CN113155792A - Ratio fluorescence detection method with ratio capable of being adjusted at will - Google Patents
Ratio fluorescence detection method with ratio capable of being adjusted at will Download PDFInfo
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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Abstract
The invention discloses a ratio fluorescence detection method with randomly adjustable ratio, which utilizes a double-excitation up-conversion material which can generate two emissions which are independent and not interfered with each other under the excitation of two different excitation lights, and uses the ratio of the fluorescence intensities at the two wavelengths as a signal parameter to detect a target object. In the detection method, the ratio of the fluorescence of the two emission wavelengths can be adjusted at will, and a high-intensity reference signal can be kept while the intensity of the detection signal is reduced, so that the detection limit can be reduced, the accuracy of reference can be ensured, and the comprehensive applicability of the detection method can be greatly improved.
Description
Technical Field
The invention relates to the field of detection, in particular to a ratio fluorescence detection method with a ratio capable of being adjusted at will.
Background
The upconversion luminescent nano material (UCNPs) is mainly prepared by doping inorganic matrixes such as oxides, fluorides, oxyhalides and the like with trivalent rare earth ions (such as Er)3+、Eu3+、Yb3+、Tm3+、Ho3+Etc.) to obtain a non-linear optical anti-stokes process, the up-conversion process can make two or more pump photons which are absorbed continuously reach a luminous energy level through radiationless relaxation, and then the pump photons are transited to a ground state to generate a high-energy emission photon. The unique frequency conversion capability of the UCNPs ensures that the UCNPs have a plurality of advantages, such as high signal-to-noise ratio, long fluorescence life, no light flicker and light bleaching, low toxicity, narrow emission band, larger penetration depth, almost no damage to biological tissues and the like, so that the UCNPs have the remarkable advantages in the biomedical fieldCorresponding diagnosis, treatment, imaging, biological monitoring, etc. have acquired revolutionary opportunities and developments. Therefore, the research on the application of the upconversion luminescent nano material in the biological direction has great scientific value.
Ratiometric fluorescence is an analytical method for determining a target by measuring the ratio of fluorescence intensities at two different wavelengths as a signal parameter. The existing probes for ratiometric fluorescence measurement all belong to single excitation materials, and mainly combine two different fluorophores on a nanoparticle, one fluorophore is used as a reference unit, the other fluorophore is used as a signal unit, and an analyte is detected by measuring the change of the fluorescence intensity ratio of two wavelengths. Most of the up-conversion luminescent nano-materials that have been developed at present also belong to single-excitation materials.
A single-excitation upconverter material is capable of producing one or more emissions under a single excitation condition, and when the excitation energy is changed, all of the emissions are simultaneously changed accordingly. However, this variation is only reflected in a uniform change in emission intensity, while the intensity ratio between different emission peaks is always constant. Research shows that the ratio of different emissions under the same excitation is an inherent property determined by self conditions such as the structure and element doping ratio of the nano material. Thus, the drawback of inconsistent ratios has created a great limitation in the use of upconverting nanomaterials as detection probes. In the previous report, in order to reduce the detection limit, researchers must reduce the detection signal as much as possible, but because the ratio is fixed, the signal used as a reference will also reduce the intensity greatly, so that the reference accuracy is reduced, the effectiveness and accuracy cannot be guaranteed, the difficulty in practical use of the probe is increased, and the development of detection application is not facilitated.
Disclosure of Invention
In view of the above problems of the prior art, it is an object of the present invention to provide a ratiometric fluorescence detection method in which the ratio can be arbitrarily adjusted in order to improve the sensitivity of ratiometric fluorescence detection.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a ratio fluorescence detection method with an arbitrarily adjustable ratio, which comprises the following steps:
the emission wavelength of the nano probe under the excitation of the first excitation light is A, and the emission wavelength under the excitation of the second excitation light is B; by adjusting the intensity of the first excitation light and/or the second excitation light, the fluorescence ratio I of the nanoprobe under the same condition, which is the intensity I of the fluorescence with the emission wavelength A generated by the nanoprobe under the excitation of the first excitation light, can be adjusted arbitrarilyAAnd the intensity I of the fluorescence with the emission wavelength B generated under the excitation of the second excitation lightBI ═ IA/IB；
and 4, calculating to obtain a fluorescence ratio according to the intensities of the two fluorescence signals collected in the step 3, and using the fluorescence ratio to represent the concentration of the object to be detected in the sample to be detected.
Furthermore, the nanoprobe is composed of rare earth up-conversion nanoparticles regulated and controlled by exciting light and a mesoporous silica layer coated outside the rare earth up-conversion nanoparticles; specific molecules capable of identifying an object to be detected are loaded in mesopores of the mesoporous silicon dioxide layer. The rare earth upconversion nanoparticles regulated by the exciting light emit red fluorescence under the excitation of 980nm laser and emit green fluorescence under the excitation of 808nm laser.
Furthermore, the rare earth up-conversion nano particle has a 3-layer shell-core structure and sequentially comprises an activator layer, an energy transfer agent layer and a sensitizer from inside to outsideA reagent layer, the chemical expression of which is: NaErF4:Yb,Tm@NaYF4:Yb@NaNdF4Yb, i.e. NaErF co-doped with Yb and Tm4Nanoparticle inner core NaErF4Yb and Tm are activator layers, and Yb-doped NaYF serving as an energy transfer agent layer is sequentially coated outside the inner core4First shell NaYF4Yb, Yb-doped NaNdF as sensitizer layer4Second shell layer of NaNdF4:Yb。
Furthermore, the invention can realize the detection of different objects to be detected which have corresponding recognition relation with the specific molecules by replacing the specific molecules in the nano probe.
Compared with the prior art, the invention has the beneficial effects that:
the ratio fluorescence detection method utilizes a double-excitation up-conversion material which can generate two types of emission which are independent and not interfered with each other under the excitation of two different excitation lights, the fluorescence intensity ratio at the two wavelengths is used as a signal parameter to detect a target object, the fluorescence ratio of the two can be adjusted at will, and a high-intensity reference signal can be still reserved while the detection signal intensity is reduced, so that the detection limit can be reduced, the accuracy of reference can be ensured, and the comprehensive applicability of the detection method can be greatly improved.
Drawings
FIG. 1 is a fluorescence spectrum of rare earth upconversion nanoparticles obtained in step 1 of example 1 under excitation of 980nm laser;
FIG. 2 is a fluorescence spectrum of rare earth upconversion nanoparticles obtained in step 1 of example 1 under the excitation of 808nm laser;
FIG. 3 is an electron micrograph of rare earth upconverting nanoparticles obtained in step 1 of example 1;
FIG. 4 is a TEM image of the nanoprobe obtained in example 1;
FIG. 5 is a photograph (converted to a gray-scale photograph) of five ratios (4: 1, 2:1, 1:2, 1:4, respectively) of the emission ratios r obtained in example 1 by adjusting the excitation light wattages of 808nm and 980nm, respectively, and photographing by a mobile phone system;
FIG. 6 is a plot of the three ratios versus quenching for the same gradient of NO concentration change in example 1;
FIG. 7 is a correlation between the concentration of 0 to 25ppb NO and the change in the ratio, which were established in the ratios of 1:4 (FIG. 7(a)) and 1:2 (FIG. 7(b)) in example 1;
FIG. 8 is a graph showing the results of example 1 in which exhaled breath was measured in asthmatic patients according to the 1:2 ratio condition.
Detailed Description
The following embodiments of the present invention will be described in detail with reference to the accompanying drawings, which are provided for implementing the technical solution of the present invention, and provide detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following embodiments.
Example 1
The embodiment provides a nano probe for detecting NO and a detection method thereof:
one, nanometer probe
The nanoprobe of the embodiment is composed of rare earth upconversion nanoparticles regulated and controlled by exciting light and a mesoporous silica layer coated outside the rare earth upconversion nanoparticles; wherein: the emission wavelength of the rare earth upconversion nanoparticles regulated and controlled by the exciting light under the excitation of 980nm laser is 660nm (red fluorescence), and the emission wavelength under the excitation of 808nm laser is 540nm (green fluorescence); photosensitive molecular rhodamine spiro lactam RdMs are loaded in mesopores of the mesoporous silicon dioxide layer. RdMs can rapidly react with NO, change the chemical structure of the RdMs, change the photosensitive property of the RdMs, and generate FRET effect with up-conversion nanoparticles under the excitation of 808nm exciting light, thereby influencing the green luminescence of the novel probe and changing the luminescence ratio.
Specifically, the rare earth upconversion nanoparticle of this embodiment has a 3-layer shell-core structure, which is sequentially an activator layer, an energy transfer agent layer, and a sensitizer layer from inside to outside, and the chemical expression of the rare earth upconversion nanoparticle is as follows: NaErF4:Yb,Tm@NaYF4:Yb@NaNdF4Yb, i.e. NaErF co-doped with Yb and Tm4Nanoparticle inner core NaErF4Yb and Tm are activator layers, and Yb-doped NaYF serving as an energy transfer agent layer is sequentially coated outside the inner core4First shell NaYF4Yb, Yb-doped NaNdF as sensitizer layer4Second shellLayer of NaNdF4Yb. Wherein, in the activator layer, Er3+、Tm3+、Yb3+The molar percentage of (A) is 80%: 0.5%: 19.5 percent; in the energy transfer agent layer, Y3+And Yb3+The molar percentage of (A) is 90%: 10 percent; in the sensitizer layer, Nd3+And Yb3+The molar percentage of (A) is 90%: 10 percent.
The preparation method of the nanoprobe comprises the following steps: firstly, preparing rare earth upconversion nanoparticles regulated and controlled by exciting light by a seed crystal method, and then coating a mesoporous silica layer outside the rare earth upconversion nanoparticles by a sol-gel method; and loading RdMs molecules in the mesopores of the mesoporous silicon dioxide layer by a physical adsorption method to obtain the nano probe. The method comprises the following specific steps:
1. preparation of rare earth upconversion nanoparticles NaErF with excitation light regulation and control by seed crystal method4:Yb,Tm@NaYF4:Yb@NaNdF4:Yb
(1) Adding 1mmol erbium acetate, thulium acetate and ytterbium acetate, 630mg sodium fluoride, 10mL oleic acid and 10mL octadecene into a 100mL three-neck flask A, heating to 110 ℃ under magnetic stirring, and vacuumizing for 10min to remove water and oxygen; removing all and then introducing N2Heating to 300 ℃, and reacting for 1h under the condition of heat preservation;
wherein, the mole percentage of erbium acetate, thulium acetate and ytterbium acetate is 80%: 0.5%: 19.5 percent.
(2) Adding 0.9mmol of yttrium acetate, 0.1mmol of ytterbium acetate, 4mL of oleic acid and 4mL of octadecene into a 50mL three-neck flask B, heating to 110 ℃ under magnetic stirring, and vacuumizing for 10min to remove water and oxygen; removing all and then introducing N2And (3) heating to 200 ℃, injecting the mixture into the three-neck flask A at the speed of 1mL/min after the reaction in the step (1) is finished, and keeping the temperature at 300 ℃ for 40 min.
(3) Adding 0.9mmol of neodymium acetate, 0.1mmol of ytterbium acetate, 4mL of oleic acid and 4mL of octadecene into a 50mL three-neck flask C, heating to 110 ℃ under magnetic stirring, and vacuumizing for 10min to remove water and oxygen; removing all and then introducing N2Heating to 200 deg.C, and injecting into the reactor at a rate of 1mL/min after the reaction in step (2)In a flask A, keeping the temperature at 300 ℃ for reaction for 40 min;
after the reaction is finished, cooling to room temperature, and then adding ethanol for centrifugal separation to obtain the target product rare earth upconversion nanoparticles.
2. Coating mesoporous silicon dioxide layer outside rare earth conversion nanoparticles by sol-gel method
(1) 200mg CTAB and 0.2mmol of rare earth upconversion nanoparticles were dispersed in a 250mL single-neck flask containing 20mL of pure water, and stirred at 50 ℃ overnight.
(2) 30mL of pure water, 5mL of ethanol, and 0.5mL of sodium hydroxide were put into the flask, and after the reaction was continued at 50 ℃ for 10 minutes, 0.2mL of TEOS and 0.1mL of APS were added, and the reaction was continued at 70 ℃ for 1 hour. And after the reaction is finished, sucking out liquid in the flask and centrifuging to obtain the rare earth upconversion nanoparticles coated with the silicon dioxide layer.
(3) And dispersing the obtained rare earth upconversion nanoparticles coated with the silicon dioxide layer into 50mL of ethanol in which 500mg of ammonium nitrate is dissolved again by using a 250mL single-neck flask, stirring for 2h at room temperature, and centrifuging after the reaction is finished to obtain the rare earth upconversion nanoparticles coated with the mesoporous silicon dioxide layer.
3. Loading RdMs molecules in mesopores of mesoporous silicon dioxide layer by physical adsorption method
(1) Preparation of RdMs by chemical Synthesis
1g of rhodamine B and 0.5g of o-phenylenediamine and 50mL of methylene chloride were added to a 250mL single-neck flask, and the rhodamine B and the o-phenylenediamine were dissolved in the methylene chloride by sonication. 1mL of triethylamine and 1mL of 1-propylphosphoric cyclic anhydride were added to the solution, and the reaction was stirred at room temperature for 24 hours. After the reaction is finished, adsorbing the obtained solution by silica gel powder, separating by using a column chromatography technology to obtain a light yellow transparent liquid, and drying to obtain a product RdMs which is white or light yellow powder.
(2) Using a 100mL single-neck flask, 20mg of RdMs were ultrasonically dissolved in 20mL of ethanol. And (3) taking 0.1mmol of rare earth upconversion nanoparticles coated with the mesoporous silica layer, dispersing the rare earth upconversion nanoparticles in the ethanol solution, carrying out ultrasonic treatment for 1 hour, and stirring for 1 day at room temperature.
(3) And centrifuging after stirring is finished, taking the precipitate, dispersing the precipitate again by using a proper amount of ethanol, and centrifuging and cleaning. And (3) drying the precipitate obtained after cleaning in an oven at 50 ℃ for 2h to obtain the mesoporous rare earth up-conversion nanoprobe loaded with the RdMs molecules.
Fig. 1 is a fluorescence spectrum of the rare earth upconversion nanoparticle obtained in step 1 of this example under excitation of 980nm laser, from which it can be seen that the main emission peak of the sample is 660nm, corresponding to red fluorescence.
Fig. 2 is a fluorescence spectrum of the rare earth up-conversion nanoparticle obtained in step 1 of this example under the excitation of 808nm laser, from which it can be seen that the main emission peak of the sample is 540nm, which corresponds to green fluorescence.
FIG. 3 is a TEM image of the rare earth up-conversion nanoparticles obtained in step 1 of this example, and it can be seen that the sample is dumbbell-shaped.
Fig. 4 is a TEM image of the nanoprobe obtained in this example, from which it can be seen that the silica layer is successfully coated, and the mesoporous channel in the coating layer is clearly visible.
The fluorescence ratio I ═ I of the nanoprobe obtained in this example under the same conditions can be arbitrarily adjusted by adjusting the wattage of the 808nm excitation light and/or the wattage of the 980nm excitation light540nm/I660nm. If the wattage of the 980nm exciting light is not changed, the fluorescence ratios I of the nanoprobes are respectively 1:1, 1:2 and 1:4 by adjusting the wattage of the 808nm exciting light; under the condition that the wattage of 808nm exciting light is unchanged, the wattage of 980nm laser is adjusted to enable the fluorescence ratio I of the nano-probe to be 2:1 and 4:1 respectively. The photographs of the green fluorescence and the red fluorescence generated under the excitation light of the corresponding intensities are respectively shown in fig. 5, in which the photographs are converted into grayscale photographs, but the intensity changes of the green fluorescence and the red fluorescence can be clearly seen from the figures.
II, NO detection
For use, firstly, the fluorescent test paper for detecting nitric oxide is constructed by using the nano-probe, which is specifically as follows:
(1) cutting filter paper into a rectangular test strip with a proper size, soaking in PBS (with the probe concentration of 1mM) dispersed with the nano probe, performing ultrasonic treatment for many times, performing vortex, and standing and soaking overnight;
(2) the oven was set to 50 ℃ and the overnight soaked paper was placed in to completely dry it for use.
The method for detecting nitric oxide by using the test paper comprises the following steps:
and 4, placing the fluorescent test paper in the gas to be detected for full reaction, then taking out, obtaining the luminous ratio of the fluorescent test paper after the fluorescent test paper reacts with the gas to be detected according to the same method in the step 2, and determining the concentration of NO in the gas to be detected according to the standard working curve in the step 3.
In specific implementation, the grey value of the photo can be obtained by analyzing Image J software, 980nm laser and 808nm laser can be generated by a portable 980nm/808nm laser, and the used optical filter can transmit light of 400-700 nm.
FIG. 6 is a graph comparing the relative quenching of the three ratios for the same gradient of NO concentration change in this example. It can be seen from the figure that: as the green-red ratio decreased, the same concentration of NO increased the relative degree of quenching of the probe, demonstrating that a decrease in the ratio can further increase the sensitivity of the assay. The relative quenching degree specifically refers to the absolute value of the ratio change caused by the change in the unit NO concentration, and here is the degree of ratio decrease. The experimental approach was to prepare three sets of experiments, testing with ratios 1:1, 1:2, 1:4, respectively, under the same NO concentration gradient, obtaining the ratio of change after each measurement, tabulating the plots, respectively, and calculating the relative quenching degree for each ratio.
FIG. 7 is a graph showing the correlation between the concentration of 0 to 25ppb NO and the change in the ratio, which are established at the ratios of 1:4 (FIG. 7(a)) and 1:2 (FIG. 7(b)) in the present example. Subject to objective conditions, 1:4 approaches the optimum range of ratios permitted by plant conditions, with a 1:4 ratio at the lowest detectable NO concentration of 0.5ppb under steady state conditions. The 1:4 ratio linear equation is-0.1542 x +25.03441, R20.99978. Linear equation of 1:2 ratio is-0.001 x +0.5, R2＝0.9931。
FIG. 8 shows the results of measuring the exhaled breath of an asthmatic patient under excitation with a 980nm laser and 808nm laser wattage at a ratio of 1:2 in this example. As is known from literature, asthma sufferers typically have exhaled breath containing amounts of NO due to respiratory inflammatory reactions, generally in the range of 35 to several hundred ppb, and the test continues with a 1:2 ratio of greater span (50% for the initial 1:2 ratio, about 2.5% for each 25ppbNO reduction, up to about 500ppbNO reduction) considering that the span at the 1:4 ratio condition (25% for the initial 1:4 ratio, about 3.85% for each 25ppbNO reduction, up to about 160ppbNO reduction detectable) may not meet experimental requirements. 11 asthmatic patients were found from the hospital as experimental volunteers, exhaled breath of the same volume of 11 patients was collected, all tests were completed by the detection system and the results were calculated, as shown in table 1 and fig. 8, all of which were in line with or close to the range of asthmatic pathological NO parameters.
TABLE 1
The present invention is not limited to the above exemplary embodiments, and any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A ratio fluorescence detection method with an arbitrarily adjustable ratio is characterized by comprising the following steps:
step 1, setting a nano probe for regulating and controlling exciting light, wherein the nano probe contains specific molecules capable of identifying an object to be detected;
the emission wavelength of the nano probe under the excitation of the first excitation light is A, and the emission wavelength under the excitation of the second excitation light is B; by adjusting the intensity of the first excitation light and/or the second excitation light, the fluorescence ratio I of the nanoprobe under the same condition, which is the intensity I of the fluorescence with the emission wavelength A generated by the nanoprobe under the excitation of the first excitation light, can be adjusted arbitrarilyAAnd the intensity I of the fluorescence with the emission wavelength B generated under the excitation of the second excitation lightBI ═ IA/IB；
Step 2, adding the nano probe into a sample to be detected, enabling the object to be detected to react with the specific molecules in the nano probe, and changing the intensity I of fluorescence generated by the nano probe under the excitation of the first exciting lightA；
Step 3, determining the intensities of the first excitation light and the second excitation light according to the required fluorescence ratio, then sequentially exciting the reacted nanoprobes by the second excitation light and the first excitation light according to the determined intensities, and collecting the intensities of corresponding fluorescence signals;
and 4, calculating to obtain a fluorescence ratio according to the intensities of the two fluorescence signals collected in the step 3, and using the fluorescence ratio to represent the concentration of the object to be detected in the sample to be detected.
2. The ratiometric fluorescence detection method of claim 1, wherein: the nano probe consists of rare earth up-conversion nano particles regulated and controlled by exciting light and a mesoporous silica layer coated outside the rare earth up-conversion nano particles; specific molecules capable of identifying an object to be detected are loaded in mesopores of the mesoporous silicon dioxide layer.
3. The ratiometric fluorescence detection method of claim 2, wherein: the rare earth up-conversion nano particles are of a 3-layer shell-core structure and sequentially comprise an activator layer, an energy transfer agent layer and a sensitizer layer from inside to outside, and the chemical expression of the rare earth up-conversion nano particles is as follows: NaErF4:Yb,Tm@NaYF4:Yb@NaNdF4Yb, i.e. NaErF co-doped with Yb and Tm4Nanoparticle inner core NaErF4Yb and Tm are activator layers, and Yb-doped NaYF serving as an energy transfer agent layer is sequentially coated outside the inner core4First shell NaYF4Yb, Yb-doped NaNdF as sensitizer layer4Second shell layer of NaNdF4:Yb。
4. The ratiometric fluorescence detection method of claim 2 or 3, wherein: the rare earth upconversion nanoparticles regulated by the exciting light emit red fluorescence under the excitation of 980nm laser and emit green fluorescence under the excitation of 808nm laser.
5. The ratiometric fluorescence detection method of claim 1 or 2, wherein: the detection of different objects to be detected can be realized by adjusting specific molecules in the nano probe.
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