CN113433087A - Rapid detection method for urea concentration, detection sensor and application - Google Patents
Rapid detection method for urea concentration, detection sensor and application Download PDFInfo
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- 239000004202 carbamide Substances 0.000 title claims abstract description 88
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 title claims abstract description 87
- 238000001514 detection method Methods 0.000 title claims abstract description 31
- 239000002131 composite material Substances 0.000 claims abstract description 57
- 229910052737 gold Inorganic materials 0.000 claims abstract description 41
- 239000010931 gold Substances 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 31
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 16
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012488 sample solution Substances 0.000 claims abstract description 8
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 6
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 6
- 210000001124 body fluid Anatomy 0.000 claims abstract description 5
- 239000010839 body fluid Substances 0.000 claims abstract description 5
- 238000000354 decomposition reaction Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 110
- 108010046334 Urease Proteins 0.000 claims description 31
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 16
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 14
- 239000011259 mixed solution Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000002245 particle Substances 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 8
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 7
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 7
- 239000011449 brick Substances 0.000 claims description 7
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 7
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 230000001678 irradiating effect Effects 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 239000008055 phosphate buffer solution Substances 0.000 claims description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 2
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 2
- 238000010998 test method Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 150000003672 ureas Chemical class 0.000 abstract 1
- 210000004369 blood Anatomy 0.000 description 11
- 239000008280 blood Substances 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 210000002700 urine Anatomy 0.000 description 10
- 238000010586 diagram Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000001338 self-assembly Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000002194 synthesizing effect Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 2
- 239000003337 fertilizer Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000007707 calorimetry Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- 235000021075 protein intake Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000002441 uremic toxin Substances 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/35—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light
- G01N21/3577—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using infrared light for analysing liquids, e.g. polluted water
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Abstract
本发明公开了一种尿素浓度快速检测方法及其应用，该方法利用脲酶‑纳米金复合材料在尿素的存在下，采用790‑820nm的红外激光的照射，使溶液升温加速脲酶催化尿素分解成氨气和二氧化碳，通过便携式气压计读取压力值。通过测量一系列已知浓度的尿素溶液，获得标准曲线对照图，然后用同样的方法检测未知浓度的样本溶液，读取压力值并与标准曲线对照图进行对比，得到待检测溶液中尿素的浓度，实现对尿素浓度的快速检测。该方法尤其适用于对人的体液中尿素浓度的即时检测，具有方便、快捷、选择性好、灵敏度高等优点。
The invention discloses a method for rapid detection of urea concentration and its application. The method utilizes urease-nano gold composite material in the presence of urea, and uses 790-820 nm infrared laser irradiation to heat the solution to accelerate the decomposition of urease-catalyzed urea into ammonia Gas and carbon dioxide, pressure readings with a portable barometer. By measuring a series of urea solutions with known concentrations, the standard curve control chart is obtained, and then the sample solution of unknown concentration is detected by the same method, and the pressure value is read and compared with the standard curve chart to obtain the concentration of urea in the solution to be detected. , to achieve rapid detection of urea concentration. The method is especially suitable for instant detection of urea concentration in human body fluids, and has the advantages of convenience, speed, good selectivity and high sensitivity.
Description
Technical Field
The invention relates to a detection method of urea concentration and application thereof.
Background
Urea is widely distributed in nature, and its analysis has great significance in clinical, food and agricultural chemistry. The concentration of urea in urine can be used to assess the nutritional status of a person, such as low protein intake and food deficiency. In addition, it is an important marker for assessing uremic toxin levels. Urea also enters the environment as a fertilizer and is decomposed into toxic ammonia, thereby polluting the environment. Normal levels of urea in serum were 15-40mg/dL (2.5-7.5mM/L), with serum urea concentrations varying between 180-480mg/dL (30-90mM/L) in patients with renal insufficiency. Accurate detection of urea is of vital importance in clinical diagnostics, dairy industry, fertilizer plants and environmental monitoring. The traditional urea detection methods mainly comprise gas chromatography, calorimetry and fluorescence analysis. These methods, while reliable, require high cost, bulky equipment and complex operations that are not suitable for on-site monitoring.
Disclosure of Invention
Aiming at the defects of the prior method for detecting urea by chromatography, ultraviolet spectrophotometry and the like, the invention aims to provide a rapid urea concentration detection method which has low cost, is suitable for on-site monitoring, is rapid and convenient to use and application thereof
The invention provides a method for rapidly detecting urea concentration, which comprises the following steps:
(1) preparing urease-nano gold composite materials, urea solutions with different concentrations, 790-820nm infrared laser and a barometer;
(2) adding a urea solution with one concentration into the urease-nanogold composite material prepared in the step (1) to form a mixed solution;
(3) irradiating the mixed solution in the step (2) by using the infrared laser in the step (1), converting light energy into heat energy by using the photo-thermal property of nano particles in the mixed solution, heating the solution, accelerating the decomposition of urea into carbon dioxide and ammonia gas under the catalysis of urease, and measuring the air pressure value by using a barometer;
(4) recording the air pressure value in the step (3);
(5) respectively repeating the steps (2) to (4) by using urea solutions with different concentrations to obtain different air pressure values;
(6) drawing a standard curve comparison graph according to the urea solutions with different concentrations respectively corresponding to the different air pressure values obtained in the step (5);
(7) and (3) adding a sample solution to be detected with unknown concentration into the urease-nanogold composite material prepared in the step (1), repeating the step (3) and the step (4) to obtain an air pressure value, and contrasting the air pressure value with the standard curve comparison chart in the step (6) to obtain the urea concentration of the solution to be detected.
The preparation method of the urease-nanogold composite material comprises the following steps:
(1.1) respectively taking 640 mu L of hydroquinone solution with the solution concentration of 100mM, 40 mu L of silver nitrate solution with the solution concentration of 60mM and 40 mu L of chloroauric acid solution with the solution concentration of 25mM, sequentially adding the hydroquinone solution with the solution concentration of 18ml and polyvinylpyrrolidone solution with the solution concentration of 90mM, shaking to change the color of the solution from colorless to blue to light red brick color, adding 200 mu L of concentrated ammonia water, and removing a silver chloride template to obtain the spherical hollow porous nanogold.
(1.2) mixing the spherical hollow porous nano-gold in the step (1.1) with a urease solution, wherein the concentration of the spherical hollow porous nano-gold is 0.05-0.10mg/mL, the concentration of the urease solution is 0.10-0.20mg/mL, the spherical hollow porous nano-gold and the urease solution are assembled according to the volume ratio of (1-2) to (3-4), urease is adsorbed on the spherical hollow porous nano-gold, and then the urease-nano-gold composite material is obtained through centrifugal separation, wherein the particle size of the spherical hollow porous nano-gold in the urease-nano-gold composite material is 100-130 nm.
In the preferable step (1.2), the concentration of the urease solution is 0.15mg/mL, the particle size of the spherical hollow porous nano-gold in the urease-nano-gold composite material is 105nm, the concentration is 0.07mg/mL, and the spherical hollow porous nano-gold and the urease solution are assembled in a volume ratio of 1: 3.
And (3) reacting the spherical hollow porous nano gold and the urease solution in the step (1.2) in a centrifugal tube at room temperature at a shaking table speed of 500r for 8-12h, centrifuging at 13000rpm for 5min, removing free urease, and dissolving with secondary water to obtain the urease-nano gold composite material. The urease solution is dissolved by potassium dihydrogen phosphate buffer solution with pH between 4-6.
The pH value of the urease-nanogold composite material adopted in the step (1) is 4-6, the air pressure is measured by using an air pressure gauge under the condition that the temperature of the urease-nanogold composite material is 60-70 ℃ under the irradiation of infrared laser, and the laser irradiation power is 1.5-2.5W/cm2And reading the barometer after the laser irradiation of 790-820nm for 10-20 min. Preferably, the urease-nanogold composite used in the step (1) has a pH of 5.4, a reaction time of 12 hours, and a gas pressure is measured using a barometer under infrared laser irradiation at a temperature of 60 ℃. The laser irradiation power was 2.0W/cm2Reading the barometer after irradiating for 15min by using laser with wavelength of 808nm
As a further object of the invention is that the method is used for detecting urea concentration in a body fluid of a human.
The invention also aims to provide a urea concentration detection sensor directly obtained by the rapid detection method for the urea concentration, which comprises a urease-nanogold composite material, 790-820nm infrared laser, a barometer and the standard curve comparison chart. The barometer is a portable barometer.
The invention has strong photo-thermal efficiency of the urease-nano gold composite material, can rapidly raise the temperature in a short time, and accelerate the urease to catalyze the urea to decompose into carbon dioxide and ammonia gas, the inventor further discovers that when the urease-nano gold composite material is measured by an air pressure gauge at the pH value of 4-6 and the temperature of 60-70 ℃, the urea concentration and the air pressure value form a very stable direct proportion relation, and by utilizing the corresponding relation, the method can accurately detect the urea concentration, and experiments prove that the method can detect the urea concentration with the sensitivity of 1.00-100mM, and the normal contents of the urea in the urine and the blood of a normal human body are respectively 55-388mM (9.3-23.3g/L) and 2.5-7.5mM (0.15-0.4g/L), and the urea concentration in sweat is 1.8-46 mM, therefore, the detection range of the method meets the detection of urea in body fluid, and can be used for detecting the concentration of urea in body fluid of a human. The method does not need to use precise instruments and equipment, the instant detection effect of the detection sensor directly obtained by the method is very obvious on site, the detection is rapid, the operation is convenient, the sensitivity is high, the detection range is wide, and the detection cost is low, which cannot be compared with the existing equipment.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2 is a comparison of the standard curves for different concentrations of urea in example 1.
Detailed Description
In order to facilitate an understanding of the invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
The following is a list of some data from the experiments of the present invention:
TABLE 1 Experimental data List
The concentration of the spherical hollow porous nano gold is within the range of 0.05-0.10mg/mL, the particle size is 100-130nm, the concentration of the urease solution is 0.10-0.20mg/mL, and the volume ratio of the spherical hollow porous nano gold to the urease solution is (1-2) to (3-4); the PBS solution is potassium dihydrogen phosphate, the pH value is between 4 and 6, the spherical hollow porous nano-gold and the urease solution react for 8 to 12 hours under the specified conditions, the pH value of the adopted urease-nano-gold composite material is 4 to 6, and the air pressure is measured by using a barometer under the condition that the temperature of the urease-nano-gold composite material is 60 to 70 ℃ under the irradiation of infrared laser.The laser irradiation power is 1.5-2.5W/cm2And reading the barometer after the laser irradiation of 790-820nm for 10-20 min.
Example 1
An embodiment of the method for rapidly detecting the urea concentration is shown in fig. 1, and a schematic diagram of the principle of the method is shown in the figure. The detection method specifically comprises the following steps:
the first step is as follows: preparing urease-nano gold composite material, urea solution with different concentrations, infrared laser with wavelength of 808nm and a portable barometer.
(1) And (3) synthesizing the urease-nanogold composite material as shown in figure 1.
(1.1) preparation of spherical hollow porous Nanogold
To a round bottom flask containing 18mL of 90mM PVP solution was added 640. mu.L of 100mM hydroquinone, 40. mu.L of 60mM AgNO3The solution is protected from light, 40 mu L of 25mM chloroauric acid is added dropwise while shaking (500r), the solution is placed on a shaking table for continuous reaction for one hour after shaking vigorously for 5min (1000r), and the color of the solution is changed from colorless to blue to light red brick color. Adding 200 mu L of concentrated ammonia water, reacting for 2 hours at 500r under a shaking table, and then deepening the color of the solution; after the reaction, the supernatant was removed by centrifugation and dispersed in 2mL of water to obtain spherical hollow porous nanogold having a particle size of 105nm and a concentration of 0.07 mg/mL.
(1.2) Synthesis of urease-Nanogold composite Material
In this example, the PBS solution is potassium dihydrogen phosphate, pH is 5.4, the concentration of the dissolved urease solution is 0.15mg/mL, 5 μ L of the spherical hollow porous nanogold is taken, and 15 μ L of the urease solution is assembled according to a ratio of 1: 3. And (3) carrying out self-assembly at room temperature at a shaking table speed of 500 r/shaking for 12h, carrying out centrifugal treatment at a rotating speed of 13000rpm for 5min after the assembly is finished, and dissolving with secondary water to obtain the urease-nanogold composite material.
The second step is that: obtaining a standard curve map
(2) Adding a urea solution with one concentration into the urease-nanogold composite material prepared in the step (1) to form a mixed solution;
(3) irradiating the mixed solution in the step (2) by using infrared laser with wavelength of 808nm, heating the mixed solution to release carbon dioxide and ammonia gas, and measuring the air pressure value by using an air pressure meter;
(4) reading the air pressure value in the step (3);
(5) respectively repeating the steps (2) to (4) by using urea solutions with different concentrations to obtain different air pressure values;
(6) and (5) respectively drawing a standard curve comparison graph according to the urea solutions with different concentrations corresponding to the different air pressure values obtained in the step (5), and showing the standard curve comparison graph in a figure 2.
The third step: in actual practice, the urea content in a blood sample of unknown concentration is measured.
(7) Taking a blood sample solution with unknown concentration, adding 80 mu L of solution to be detected into the urease-nanogold composite material prepared in the step (1), and repeating the step (3) and the step (4), wherein the infrared laser with the wavelength of 808nm in the step (3) is used, and the infrared laser with the wavelength of 2.0W/cm is used2Irradiating the solution for 15min, reading the air pressure value, and comparing the air pressure value reading with the standard value comparison chart in the step (6) to quickly obtain the concentration of the urea in the solution to be detected. Knowing that the urea concentration in normal blood is in the range of 2.5-7.5mM, our test range is satisfactory for determining the urea concentration in blood.
Example two
An embodiment of the method for rapidly detecting the urea concentration is shown in fig. 1, and a schematic diagram of the principle of the method is shown in the figure. The detection method specifically comprises the following steps:
the first step is as follows: preparing urease-nano gold composite material, urea solution with different concentrations, infrared laser with the wavelength of 790nm and a portable barometer.
(1) And (3) synthesizing the urease-nanogold composite material as shown in figure 1.
(1.1) preparation of spherical hollow porous Nanogold
To a round bottom flask containing 18mL of 90mM PVP solution was added 640. mu.L of 100mM hydroquinone, 40. mu.L of 60mM AgNO3The solution is protected from light, 40 mu L of 25mM chloroauric acid is added dropwise while shaking (500r), the solution is placed on a shaking table for continuous reaction for one hour after shaking vigorously for 5min (1000r), and the color of the solution is changed from colorless to blue to light red brick color. Adding 200 mu L of concentrated ammonia water, reacting for 2 hours at 500r under a shaking table, and obtaining a solutionDeepening the color; after the reaction, the supernatant was removed by centrifugation and dispersed in 2mL of water to obtain spherical hollow porous nanogold having a particle size of 100nm and a concentration of 0.05 mg/mL.
(1.2) Synthesis of urease-Nanogold composite Material
The spherical hollow porous nanogold obtained in step (1.1) and a urease solution (0.10mg/mL) dissolved in PBS solution (potassium dihydrogen phosphate, pH 4) were each assembled in the following proportions. Shaking for 8h (500r) at room temperature for self-assembly, centrifuging at 13000rpm for 5min after the assembly is finished, and dissolving with secondary water to obtain the urease-nanogold composite material.
And (3) taking 4 mu L of the spherical hollow porous nano gold, and assembling 16 mu L of the urease solution according to the volume ratio of 1: 4.
The second step is that: obtaining a standard curve map
(2) Adding a urea solution with one concentration into the urease-nanogold composite material prepared in the step (1) to form a mixed solution;
(3) irradiating the mixed solution in the step (2) by using 790nm infrared laser, heating the mixed solution to release carbon dioxide and ammonia gas, and measuring the air pressure value by using an air pressure meter;
(4) reading the air pressure value in the step (3);
(5) respectively repeating the steps (2) to (4) by using urea solutions with different concentrations to obtain different air pressure values;
(6) and (5) respectively drawing a standard curve comparison graph according to the urea solutions with different concentrations corresponding to the different air pressure values obtained in the step (5), and showing the standard curve comparison graph in a figure 2.
The third step: in actual practice, the urea content in a blood sample of unknown concentration is measured.
(7) Taking a blood sample solution with unknown concentration, adding 80 mu L of solution to be detected into the urease-nanogold composite material prepared in the step (1), and repeating the step (3) and the step (4), wherein 790nm infrared laser in the step (3) is used, and 1.5W/cm is used2Irradiating the solution for 10min, reading the air pressure value, and comparing the air pressure value reading with the standard value comparison chart in the step (6) to quickly obtain the concentration of the urea in the solution to be detected.Knowing that the urea concentration in normal blood is in the range of 2.5-7.5mM, our test range is satisfactory for determining the urea concentration in blood.
Example three
An embodiment of the method for rapidly detecting the urea concentration is shown in fig. 1, and a schematic diagram of the principle of the method is shown in the figure. The detection method specifically comprises the following steps:
the first step is as follows: preparing urease-nano gold composite material, urea solution with different concentrations, 820nm infrared laser and portable barometer.
(1) And (3) synthesizing the urease-nanogold composite material as shown in figure 1.
(1.1) preparation of spherical hollow porous Nanogold
To a round bottom flask containing 18mL of 90mM PVP solution was added 640. mu.L of 100mM hydroquinone, 40. mu.L of 60mM AgNO3The solution is protected from light, 40 mu L of 25mM chloroauric acid is added dropwise while shaking (500r), the solution is placed on a shaking table for continuous reaction for one hour after shaking vigorously for 5min (1000r), and the color of the solution is changed from colorless to blue to light red brick color. Adding 200 mu L of concentrated ammonia water, reacting for 2 hours at 500r under a shaking table, and then deepening the color of the solution; after the reaction, the supernatant was removed by centrifugation and dispersed in 2mL of water to obtain spherical hollow porous nanogold having a particle size of 130nm and a concentration of 0.10 mg/mL.
(1.2) Synthesis of urease-Nanogold composite Material
To 13.3. mu.L of urease dissolved in PBS (potassium dihydrogen phosphate, pH 4) at a ratio of 1:2 was added 6.7. mu.L of the spherical hollow porous nanogold obtained in step (1.1) at 0.20 mg/mL. Shaking for 12h (500r) at room temperature for self-assembly, centrifuging at 13000rpm for 5min after the assembly is finished, and dissolving with secondary water to obtain the urease-nanogold composite material.
The second step is that: and detecting the urea content in the blood sample with unknown concentration.
Taking a blood sample solution with unknown concentration, adding 80 mu L of solution to be detected into the urease-nanogold composite material prepared in the step (1), and repeating the step (3) and the step (4), wherein 820nm infrared laser in the step (3) is used, and 2.5W/cm is used2Irradiating the solution for 20min at the power of (1), and reading gasAnd (4) reading the pressure value, and comparing the pressure value reading with a standard value comparison map to quickly obtain the concentration of the urea in the solution to be detected.
Example four
An embodiment of the method for rapidly detecting the urea concentration is shown in fig. 1, and a schematic diagram of the principle of the method is shown in the figure. The detection method specifically comprises the following steps:
the first step is as follows: preparing urease-nano gold composite material, urea solution with different concentrations, infrared laser with the wavelength of 810nm and a portable barometer.
(1) And (3) synthesizing the urease-nanogold composite material as shown in figure 1.
(1.1) preparation of spherical hollow porous Nanogold
To a round bottom flask containing 18mL of 90mM PVP solution was added 640. mu.L of 100mM hydroquinone, 40. mu.L of 60mM AgNO3The solution is protected from light, 40 mu L of 25mM chloroauric acid is added dropwise while shaking (500r), the solution is placed on a shaking table for continuous reaction for one hour after shaking vigorously for 5min (1000r), and the color of the solution is changed from colorless to blue to light red brick color. Adding 200 mu L of concentrated ammonia water, reacting for 2 hours at 500r under a shaking table, and then deepening the color of the solution; and after the reaction is finished, centrifuging to remove the supernatant, and dispersing into 2mL of water to obtain the spherical hollow porous nanogold with the particle size of 120nm and the concentration of 0.08 mg/mL.
(1.2) Synthesis of urease-Nanogold composite Material
To 12 μ L of urease dissolved in PBS solution (potassium dihydrogen phosphate, pH 4.5) and 0.17mg/mL, 8 μ L of the spherical hollow porous nanogold obtained in step (1.1) was added, and the assembly ratio was 2: 3. Shaking for 10h (500r) at room temperature for self-assembly, centrifuging at 13000rpm for 5min after the assembly is finished, and dissolving with secondary water to obtain the urease-nanogold composite material.
The third step: and detecting the urea content in the urine sample with unknown concentration.
Taking a urine sample solution with unknown concentration, adding 80 mu L of solution to be detected into the urease-nanogold composite material prepared in the step (1), and repeating the step (3) and the step (4), wherein the solution with the concentration of 810nm and the concentration of 1.6W/cm in the step (3) is used2Irradiating the solution with infrared laser at power for 13min, and reading air pressureAnd (4) reading the value, and comparing the pressure value reading with a standard value comparison map to quickly obtain the concentration of the urea in the solution to be detected. The urea concentration of normal urine is known to be in the range of 55-388mM (9.3-23.3g/L), and the detection range meets the determination of the urea concentration in urine.
Example five
An embodiment of the method for rapidly detecting the urea concentration is shown in fig. 1, and a schematic diagram of the principle of the method is shown in the figure. The detection method specifically comprises the following steps:
the first step is as follows: preparing urease-nano gold composite material, urea solution with different concentrations, infrared laser of 800nm and portable barometer.
(1) And (3) synthesizing the urease-nanogold composite material as shown in figure 1.
(1.1) preparation of spherical hollow porous Nanogold
To a round bottom flask containing 18mL of 90mM PVP solution was added 640. mu.L of 100mM hydroquinone, 40. mu.L of 60mM AgNO3The solution is protected from light, 40 mu L of 25mM chloroauric acid is added dropwise while shaking (500r), the solution is placed on a shaking table for continuous reaction for one hour after shaking vigorously for 5min (1000r), and the color of the solution is changed from colorless to blue to light red brick color. Adding 200 mu L of concentrated ammonia water, reacting for 2 hours at 500r under a shaking table, and then deepening the color of the solution; and after the reaction is finished, centrifuging to remove the supernatant, and dispersing into 2mL of water to obtain the spherical hollow porous nanogold with the particle size of 115nm and the concentration of 0.06 mg/mL.
(1.2) Synthesis of urease-Nanogold composite Material
mu.L of the spherical hollow porous nanogold obtained in step (1.1) was added to 15. mu.L of urease dissolved in PBS (potassium dihydrogen phosphate, pH 5.5) at an assembly ratio of 1:3, at 0.20 mg/mL. Shaking for 11h (500r) at room temperature for self-assembly, centrifuging at 13000rpm for 5min after the assembly is finished, and dissolving with secondary water to obtain the urease-nanogold composite material.
The third step: and detecting the urea content in the urine sample with unknown concentration.
Taking a urine sample solution with unknown concentration, adding 80 mu L of solution to be detected into the urease-nanogold composite material prepared in the step (1), and repeating the step (a)3) And a step (4) of using the infrared laser of 800nm in the step (3) with 1.7W/cm2The solution is irradiated for 18min, the reading of the air pressure value is read, and the comparison is carried out by using the reading of the air pressure value and a standard value comparison map, so that the concentration of the urea in the solution to be detected can be quickly obtained. Knowing that the urea concentration of normal urine is in the range of 55-388mM (9.3-23.3g/L), our assay range is satisfactory for determining the urea concentration in urine.
Claims (10)
1. A urea concentration rapid detection method comprises the following steps:
(1) preparing urease-nano gold composite materials, urea solutions with different concentrations, 790-820nm infrared laser and a barometer;
(2) adding a urea solution with one concentration into the urease-nanogold composite material prepared in the step (1) to form a mixed solution;
(3) irradiating the mixed solution in the step (2) by using the infrared laser in the step (1), converting light energy into heat energy by using the photo-thermal property of nano particles in the mixed solution, heating the solution, accelerating the decomposition of urea into carbon dioxide and ammonia gas under the catalysis of urease, and measuring the air pressure value by using a barometer;
(4) recording the air pressure value in the step (3);
(5) respectively repeating the steps (2) to (4) by using urea solutions with different concentrations to obtain different air pressure values;
(6) drawing a standard curve comparison graph according to the urea solutions with different concentrations respectively corresponding to the different air pressure values obtained in the step (5);
(7) and (3) adding a sample solution to be detected with unknown concentration into the urease-nanogold composite material prepared in the step (1), repeating the step (3) and the step (4) to obtain an air pressure value, and contrasting the air pressure value with the standard curve comparison chart in the step (6) to obtain the urea concentration of the solution to be detected.
2. The method for rapidly detecting the concentration of urea according to claim 1, wherein the preparation of the urease-nanogold composite material comprises the following steps:
(1.1) respectively and sequentially adding 640 mu L of hydroquinone solution with the solution concentration of 100mM, 40 mu L of silver nitrate solution with the solution concentration of 60mM and 40 mu L of chloroauric acid solution with the solution concentration of 25mM into 18ml of polyvinylpyrrolidone solution with the solution concentration of 90mM, shaking to change the color of the solution from colorless to blue to light red brick color, adding 200 mu L of concentrated ammonia water, and removing a silver chloride template to obtain spherical hollow porous nanogold;
(1.2) mixing the spherical hollow porous nano-gold in the step (1.1) with a urease solution, wherein the concentration of the spherical hollow porous nano-gold is 0.05-0.10mg/mL, the concentration of the urease solution is 0.10-0.20mg/mL, the spherical hollow porous nano-gold and the urease solution are assembled according to the volume ratio of (1-2) to (3-4), urease is adsorbed on the spherical hollow porous nano-gold, and then the urease-nano-gold composite material is obtained through centrifugal separation, wherein the particle size of the spherical hollow porous nano-gold in the urease-nano-gold composite material is 100-130 nm.
3. The method for rapidly detecting the urea concentration according to claim 2, wherein in the step (1.2), the concentration of the urease solution is 0.15mg/mL, the particle size of the spherical hollow porous nano-gold in the urease-nano-gold composite material is 105nm, the concentration is 0.07mg/mL, and the spherical hollow porous nano-gold and the urease solution are assembled in a volume ratio of 1: 3.
4. The method for rapidly detecting the urea concentration according to claim 2 or 3, wherein in the step (1.2), the spherical hollow porous nano-gold and the urease solution react for 8-12h in a centrifuge tube at room temperature and at a shaker speed of 500r, then the solution is centrifuged for 5min at 13000rpm to remove free urease, and the urease-nano-gold composite material is obtained by dissolving the solution with secondary water.
5. The method for rapid detection of urea concentration according to claim 2, wherein in step (1.2) the urease solution is dissolved by potassium dihydrogen phosphate buffer solution with pH between 4-6.
6. The method for rapidly detecting urea concentration according to claim 1,the method is characterized in that: the pH value of the urease-nanogold composite material adopted in the step (1) is 4-6, the air pressure is measured by using an air pressure gauge under the condition that the temperature of the urease-nanogold composite material is 60-70 ℃ under the irradiation of infrared laser, and the laser irradiation power is 1.5-2.5W/cm2And reading the barometer after the laser irradiation of 790-820nm for 10-20 min.
7. The method for rapidly detecting the concentration of urea according to claim 6, characterized in that: and (2) measuring the air pressure by using an air pressure gauge under the condition that the pH value of the urease-nanogold composite material adopted in the step (1) is 5.4 and the temperature of the urease-nanogold composite material is 60 ℃ under the irradiation of infrared laser. The laser irradiation power was 2.0W/cm2And reading the barometer after irradiating for 15min by using laser at 808 nm.
8. A rapid test method for urea concentration according to claim 1, which is used for testing the urea concentration in human body fluid.
9. The detection sensor obtained by the rapid detection method of urea concentration according to claim 1, which comprises urease-nano gold composite material, 790-820nm infrared laser, barometer and the standard curve comparison chart.
10. The test sensor of claim 9, wherein the barometer is a portable barometer.
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