CN110927131A - Urease sensing device based on whispering gallery mode laser and manufacturing method thereof - Google Patents
Urease sensing device based on whispering gallery mode laser and manufacturing method thereof Download PDFInfo
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Abstract
The invention belongs to the field of optical micro-sensing, and particularly relates to a urease sensing device based on whispering gallery mode laser and a manufacturing method thereof, wherein the urease sensing device greatly improves the detection sensitivity of urease. The invention comprises a 532nm pump pulse laser 1, a spectrum analyzer 2, a charge coupled device CCD3, a functionalized 5CB microdroplet 4 and a reaction solution 5; the functionalized 5CB4 microdroplet is co-doped with pH sensitive molecule stearic acid and fluorescent dye nile red, and is generated in a reaction solution 5 through a capillary microtube 7. The invention has the advantages that: 1. the 5CB microsphere cavity formed by surface tension has extremely high surface smoothness and geometric uniformity, and surface scattering loss is reduced. The excited whispering gallery mode laser has extremely high Q value and ultralow laser threshold, so that the detection sensitivity of urease is greatly improved.
Description
Technical Field
The invention belongs to the field of optical micro-sensing, and particularly relates to a urease sensing device based on whispering gallery mode laser and a manufacturing method thereof, wherein the urease sensing device greatly improves the detection sensitivity of urease.
Background
The detection of urease has great significance for guaranteeing the life health of human beings. The content of urease in the body can be detected to provide important basis for determining gastritis, gastric ulcer and intestinal infection. However, the conventional urease detection methods have various disadvantages such as low sensitivity, complicated operation, or long detection time, and thus research on urease sensors is being continuously conducted. Liquid crystals have been widely used for the detection of biochemical molecules as a sensitive material due to the amplification and transduction of chemical and biomolecular events at the water/liquid phase interface. But existing liquid crystal sensor readout methods are still based on inaccurate naked eye observations under optical microscopy. Under the observation mode, the reaction process to be measured is difficult to be quantitatively and accurately characterized, so that the further development of the liquid crystal sensing device in real-time monitoring is limited. Here we excite whispering gallery mode laser in the functionalized liquid crystal droplets and monitor urease concentration real-time, sensitively and quantitatively by spectral response.
Disclosure of Invention
The invention aims to provide a urease sensing device based on whispering gallery mode laser.
The invention also aims to provide a manufacturing method of the urease sensing device based on the whispering gallery mode laser.
The purpose of the invention is realized as follows:
a urease sensing device based on whispering gallery mode laser comprises a 532nm pump pulse laser 1, a spectrum analyzer 2, a charge coupled device CCD3, functionalized 5CB microdroplets 4 and a reaction solution 5; the functionalized 5CB4 microdroplet is co-doped with pH sensitive molecule stearic acid and fluorescent dye nile red and is generated in a reaction solution 5 through a capillary microtubule 7; the reaction solution 5 contains urease and a substrate urea for the urease, light 11 emitted by a 532nm pump pulse laser sequentially passes through a polarization controller 8 and a semi-reflecting and semi-transparent mirror 9, and is finally focused on the surface of a functionalized 5CB microdroplet 4 by a 10x optical objective 10 and excites dye nile red to generate fluorescence; the working frequency and the working voltage of the 532nm pump pulse laser are controlled by a frequency controller 16 and a voltage stabilizer 17, the controlled working frequency is 6Hz, and the voltage is 220V; whispering gallery mode lasers generated by 5CB microdroplets are collected through a 10x optical objective lens 10 and sequentially pass through a half-reflecting and half-transmitting lens 9 and a beam splitter 12; a beam of light is filtered by a 532nm filter and then transmitted to a spectrum analyzer 2 for spectrum analysis; the other beam of light enters a charge coupled device CCD3 for droplet imaging; the obtained data are all transmitted to the computer 4 for information processing and display.
Functionalized 5CB droplet 4 was co-doped with 0.05 wt.% stearic acid and 0.01 wt.% nile red, functionalized 5CB droplet 4 being produced by controlling the injection rate of micro syringe pump 14, which was controlled to a diameter of 50 μm.
The reaction solution 5 is placed in a reaction vessel 6, and a micro heating platform 18 is arranged at the bottom; the reaction vessel 6 is made of silicon dioxide, the radius of the bottom of the reaction vessel is 2cm, and the height of the reaction vessel is 3 cm.
The micro-syringe pump 14 and the functionalized 5CB microdroplets 4 are precisely position controlled by an X-Y-Z three-dimensional electric adjusting frame 15.
A manufacturing method of a urease sensing device based on whispering gallery mode laser comprises the following steps:
step 1: stearic acid and nile red with the mass fractions of 0.05 percent and 0.01 percent are co-doped in 1mL of nematic liquid crystal 5 CB;
step 2: placing the mixed solution in an ultrasonic machine for ultrasonic treatment for 30 minutes to fully and uniformly mix the mixed solution, wherein the working temperature of the ultrasonic machine is set to be 25 ℃;
and step 3: drawing a quartz capillary tube with the outer diameter of 150 mu m and the inner diameter of 75 mu m into a capillary microtube with the inner diameter of 5 mu m by using a flame tapering method; the other end of the capillary micro-tube is connected with a micro injection pump with controllable injection rate;
and 4, step 4: by controlling the injection rate of a micro syringe pump, functionalized 5CB microdroplets with the diameter of 50 mu m are generated in the reaction solution;
and 5: the temperature of the reaction solution is monitored in real time by a miniature temperature probe, and the temperature is ensured to be constant at 25 ℃ by a feedback adjusting device;
step 6: light emitted by a 532nm pump pulse laser sequentially passes through a polarization controller and a semi-reflecting and semi-transparent mirror, is focused on the surface of a functionalized 5CB microdroplet by a 10x optical objective lens and excites dye nile red to generate fluorescence;
and 7: the reaction solution contains urease and an acting substrate urea thereof, and ammonium ions and hydroxide ions are released by enzymolysis so as to increase the pH value of the solution;
and 8: the whispering gallery mode laser generated by 5CB microdroplets is collected through a 10x optical objective lens and sequentially passes through a half-reflecting and half-transmitting lens and a beam splitter, and one beam of laser is transmitted to a spectrum analyzer after being filtered by a 532nm filter for spectrum analysis; the other beam of light enters a charge coupled device CCD for droplet imaging, and the obtained data are transmitted to a computer for information processing and display.
The invention has the advantages that:
first, the 5CB microsphere shaped cavity formed by surface tension has extremely high surface smoothness and geometry uniformity, reducing surface scattering losses. The excited whispering gallery mode laser has extremely high Q value and ultralow laser threshold, so that the detection sensitivity of urease is greatly improved.
Secondly, the liquid crystal sensing technology based on the whispering gallery mode laser can convert the biochemical reaction process of urease into spectral information in real time, so that the whole detection process can be accurately quantified and monitored in real time. The defect that the traditional liquid crystal sensor can only carry out rough evaluation through optical patterns is completely overcome.
The sensing device of the invention has a complete temperature feedback system, effectively avoids disturbance errors caused by temperature changes of the operating environment, and enables the detection result to be more accurate and reliable.
And the doped functional material stearic acid is sensitive to the change of the external pH value, so that the detection limit of the sensing device on urease can be effectively reduced.
Drawings
FIG. 1 is a schematic diagram of a whispering gallery mode laser based urease sensing system;
FIG. 2 is a schematic view of a functionalized 5CB droplet sensing probe;
FIG. 3 is the results of a urease sensing system based on whispering gallery mode laser testing different concentrations of urease solution.
Detailed Description
The invention will be further described with reference to the accompanying drawings.
The invention relates to a urease main sensing device based on whispering gallery mode laser, which comprises a 532nm pump pulse laser, a spectrum analyzer, a charge coupled device CCD, functionalized 5CB microdroplets and a reaction solution. The functionalized 5CB microdroplets are doped with stearic acid and fluorescent dye nile red and are generated in the reaction solution through capillary microtubes. The reaction solution contains urease and its acting substrate urea. Light emitted by a 532nm pump pulse laser sequentially passes through a polarization controller and a semi-reflecting and semi-transparent mirror, is focused on the surface of a functionalized 5CB microdroplet by a 10x optical objective lens and excites dye nile red to generate fluorescence. Because the effective refractive index of the droplets is higher than that of the reaction solution, the generated fluorescence is continuously and totally reflected at the interface of the droplets and the reaction solution, and finally forms whispering gallery mode laser. The working frequency and the working voltage of the 532nm pump pulse laser are controlled by a frequency controller and a voltage stabilizer. The controlled working frequency is 6Hz, and the voltage is 220V; whispering gallery mode lasers generated by 5CB droplets are collected through a 10x optical objective lens and sequentially pass through a half-reflecting and half-transmitting lens and a beam splitter. A beam of light is filtered by a 532nm filter and then transmitted to a spectrum analyzer for spectrum analysis; the other beam of light enters a Charge Coupled Device (CCD) for droplet imaging. The obtained data are all transmitted to a computer for information processing and display.
The dye-doped liquid crystal microsphere temperature sensor of the invention may further comprise:
1. the functionalized 5CB microdroplets were doped with 0.05 wt.% stearic acid and 0.01 wt.% nile red.
2. The reaction solution is placed in a reaction vessel, and a micro heating table is arranged at the bottom of the reaction vessel.
(1) The reaction solution is led into the reaction solution through a capillary micro-tube connected with a micro injection pump;
(2) the reaction vessel is made of silicon dioxide, the radius of the bottom of the reaction vessel is 2cm, and the height of the reaction vessel is 3 cm;
(3) the temperature change of the micro heating platform is adjusted by a temperature controller.
3. The temperature of the reaction solution is monitored in real time by a miniature temperature probe and fed back to a control computer.
4. The functionalized 5CB microdroplets are generated by controlling the injection rate of a micro injection pump, and the diameter of the microdroplets is controlled to be about 50 mu m;
5. the micro injection pump and the functionalized 5CB microdroplet are precisely controlled in position by an X-Y-Z three-dimensional electric adjusting frame.
The invention relates to a manufacturing method of a urease sensing device based on whispering gallery mode laser, which comprises the following steps:
(1) stearic acid and nile red with the mass fractions of 0.05 percent and 0.01 percent are co-doped in 1mL of nematic liquid crystal 5 CB;
(2) placing the mixed solution in an ultrasonic machine for ultrasonic treatment for 30 minutes to fully and uniformly mix the mixed solution, wherein the working temperature of the ultrasonic machine is set to be 25 ℃;
(3) a quartz capillary having an outer diameter of 150 μm and an inner diameter of 75 μm was drawn into a capillary microtube having an inner diameter of 5 μm by a flame tapering method. The other end of the capillary micro-tube is connected with a micro injection pump with controllable injection rate;
(4) by controlling the injection rate of a micro syringe pump, functionalized 5CB microdroplets with the diameter of 50 mu m are generated in the reaction solution;
(5) the temperature of the reaction solution is monitored in real time by a miniature temperature probe, and the temperature is ensured to be constant at 25 ℃ by a feedback adjusting device;
(6) light emitted by a 532nm pump pulse laser sequentially passes through a polarization controller and a semi-reflecting and semi-transparent mirror, is focused on the surface of a functionalized 5CB microdroplet by a 10x optical objective lens and excites dye nile red to generate fluorescence. Because the effective refractive index of the microdroplet is higher than that of the reaction solution, the generated fluorescence can be subjected to continuous total reflection at the interface of the microdroplet and the reaction solution, and finally forms whispering gallery mode laser;
(7) the reaction solution contains urease and urea as acting substrate, and the enzymolysis releases ammonium ion and hydroxyl ion to raise the pH value of the solution. Due to the pH sensitivity of stearic acid, the deprotonation behavior of stearic acid caused by an increase in solution pH induces a change in the alignment state of the 5CB molecule. This change is sensitively captured by the whispering gallery mode laser excited in the droplet and dynamically characterized in real time in a spectrally shifted manner.
(8) Whispering gallery mode lasers generated by 5CB droplets are collected through a 10x optical objective lens and sequentially pass through a half-reflecting and half-transmitting lens and a beam splitter. A beam of light is filtered by a 532nm filter and then transmitted to a spectrum analyzer for spectrum analysis; the other beam of light enters a Charge Coupled Device (CCD) for droplet imaging. The obtained data are all transmitted to a computer for information processing and display.
As shown in figures 1 and 2, the urease sensing device based on whispering gallery mode laser mainly comprises a 532nm pump pulse laser (1), a spectrum analyzer (2), a charge coupled device CCD (3), functionalized 5CB microdroplets (4) and a reaction solution (5).
The functionalized 5CB microdroplets are doped with stearic acid and fluorescent dye Nile Red, and are generated in a reaction solution (5) through capillary microtubes (7). The reaction solution contains urease and its acting substrate urea
Light (11) emitted by a 532nm pump pulse laser sequentially passes through a polarization controller (8) and a semi-reflecting and semi-transparent mirror (9), and is finally focused on the surface of a functionalized 5CB microdroplet (4) by a 10X optical objective (10) and excites dye nile red to generate fluorescence. Because the effective refractive index of the droplets is higher than that of the reaction solution (5), the generated fluorescence is continuously and totally reflected at the interface of the droplets and the reaction solution, and finally forms whispering gallery mode laser. The working frequency and the working voltage of the 532nm pump pulse laser are controlled by a frequency controller (16) and a voltage stabilizer (17). The controlled working frequency is 6Hz, and the voltage is 220V;
whispering gallery mode lasers generated by 5CB droplets are collected through a 10x optical objective (10) and sequentially pass through a half-reflecting and half-transmitting mirror (9) and a beam splitter (12). One beam of light is filtered by a 532nm filter and then transmitted to a spectrum analyzer (2) for spectrum analysis; the other beam of light enters a charge coupled device CCD (3) for droplet imaging. The obtained data are all transmitted to a computer (4) for information processing and display.
The reaction solution (5) is contained in a reaction vessel (6) and a micro heating stage (18) is provided at the bottom. The reaction solution (5) is introduced into the reaction solution through a capillary micro-tube connected with a micro injection pump; the reaction vessel (6) is made of silicon dioxide, the radius of the bottom of the reaction vessel is 2cm, and the height of the reaction vessel is 3 cm. The temperature change of the micro heating platform (18) is adjusted by a temperature controller (20). The micro temperature probe (21) monitors the temperature of the reaction solution (5) in real time and feeds back the temperature to the control computer (4).
The functionalized 5CB microdroplets were doped with 0.05 wt.% stearic acid and 0.01 wt.% nile red. The functionalized 5CB microdroplets are generated by controlling the injection rate of a micro injection pump (14), and the diameter of the microdroplets is controlled to be about 50 mu m; the micro injection pump (14) and the functionalized 5CB microdroplets are precisely controlled in position through an X-Y-Z three-dimensional electric adjusting frame (15).
The manufacturing method of the urease sensing device based on the whispering gallery mode laser comprises the following steps:
(1) stearic acid and nile red with the mass fractions of 0.05 percent and 0.01 percent are co-doped in 1mL of nematic liquid crystal 5 CB;
(2) placing the mixed solution in an ultrasonic machine for ultrasonic treatment for 30 minutes to fully and uniformly mix the mixed solution, wherein the working temperature of the ultrasonic machine is set to be 25 ℃;
(3) a quartz capillary having an outer diameter of 150 μm and an inner diameter of 75 μm was drawn into a capillary microtube having an inner diameter of 5 μm by a flame tapering method. The other end of the capillary micro-tube is connected with a micro injection pump with controllable injection rate;
(4) by controlling the injection rate of a micro syringe pump, functionalized 5CB microdroplets with the diameter of 50 mu m are generated in the reaction solution;
(5) the temperature of the reaction solution is monitored in real time by a miniature temperature probe, and the temperature is ensured to be constant at 25 ℃ by a feedback adjusting device;
(6) light emitted by a 532nm pump pulse laser sequentially passes through a polarization controller and a semi-reflecting and semi-transparent mirror, is focused on the surface of a functionalized 5CB microdroplet by a 10x optical objective lens and excites dye nile red to generate fluorescence. Because the effective refractive index of the microdroplet is higher than that of the reaction solution, the generated fluorescence can be subjected to continuous total reflection at the interface of the microdroplet and the reaction solution, and finally forms whispering gallery mode laser;
(7) the reaction solution contains urease and urea as acting substrate, and the enzymolysis releases ammonium ion and hydroxyl ion to raise the pH value of the solution. Due to the pH sensitivity of stearic acid, the deprotonation behavior of stearic acid caused by an increase in solution pH induces a change in the alignment state of the 5CB molecule. This change is sensitively captured by the whispering gallery mode laser excited in the droplet and dynamically characterized in real time in a spectrally shifted manner.
(8) Whispering gallery mode lasers generated by 5CB droplets are collected through a 10x optical objective lens and sequentially pass through a half-reflecting and half-transmitting lens and a beam splitter. A beam of light is filtered by a 532nm filter and then transmitted to a spectrum analyzer for spectrum analysis; the other beam of light enters a Charge Coupled Device (CCD) for droplet imaging. The obtained data are all transmitted to a computer for information processing and display.
The tuning principle of the all-optical tuning echo wall mode micro laser is as follows:
Δ λ is a wavelength change value, λ0For emitting the wavelength of the laser, n0In order to obtain the liquid crystal refractive index, Δ n is the liquid crystal refractive index change value, Γ is the action factor, and generally Γ is much smaller than 1. Wherein λ0，n0And Γ is a constant. It is apparent that Δ λ is consistent with Δ n in sign, increasing or decreasing.
When only urea is present in the reaction solution, the pH of the solution does not change, and the 5CB molecules in the droplets are arranged parallel to the droplet surface. The effective refractive index of the liquid crystal molecules perceived by the whispering gallery mode at this time is 1.71. When urease exists in the reaction solution to be detected, the urease hydrolyzes urea to release hydroxide ions and ammonium ions, so that the pH value of the solution is increased. The pH change causes deprotonation behavior of stearic acid, thereby inducing a change in the arrangement of the 5CB molecules from parallel to perpendicular surfaces. The effective refractive index of the liquid crystal molecules perceived by the whispering gallery modes at this time is between 1.71 and 1.54. That is, as the hydrolysis reaction proceeds, the refractive index perceived by the whispering gallery modes decreases. As can be seen from equation (1), the whispering gallery mode resonance wavelength will decrease accordingly, i.e., a blue shift will occur.
The results of the urease sensing system based on whispering gallery mode laser testing different concentrations of urease solution are shown in fig. 3. Whispering gallery mode laser spectroscopy produces a concentration-dependent blue-shifted response under the action of urease. This is shown in particular by the increase in the amount of spectral blue shift with increasing urease concentration.
The above-mentioned embodiments further illustrate the specific preparation method of the present invention. The invention converts the biochemical reaction process of urease into spectral information in real time, so that the whole detection process can be accurately quantified and monitored in real time, and the accuracy and reliability of results are ensured.
In summary, the present invention relates to the field of optical micro-sensing, and more particularly to a urease sensing device based on whispering gallery mode laser and a method for manufacturing the same. And the pH sensitive molecules stearic acid and fluorescent dye Nile Red are codoped into the nematic liquid crystal 5CB to functionalize the nematic liquid crystal. Micron-sized 5CB microdroplets are prepared by accurately controlling the injection rate of a micro injection pump, and are used as an optical resonance microcavity and a biosensing probe. The reaction solution contains urease and a substrate urea for the action of the urease, and the enzymolysis promotes the deprotonation of stearic acid so as to induce 5CB molecules to generate the change of the arrangement state. The change is sensitively captured by whispering gallery mode laser excited in the microdroplet and accurately characterized in real time in a spectral shift mode, thereby realizing urease detection with high sensitivity and low detection limit.
Claims (5)
1. A urease sensing device based on whispering gallery mode laser is characterized by comprising a 532nm pump pulse laser (1), a spectrum analyzer (2), a charge coupled device CCD (3), functionalized 5CB microdroplets (4) and a reaction solution (5); the functionalized 5CB (4) microdroplet is co-doped with pH sensitive molecule stearic acid and fluorescent dye nile red and is generated in a reaction solution (5) through a capillary microtube (7); the reaction solution (5) contains urease and a substrate urea for the urease, light (11) emitted by a 532nm pump pulse laser sequentially passes through a polarization controller (8) and a semi-reflecting and semi-transparent mirror (9), and is finally focused on the surface of a functionalized 5CB microdroplet (4) by a 10x optical objective (10) and excites dye nile red to generate fluorescence; the working frequency and the working voltage of the 532nm pump pulse laser are controlled by a frequency controller (16) and a voltage stabilizer (17), the controlled working frequency is 6Hz, and the voltage is 220V; whispering gallery mode lasers generated by 5CB microdroplets are collected through a 10x optical objective (10) and sequentially pass through a half-reflecting and half-transmitting mirror (9) and a beam splitter (12); one beam of light is filtered by a 532nm filter and then transmitted to a spectrum analyzer (2) for spectrum analysis; the other beam of light enters a charge coupled device CCD (3) for microdroplet imaging; the obtained data are all transmitted to a computer (4) for information processing and display.
2. A whispering gallery mode laser based urease sensing apparatus according to claim 1 wherein the functionalized 5CB droplets (4) are co-doped with 0.05 wt.% stearic acid and 0.01 wt.% nile red, the functionalized 5CB droplets (4) being produced by controlling the injection rate of the micro syringe pump (14) with a diameter controlled at 50 μm.
3. The urease sensing apparatus based on whispering gallery mode laser according to claim 1, wherein the reaction solution (5) is contained in the reaction vessel (6) and the bottom is equipped with a micro heating stage (18); the reaction vessel (6) is made of silicon dioxide, the radius of the bottom of the reaction vessel is 2cm, and the height of the reaction vessel is 3 cm.
4. The whispering gallery mode laser-based urease sensing apparatus of claim 1, wherein the micro syringe pump (14) and functionalized 5CB microdroplets (4) are precisely position controlled by an X-Y-Z three-dimensional motorized adjustment stage (15).
5. A manufacturing method of a urease sensing device based on whispering gallery mode laser is characterized by comprising the following steps:
step 1: stearic acid and nile red with the mass fractions of 0.05 percent and 0.01 percent are co-doped in 1mL of nematic liquid crystal 5 CB;
step 2: placing the mixed solution in an ultrasonic machine for ultrasonic treatment for 30 minutes to fully and uniformly mix the mixed solution, wherein the working temperature of the ultrasonic machine is set to be 25 ℃;
and step 3: drawing a quartz capillary tube with the outer diameter of 150 mu m and the inner diameter of 75 mu m into a capillary microtube with the inner diameter of 5 mu m by using a flame tapering method; the other end of the capillary micro-tube is connected with a micro injection pump with controllable injection rate;
and 4, step 4: by controlling the injection rate of a micro syringe pump, functionalized 5CB microdroplets with the diameter of 50 mu m are generated in the reaction solution;
and 5: the temperature of the reaction solution is monitored in real time by a miniature temperature probe, and the temperature is ensured to be constant at 25 ℃ by a feedback adjusting device;
step 6: light emitted by a 532nm pump pulse laser sequentially passes through a polarization controller and a semi-reflecting and semi-transparent mirror, is focused on the surface of a functionalized 5CB microdroplet by a 10x optical objective lens and excites dye nile red to generate fluorescence;
and 7: the reaction solution contains urease and an acting substrate urea thereof, and ammonium ions and hydroxide ions are released by enzymolysis so as to increase the pH value of the solution;
and 8: the whispering gallery mode laser generated by 5CB microdroplets is collected through a 10x optical objective lens and sequentially passes through a half-reflecting and half-transmitting lens and a beam splitter, and one beam of laser is transmitted to a spectrum analyzer after being filtered by a 532nm filter for spectrum analysis; the other beam of light enters a charge coupled device CCD for droplet imaging, and the obtained data are transmitted to a computer for information processing and display.
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| CN106053389A (en) * | 2016-05-25 | 2016-10-26 | 哈尔滨工程大学 | Micro-droplet sensing device and method using same to measure refractivity |
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