage solution on the long-period fiber grating, and then sealing to obtain a long-period fiber grating biosensor; clamping the long-period fiber bragg grating biosensor into a chip to obtain a long-period fiber bragg grating biosensor coupling micro-fluidic chip;
the chip comprises a glass plate and a polydimethylsiloxane template;
the grating period of the long period fiber grating is 320-400 mu m, the total grating length is 15-20 mm, the grating diameter is 100-120 mu m, and the loss peak intensity is 25-35 dB.
In the invention, the grating period of the long period fiber grating is preferably 330 to 390 μm, more preferably 360 to 380 μm; the total grating length is preferably 16 to 19mm, more preferably 17 to 18mm; the grating diameter is preferably 110 to 119. Mu.m, more preferably 114 to 116. Mu.m; the loss peak intensity is preferably 28 to 32dB, more preferably 30dB.
In the invention, the preparation of the long-period fiber grating comprises the following steps: the single-mode fiber is inscribed to obtain a long-period fiber grating; the method for burning is a femtosecond laser direct writing technology; in the recording, the repetition rate of the femtosecond laser pulse is preferably 0.5 to 1.5kHz, more preferably 1kHz, and the energy is preferably 0.6 to 0.9. Mu.J, more preferably 0.8. Mu.J.
In the invention, the specific steps of the surface silanization treatment are as follows: soaking the long-period fiber bragg grating in a hydrochloric acid methanol solution, a sodium hydroxide solution and a 3-aminopropyl trimethoxy silane acetonitrile solution in sequence; the volume ratio of hydrochloric acid to methanol in the hydrochloric acid methanol solution is preferably 0.5-1.5: 0.5 to 1.5, more preferably 0.6 to 1:0.8 to 1; the mass fraction of hydrochloric acid is preferably 35 to 38%, more preferably 36 to 37%; the mass fraction of the methanol is 99.6-99.9%, and more preferably 99.8%; the concentration of the sodium hydroxide solution is preferably 0.5 to 1.2mol/L, more preferably 0.6 to 1.1mol/L; the volume concentration of the 3-aminopropyl trimethoxysilane acetonitrile solution is preferably 1.5 to 2.5%, and more preferably 1.6 to 2.2%.
In the present invention, the soaking temperature is preferably 15 to 35 ℃, and more preferably 20 to 30 ℃; the soaking time in the hydrochloric acid methanol solution is preferably 1 to 5 hours, more preferably 2 to 4 hours; the soaking time in the sodium hydroxide solution is preferably 5 to 12 hours, more preferably 8 to 10 hours; the time for immersing in the acetonitrile solution of 3-aminopropyl trimethoxysilane is preferably 12 to 20 hours, more preferably 14 to 18 hours.
In the invention, the reagent used for modifying the polydopamine is a dopamine hydrochloride solution; the dopamine hydrochloride solution comprises dopamine hydrochloride and Tris-HCl buffer solution, and the mass volume ratio of the dopamine hydrochloride to the Tris-HCl buffer solution is preferably 8-12 mg:0.8 to 1.2mL, more preferably 9 to 10mg: 0.9-1 mL; the concentration of Tris-HCl buffer is preferably 8 to 12mmol/L, more preferably 9 to 10mmol/L; the pH is preferably 7.5 to 8.5, more preferably 7.8 to 8; the temperature of the modified polydopamine is preferably 15-35 ℃, and more preferably 20-30 ℃; the time for modifying the polydopamine is preferably 1 to 2 hours, more preferably 1.5 hours.
In the invention, the dopamine hydrochloride solution is obtained by dissolving dopamine hydrochloride in Tris-HCl buffer solution; the specific steps of the modified polydopamine are as follows: and (3) coating the surface of the long-period fiber grating subjected to surface silanization with dopamine hydrochloride solution, and coating the dopamine hydrochloride solution on the surface of the long-period fiber grating subjected to surface silanization.
In the invention, before the polydopamine is modified, the long-period fiber bragg grating subjected to surface silanization treatment is washed by water and then dried; the number of times of washing is preferably 2 to 4 times, more preferably 3 times; the drying temperature is preferably 20 to 40 ℃, more preferably 25 to 35 ℃.
In the present invention, the concentration of the phage solution is preferably 10 4 ～10 7 CFU/mL, more preferably 10 5 ～10 6 CFU/mL; the temperature of the modified phage solution is preferably 15-35 ℃, and more preferably 20-30 ℃; the time for modifying the phage solution is preferably 20 to 50 minutes, more preferably 30 to 40 minutes.
In the present invention, the specific steps for modifying phage solution are: coating phage solution on the surface of the long-period fiber bragg grating modified with polydopamine, and coating the phage solution on the surface of the long-period fiber bragg grating fully covered with polydopamine; during the modification of the phage solution, the phage solution is preferably repeatedly applied 3 to 5 times, and more preferably, the phage solution is repeatedly applied 4 times.
In the invention, before the phage solution is modified, the long-period fiber bragg grating of the modified polydopamine is washed by water and then dried; the number of times of washing is preferably 2 to 4 times, more preferably 3 times; the drying temperature is preferably 20 to 40 ℃, more preferably 25 to 35 ℃.
In the present invention, the blocking is preferably performed in a bovine serum albumin solution, and the mass concentration of the bovine serum albumin solution is preferably 3 to 7%, and more preferably 4 to 5%; the sealing temperature is preferably 15-25 ℃, and more preferably 18-20 ℃; the blocking time is preferably 5 to 12 hours, more preferably 6 to 10 hours.
In the invention, after the end of the sealing, the obtained product is washed and dried in sequence; the reagent used for washing is preferably PBS buffer solution, and the pH value of the PBS buffer solution is preferably 7.2-7.4, and more preferably 7.4; the number of times of washing is preferably 2 to 4 times, more preferably 3 times; the drying temperature is preferably 20 to 40 ℃, more preferably 25 to 35 ℃.
In the invention, the preparation of the PBS buffer solution comprises the following steps: weigh 8g NaCl, 0.2g KCl, 1.44g Na 2 HPO 4 、0.24gKH 2 PO 4 Dissolving in 800mL of distilled water, regulating the solution to 7.2-7.4 by using HCl, and finally adding distilled water to a volume of 1L to obtain the PBS buffer solution with the concentration of 0.01M.
In the present invention, the polydimethylsiloxane-based template preferably includes 4 independent units, each unit including a sample introduction region, a microfluidic channel, a reaction region, and a test region; the sample injection area is sequentially communicated with the reaction area and the test area through the microfluidic channel; the sample injection area and the test area are hollow, and the microfluidic channel and the reaction area are hollow and sealed.
In the invention, the preparation of the polydimethylsiloxane-based template comprises the following steps:
(1) Preparing a chip die: adopting AI drawing, leading the designed graph into jdprint software, setting the carving path and depth, and carving by a carving machine to obtain a chip mould;
(2) And (3) carrying out hydrophobic treatment on a chip die: placing the chip die in octadecyl trichlorosilane toluene solution with the mass concentration of 1-4% for hydrophobic treatment for 20-30 min to obtain the hydrophobic treated chip die;
(3) Preparing a polydimethylsiloxane template: the mass ratio is 10: pouring the mixed solution of 0.5-1.5 of polydimethylsiloxane and curing agent into a chip die subjected to hydrophobic treatment, curing, and taking out the polydimethylsiloxane template.
In the step (2) of the present invention, the mass concentration of the octadecyltrichlorosilane toluene solution is preferably 1.5 to 3.5%, and more preferably 2 to 3%; the time for the hydrophobic treatment is preferably 22 to 28 minutes, more preferably 24 to 26 minutes.
In the step (3), before the polydimethylsiloxane-based template is prepared, the chip die subjected to hydrophobic treatment is washed by toluene and then dried; the number of times of washing is preferably 2 to 4 times, more preferably 3 times; the drying is preferably carried out at room temperature.
In step (3) of the present invention, the curing agent is preferably poly (dimethylmethylhydrosiloxane); in the mixed solution, the mass ratio of the polydimethylsiloxane to the curing agent is preferably 10:0.7 to 1.3, more preferably 10:0.9 to 1.1; the specific steps of the curing treatment are as follows: firstly, standing a chip mould which is filled with the mixed solution and subjected to hydrophobic treatment, and then drying at 55-65 ℃; the temperature of the standing is preferably 15 to 35 ℃, more preferably 20 to 30 ℃, and the time of the standing is preferably 3 to 4 hours, more preferably 3.5 hours; the drying temperature is preferably 56 to 64℃and more preferably 58 to 62 ℃.
In the invention, the preparation of the chip comprises the following steps: and (3) plasma treating the polydimethylsiloxane template and the glass plate, and then bonding the polydimethylsiloxane template and the glass plate at 75-85 ℃ for 1-3 hours to obtain the chip.
In the present invention, the temperature of the plasma treatment is preferably 15 to 35 ℃, and more preferably 20 to 30 ℃; the plasma treatment time is preferably 0.5 to 1.5min, more preferably 1min; the bonding temperature is preferably 76-84 ℃, and more preferably 78-82 ℃; the bonding time is preferably 1.5 to 2.5 hours, more preferably 2 hours.
In the present invention, the surface of the glass plate facing upward during the plasma treatment is bonded to the polydimethylsiloxane-based template during the bonding.
In the invention, the sensing area of the long-period fiber bragg grating biosensor is clamped into a testing area in a chip.
The invention also provides the long-period fiber grating biosensor coupling microfluidic chip prepared by the preparation method of the long-period fiber grating biosensor coupling microfluidic chip.
According to the coupling microfluidic chip of the long-period fiber grating biosensor, the virulent strain and the antibiotic drug are subjected to micro-culture in the microfluidic chip, and the virulent strain is specifically captured by using the phage on the surface of the long-period fiber grating biosensor, so that the shift of resonance wavelength is shown. If the virulent strain is sensitive to the antibiotic drug, the virulent strain dies or inhibits propagation in the culture process, compared with the virulent strain without the antibiotic drug, the resonant wavelength offset is obviously different, and the sensitivity degree of the pathogenic strain to the antibiotic drug can be judged according to the different concentrations of the antibiotic drug; if the virulent strain has drug resistance to the antibiotic drug, the virulent strain can not inhibit propagation or inhibit propagation of the high-concentration antibiotic drug because of the drug in the culture process, and compared with the virulent strain without the antibiotic drug, the virulent strain has almost the same resonance wavelength offset or has different resonance wavelength offset because of the high-concentration antibiotic drug.
The invention also provides application of the long-period fiber grating biosensor coupling microfluidic chip in preparing a virulent strain drug sensitivity test product.
In the present invention, the virulent strain is preferably a staphylococcus aureus strain.
In the invention, the method for testing the drug sensitivity of the virulent strain comprises the following steps:
(1) Preparing a solution of virulent strain with a concentration of 10 3 ～10 5 CFU/mL, preparing a sterile physiological saline with mass concentration of 0.85-1.0%; preparing four antibiotic drug solutions with the antibiotic drug concentration of 0 mug/mL, 0.001-0.01 mug/mL, 0.05-0.1 mug/mL and 1-3 mug/mL respectively, and preparing a MH liquid broth culture medium as a solvent;
(2) In 4 independent units of each sterilized long-period fiber bragg grating biosensor coupling microfluidic chip, 50-150 mu L of antibiotic drug solutions with different concentrations are respectively injected by an injector, the solution enters the reaction area, 50-150 mu L of virulent strain solution is injected by the injector, and the solution enters the reaction area, and then the solution is cultured;
(3) Immersing the sensing region of the long-period fiber grating biosensor in PBS buffer solution with pH of 7.2-7.4, recording the transmission spectrum positions, and recording as the initial signal position (lambda) 0-i )；
(4) After the chip culture in the step (2) is completed, clamping the sensing area of the long-period fiber bragg grating biosensor dried in the step (3) into the position of a testing area of the microfluidic chip, clamping each testing area into the sensing area of one long-period fiber bragg grating biosensor, enabling liquid in the reaction area to enter the testing area of the chip in a pressing mode, and testing the sensing area of the long-period fiber bragg grating biosensor;
(5) After the test is finished, the long-period fiber grating biosensor in the step (4) is separated from the microfluidic chip, the sensing area is washed by PBS buffer solution with pH of 7.2-7.4, then the sensing area is immersed in the PBS buffer solution with pH of 7.2-7.4, and the transmission spectrum position is recorded and recorded as the target object signal position (lambda) i-i ) Lambda is taken as i-i And lambda is 0-i Corresponding to the subtraction, the resonance wavelength offset (Deltalambda) is obtained i-i )；
(6) Immersing the sensing area of the long-period fiber grating biosensor in the step (5) in NH with pH of 10-11 3 ·H 2 Washing the sensing area with PBS buffer solution with pH of 7.2-7.4 after soaking in O solution, and drying;
(7) Repeating the steps (3), (4), (5) and (6), and testing and recording data in each microfluidic chip testing area in sequence. And judging the drug sensitivity of the virulence strain to each antibiotic drug according to the obtained resonance wavelength offset.
In the step (2) of the present invention, the temperature of the culture is preferably 37℃and the time of the culture is preferably 2 to 4 hours, more preferably 3 hours.
In the step (4) of the present invention, the test time is preferably 10 to 15 minutes, more preferably 11 to 14 minutes.
In the step (5) of the present invention, the number of times of flushing is preferably 3 to 5 times, more preferably 4 times; the volume of PBS buffer used in washing is preferably 5 to 10mL, more preferably 7 to 9mL.
In the step (6) of the present invention, NH 3 ·H 2 The volume of the O solution is preferably 5 to 10mL, more preferably 6 to 9mL; immersed in NH 3 ·H 2 The time in the O solution is preferably 5 to 10 minutes, more preferably 6 to 9 minutes; the volume of the PBS buffer is preferably 5 to 10mL, more preferably 6 to 8mL; the number of times of flushing is preferably 3 to 5 times, more preferably 4 times; the drying temperature is preferably 20 to 40 ℃, more preferably 25 to 35 ℃.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method of the long-period fiber grating biosensor comprises the following steps:
(1) The method comprises the steps of (1) taking a single-mode fiber as a raw material, and obtaining a long-period fiber bragg grating after burning by adopting a femtosecond laser direct writing technology; during recording, the repetition frequency of the femtosecond laser pulse is 1kHz, and the energy is 0.8 mu J; the period of the long period fiber grating is 360 μm, the total grating length is 18mm, the grating diameter is 114 μm, and the loss peak intensity is 30dB. At 25 ℃, the volume ratio of the long-period fiber grating to hydrochloric acid to methanol is 1:1 (the mass fraction of hydrochloric acid is 37 percent, the mass fraction of methanol is 99.8 percent), 1mol/L sodium hydroxide solution and 3-aminopropyl trimethoxysilane acetonitrile solution with the volume concentration of 2 percent for 2 hours, 10 hours and 12 hours; washing 3 times with water after soaking, and drying at 25 ℃ to obtain the long-period fiber bragg grating subjected to surface silanization treatment;
(2) 10mg of dopamine hydrochloride is dissolved in 1mL of 10mmol/L Tris-HCl (pH=8) buffer solution to obtain dopamine hydrochloride solution; coating the solution on a long-period fiber grating subjected to surface silanization treatment, after reacting for 1h, washing with water for 3 times, and drying at 25 ℃ to obtain a long-period fiber grating of modified polydopamine;
(3) Coating the surface of the long-period fiber grating of the modified polydopamine with the concentration of 10 5 CFU/mL of Staphylococcus aureus phage solution inIncubating for 30min at 20 ℃ to obtain a long-period fiber grating of the modified phage; repeated coating concentration during incubation was 10 5 4 times of CFU/mL staphylococcus aureus strain phage solution, wherein the interval time is the same every two times;
(4) The long-period fiber grating of the modified phage was incubated with a 5% strength by mass solution of bovine serum albumin at 20℃for 6h. And (3) cleaning the fiber grating biosensor for 3 times by using PBS buffer solution with pH value of 7.4, and drying the fiber grating biosensor at 25 ℃ to obtain the long-period fiber grating biosensor.
The preparation method of the chip comprises the following steps:
(1) And (3) setting a chip die by adopting AI drawing, importing the designed graph into jdprint software, setting the carving path and depth of the graph, and carving by using a carving machine to obtain the chip die. Performing hydrophobic treatment on the chip mold by using an analytical pure octadecyl trichlorosilane toluene solution with the mass concentration of 3% for 25min, washing the chip mold for 3 times by using the analytical pure toluene, and airing the chip mold at room temperature to obtain a chip mold subjected to the hydrophobic treatment;
(2) The polydimethyl siloxane and the poly (dimethyl methyl hydrogen siloxane) are fully mixed and uniformly stirred, wherein the mass ratio of the polydimethyl siloxane to the poly (dimethyl methyl hydrogen siloxane) is 10:1.2, pouring the mixed solution into a chip die subjected to hydrophobic treatment, vacuumizing to remove bubbles, standing for 3.5 hours at room temperature, and then putting the mixed solution into a 55 ℃ oven for complete solidification to obtain a polydimethylsiloxane-based template;
(3) And placing the polydimethylsiloxane-based template and the glass sheet into a plasma cleaner for plasma treatment at 15 ℃ for 1.5min (the surface to be bonded of the glass sheet is upward), and then bonding the two in a 75 ℃ oven for 2.5h to obtain the chip.
The preparation method of the long-period fiber grating biosensor coupling micro-fluidic chip comprises the following steps:
and clamping the sensing area of the prepared long-period fiber bragg grating biosensor into the position of the testing area of the prepared microfluidic chip to obtain the coupling microfluidic chip of the long-period fiber bragg grating biosensor.
Example 2
The preparation method of the long-period fiber grating biosensor comprises the following steps:
(1) The method comprises the steps of (1) taking a single-mode fiber as a raw material, and obtaining a long-period fiber bragg grating after burning by adopting a femtosecond laser direct writing technology; during recording, the repetition frequency of the femtosecond laser pulse is 1.2kHz, and the energy is 0.7 mu J; the period of the long period fiber grating is 350 μm, the total grating length is 16mm, the grating diameter is 115 μm, and the loss peak intensity is 32dB. At 28 ℃, the volume ratio of the long-period fiber bragg grating to hydrochloric acid to methanol is 0.5:1 (the mass fraction of hydrochloric acid is 37 percent, the mass fraction of methanol is 99.7 percent), 0.9mol/L sodium hydroxide solution and 3-aminopropyl trimethoxysilane acetonitrile solution with the volume concentration of 2.2 percent for 3 hours, 10 hours and 15 hours; washing 3 times with water after soaking, and drying at 25 ℃ to obtain the long-period fiber bragg grating subjected to surface silanization treatment;
(2) 11mg of dopamine hydrochloride is dissolved in 1.2mL of 9mmol/L Tris-HCl (pH=7.8) buffer solution to obtain dopamine hydrochloride solution; coating the solution on a long-period fiber grating subjected to surface silanization treatment, after reacting for 1.5 hours, washing with water for 3 times, and drying at 25 ℃ to obtain a long-period fiber grating modified with polydopamine;
(3) Coating the surface of the long-period fiber grating of the modified polydopamine with the concentration of 10 6 Incubating the CFU/mL staphylococcus aureus strain phage solution at 25 ℃ for 25min to obtain a long-period fiber grating of the modified phage; repeated coating concentration during incubation was 10 6 CFU/mL staphylococcus aureus strain phage solution is 5 times, and the interval time is the same every two times;
(4) The long-period fiber grating of the modified phage was incubated with a 4% strength by mass solution of bovine serum albumin at 25℃for 8h. And (3) cleaning the fiber grating biosensor for 3 times by using PBS buffer solution with the pH value of 7.3, and drying the fiber grating biosensor at 25 ℃ to obtain the long-period fiber grating biosensor.
The preparation method of the chip comprises the following steps:
(1) And (3) setting a chip die by adopting AI drawing, importing the designed graph into jdprint software, setting the carving path and depth of the graph, and carving by using a carving machine to obtain the chip die. Performing hydrophobic treatment on the chip mold by using an analytically pure octadecyl trichlorosilane toluene solution with the mass concentration of 3.5% for 25min, washing the chip mold for 3 times by using analytically pure toluene, and airing the chip mold at room temperature to obtain a chip mold subjected to hydrophobic treatment;
(2) The polydimethyl siloxane and the poly (dimethyl methyl hydrogen siloxane) are fully mixed and uniformly stirred, wherein the mass ratio of the polydimethyl siloxane to the poly (dimethyl methyl hydrogen siloxane) is 10:1, pouring the mixed solution into a chip die subjected to hydrophobic treatment, vacuumizing to remove bubbles, standing for 3 hours at room temperature, and then putting the mixed solution into a 58 ℃ oven for complete solidification to obtain a polydimethylsiloxane-based template;
(3) And (3) putting the polydimethylsiloxane-based template and the glass sheet into a plasma cleaner, performing plasma treatment for 1min at 20 ℃ (the surface to be bonded of the glass sheet is upward), and then bonding the two in an oven at 80 ℃ for 2h to obtain the chip.
The preparation method of the long-period fiber grating biosensor coupling micro-fluidic chip comprises the following steps:
and clamping the sensing area of the prepared long-period fiber bragg grating biosensor into the position of the testing area of the prepared microfluidic chip to obtain the coupling microfluidic chip of the long-period fiber bragg grating biosensor.
Example 3
The preparation method of the long-period fiber grating biosensor comprises the following steps:
(1) The method comprises the steps of (1) taking a single-mode fiber as a raw material, and obtaining a long-period fiber bragg grating after burning by adopting a femtosecond laser direct writing technology; during recording, the repetition frequency of the femtosecond laser pulse is 1.2kHz, and the energy is 0.6 mu J; the period was 350 μm, the total grating length was 16mm, the grating diameter was 110 μm, and the loss peak intensity was 25dB. At 25 ℃, the volume ratio of the long-period fiber grating to hydrochloric acid to methanol is 0.5:1.2 (hydrochloric acid mass fraction of 38%, methanol mass fraction of 99.8%), 0.8mol/L sodium hydroxide solution and 1.8% 3-aminopropyl trimethoxysilane acetonitrile solution for 3h, 9h and 15h; washing 3 times with water after soaking, and drying at 25 ℃ to obtain the long-period fiber bragg grating subjected to surface silanization treatment;
(2) 11mg of dopamine hydrochloride is dissolved in 0.9mL of 12mmol/L Tris-HCl (pH=8) buffer solution to obtain dopamine hydrochloride solution; coating the solution on a long-period fiber grating subjected to surface silanization treatment, after reacting for 2 hours, washing with water for 3 times, and drying at 25 ℃ to obtain a long-period fiber grating of modified polydopamine;
(3) Coating the surface of the long-period fiber grating of the modified polydopamine with the concentration of 10 4 Incubating the CFU/mL staphylococcus aureus strain phage solution at 30 ℃ for 45min to obtain a long-period fiber grating of the modified phage; repeated coating concentration during incubation was 10 4 CFU/mL staphylococcus aureus strain phage solution 3 times, and the interval time is the same every two times;
(4) The long-period fiber grating of the modified phage was incubated with a 3% strength by mass solution of bovine serum albumin for 10h at 25 ℃. And (3) cleaning the fiber grating biosensor for 3 times by using PBS buffer solution with the pH value of 7.3, and drying the fiber grating biosensor at 25 ℃ to obtain the long-period fiber grating biosensor.
The preparation method of the chip comprises the following steps:
(1) And (3) setting a chip die by adopting AI drawing, importing the designed graph into jdprint software, setting the carving path and depth of the graph, and carving by using a carving machine to obtain the chip die. Performing hydrophobic treatment on the chip mold by using an analytically pure octadecyl trichlorosilane toluene solution with the mass concentration of 2% for 28min, washing the chip mold for 3 times by using analytically pure toluene, and airing the chip mold at room temperature to obtain a chip mold subjected to hydrophobic treatment;
(2) The polydimethyl siloxane and the poly (dimethyl methyl hydrogen siloxane) are fully mixed and uniformly stirred, wherein the mass ratio of the polydimethyl siloxane to the poly (dimethyl methyl hydrogen siloxane) is 10: and 0.9, pouring the mixed solution into a chip die subjected to hydrophobic treatment, vacuumizing to remove bubbles, standing for 4 hours at room temperature, and then putting the mixed solution into a 62 ℃ oven for complete solidification to obtain the polydimethylsiloxane-based template.
(3) And (3) putting the polydimethylsiloxane-based template and the glass sheet into a plasma cleaner, performing plasma treatment for 0.5min at 25 ℃ (the surface to be bonded of the glass sheet is upward), and then bonding the two in an oven at 82 ℃ for 2h to obtain the chip.
The preparation method of the long-period fiber grating biosensor coupling micro-fluidic chip comprises the following steps:
and clamping the sensing area of the prepared long-period fiber bragg grating biosensor into the position of the testing area of the prepared microfluidic chip to obtain the coupling microfluidic chip of the long-period fiber bragg grating biosensor.
Application example 1
The method for testing the drug sensitivity of staphylococcus aureus strains to ampicillin and cefoxitin by adopting the long-period fiber grating biosensor coupling micro-fluidic chip prepared in the embodiment 1 comprises the following steps:
(1) Preparing staphylococcus aureus strain solution with concentration of 10 4 CFU/mL, preparing a sterile physiological saline with mass concentration of 0.85%; respectively preparing ampicillin solution and cefoxitin solution, wherein the prepared solvent is MH liquid broth culture medium, and the ampicillin solution and the cefoxitin solution are respectively prepared into four different concentrations, namely 0 mug/mL, 0.005 mug/mL, 0.08 mug/mL and 2.5 mug/mL;
(2) And (3) injecting 100 mu L of two antibiotic medicine solutions with different concentrations into each of 4 independent units of the two sterilized chips by using an injector, entering the reaction area position, injecting 100 mu L of staphylococcus aureus strain solution by using the injector, entering the reaction area position, and culturing for 3 hours at 37 ℃.
(3) Immersing the sensing areas of the prepared long-period fiber grating biosensors (4) in PBS buffer solution with pH value of 7.3, and recording the transmission spectrum positions respectively, which are recorded as initial signal positions (lambda) 0-i )；
(4) After the chip culture in the step (2) is completed, clamping the sensing area of the long-period fiber bragg grating biosensor dried in the step (3) into the position of a testing area of a microfluidic chip cultured by ampicillin, clamping each testing area into one long-period fiber bragg grating biosensor, enabling liquid in the reaction area to enter the chip testing area in a pressing manner, and testing for 10 minutes without the long-period fiber bragg grating biosensor;
(5) After the test is finished, the long-period fiber grating biosensor in the step (4) is processed by the following stepsSeparating in microfluidic chip, washing sensing region with 5ml PBS buffer solution with pH value of 7.3 for 5 times, soaking sensing region in PBS buffer solution, recording transmission spectrum position, and recording as target signal position (lambda) i-i ) Lambda is taken as i-i And lambda is 0-i Corresponding to the subtraction, the resonance wavelength offset (Deltalambda) is obtained i-i ). Namely, the wavelength offset corresponding to ampicillin with different concentrations is delta lambda respectively 1-1 、Δλ 1-2 、Δλ 1-3 、Δλ 1-4 The method comprises the steps of carrying out a first treatment on the surface of the The wavelength offset of the cefoxitin Ding Duiying with different concentrations is delta lambda respectively 2-1 、Δλ 2-2 、Δλ 2-3 、Δλ 2-4 ；
(6) Immersing the sensing area of the long-period fiber grating biosensor in the step (5) in NH with pH of 11 at 5mLpH 3 ·H 2 Washing the sensing area with 5ml PBS buffer solution with pH value of 7.3 for 5min in O solution, and drying at 30deg.C;
(7) Repeating the steps (3), (4), (5) and (6), testing and recording data in a microfluidic chip test area cultivated by cefoxitin, wherein the corresponding wavelength offset of the two antibiotics is shown in figure 3.
As can be seen from fig. 3, the staphylococcus aureus strain was resistant to the antibiotic ampicillin and susceptible to the antibiotic cefoxitin. The comparison of the traditional K-B test paper method and the drug sensitivity anastomosis prove that the method has high accuracy in drug sensitivity test of the virulent strain. The invention provides a long-period fiber grating biosensor coupling microfluidic chip, a preparation method and application thereof, wherein the long-period fiber grating biosensor coupling microfluidic chip can be used for drug sensitivity test of a virulent strain, and has the advantages of high accuracy, simplicity and convenience in operation and strong specificity.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the long-period fiber grating biosensor coupling micro-fluidic chip is characterized by comprising the following steps of:
sequentially carrying out surface silanization treatment, modification of polydopamine and modification of phage solution on the long-period fiber grating, and then sealing to obtain a long-period fiber grating biosensor; clamping the long-period fiber bragg grating biosensor into a chip to obtain a long-period fiber bragg grating biosensor coupling micro-fluidic chip;
the chip comprises a glass plate and a polydimethylsiloxane template;
the grating period of the long period fiber grating is 320-400 mu m, the total grating length is 15-20 mm, the grating diameter is 100-120 mu m, and the loss peak intensity is 25-35 dB.
2. The method for preparing the long-period fiber grating biosensor coupling micro-fluidic chip according to claim 1, wherein the specific steps of the surface silanization treatment are as follows: soaking the long-period fiber bragg grating in a hydrochloric acid methanol solution, a sodium hydroxide solution and a 3-aminopropyl trimethoxy silane acetonitrile solution in sequence; the volume ratio of hydrochloric acid to methanol in the hydrochloric acid methanol solution is 0.5-1.5: 0.5 to 1.5; the concentration of the sodium hydroxide solution is 0.5-1.2 mol/L; the volume concentration of the 3-aminopropyl trimethoxy silane acetonitrile solution is 1.5-2.5%.
3. The method for preparing the coupling micro-fluidic chip of the long-period fiber bragg grating biosensor according to claim 2, wherein the time for soaking in the hydrochloric acid methanol solution is 1-5 h; soaking in the sodium hydroxide solution for 5-12 h; the soaking time in the 3-aminopropyl trimethoxy silane acetonitrile solution is 12-20 h.
4. The method for preparing the long-period fiber grating biosensor coupling microfluidic chip according to any one of claims 1-3, wherein the reagent used for modifying polydopamine is dopamine hydrochloride solution; the dopamine hydrochloride solution comprises dopamine hydrochloride and Tris-HCl buffer solution, and the mass volume ratio of the dopamine hydrochloride to the Tris-HCl buffer solution is 8-12 mg: 0.8-1.2 mL; the concentration of the Tris-HCl buffer solution is 8-12 mmol/L, and the pH value is 7.5-8.5; the time for modifying the polydopamine is 1-2 h.
5. The method for preparing the coupling microfluidic chip of the long-period fiber grating biosensor according to claim 4, wherein the concentration of the phage solution is 10 4 ～10 7 CFU/mL; the temperature of the modified phage solution is 15-35 ℃, and the time for modifying the phage solution is 20-50 min.
6. The method for preparing the coupling microfluidic chip of the long-period fiber grating biosensor according to claim 1 or 5, wherein the sealing is performed in a bovine serum albumin solution, and the mass concentration of the bovine serum albumin solution is 3-7%; the sealing temperature is 15-25 ℃, and the sealing time is 5-12 h.
7. The method for preparing the long-period fiber grating biosensor coupled microfluidic chip according to claim 6, wherein the polydimethylsiloxane-based template comprises 4 independent units, and each unit comprises a sample injection area, a microfluidic channel, a reaction area and a test area.
8. The method for manufacturing a long-period fiber grating biosensor coupled microfluidic chip according to claim 7, wherein the sensing area of the long-period fiber grating biosensor is clamped into a test area in the chip.
9. The long-period fiber grating biosensor coupling microfluidic chip prepared by the preparation method of the long-period fiber grating biosensor coupling microfluidic chip according to any one of claims 1 to 8.
10. The use of the long-period fiber grating biosensor coupling microfluidic chip according to claim 9 in the preparation of drug sensitivity test products of virulent strains.
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