A smartphone-based ratiometric fluorescent sensor and its preparation method and application

Abstract

The application discloses a kind of ratio fluorescent sensor based on smart phone and its preparation method and application, the preparation method includes: after mixing chloroauric acid, bovine serum albumin, sodium hydroxide, gold cluster reaction liquid is obtained;Sodium citrate, 3-aminopropyl triethoxysilane, glycerol are mixed to obtain silicon quantum dot solution;Silicon quantum dot solution, lipopolysaccharide aptamer, EDC, NHS are reacted in ultrapure water to obtain aptamer modified silicon quantum dots;Lipopolysaccharide aptamer modified silicon quantum dots and gold cluster are mixed to obtain ratio fluorescent sensor.The ratio fluorescent sensor based on smart phone prepared by the application has good stability, strong specificity, can exclude fluorescent interference in environmental matrix, and is used for lipopolysaccharide analysis and detection by combining the colorimetric analysis of color picker of smart phone, so that in-vitro naked-eye visible rapid quantitative detection of lipopolysaccharide can be realized, to meet the demand of on-site monitoring.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analysis and detection, and particularly relates to a ratio fluorescent sensor based on a smart phone, a preparation method thereof and application in detection of lipopolysaccharide. BACKGROUND

[0002] Hepatocarcinoma, i.e. liver malignant tumor, is one of the most common malignant tumors in clinic, and the morbidity is still at a high level, which has attracted close attention. More and more evidence shows that the level of lipopolysaccharide is closely related to the development stage of hepatocarcinoma. Lipopolysaccharide, also known as bacterial endotoxin, is the main component of the cell wall of gram-negative bacteria. A trace amount of lipopolysaccharide entering the body will cause target organ infection shock and organ failure. Therefore, it is urgent to detect lipopolysaccharide quickly, which is of great significance to health care and food and drug quality control. The detection of lipopolysaccharide in clinic mainly depends on limulus reagent. However, the reagent is not only expensive, but also greatly affected by environmental factors such as temperature and pH, resulting in low sensitivity of detection, which greatly limits its application in the field of biomedicine. Compared with the traditional detection method, it is necessary to develop a simple, efficient and sensitive method for rapid detection of lipopolysaccharide.

[0003] The development of fluorescent sensing technology makes it possible to develop a sensor that does not depend on limulus reagent. Quantum dots are considered as a new type of fluorescent nanomaterials, which have a narrow emission spectrum, adjustable, good chemical stability, fast response speed, and gradually become a new type of fluorescent sensor. However, many quantum dot materials contain heavy metal ions, which have a serious impact on the environment. Therefore, the development of fluorescent probes with good biocompatibility and strong stability has attracted widespread interest. Silicon quantum dots and gold nanoclusters have low toxicity and stable optical properties, which have potential application value in optical detection and biological labeling. In addition, the ratio fluorescent probe with double emission in this research has a self-calibration effect, which can effectively improve the specificity of detection. In addition, the combination of the ratio fluorescent probe and the detection device of the smart phone introduces a simple, fast and convenient reading device for signal output, which is easy for on-site rapid lipopolysaccharide analysis. This research not only has important basic scientific research value, but also has important significance in the fields of food safety, environmental monitoring and clinical diagnosis, and has certain economic benefit potential. SUMMARY

[0004] Invention purposes: In view of the problems existing in the prior art, the present application provides a ratio fluorescent sensor based on a smart phone, which improves the sensitivity of detection through ratio fluorescent technology and plays a self-calibration role; meanwhile, the fluorescent sensor has good biocompatibility and light stability, has a lipopolysaccharide concentration-dependent 'on-off' characteristic, can be used as a specific lipopolysaccharide concentration indicator, and its preparation process is simple, combined with the color picker of a smart phone, the concentration of lipopolysaccharide can be quickly detected, especially the specific recognition and detection of lipopolysaccharide and on-site rapid detection can be realized.

[0005] Technical scheme: In order to achieve the above-mentioned purpose, the preparation method of the ratio fluorescent sensor based on a smart phone provided by the present application comprises the following steps:

[0006] (1) mixing chloroauric acid, bovine serum albumin and sodium hydroxide to obtain a gold cluster reaction solution;

[0007] (2) mixing sodium citrate, 3-aminopropyltriethoxysilane and glycerol to obtain a silicon quantum dot solution;

[0008] (3) reacting the silicon quantum dot solution, a lipopolysaccharide aptamer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) in ultrapure water to obtain an aptamer-modified silicon quantum dot;

[0009] (3) adding the lipopolysaccharide aptamer-modified silicon quantum dot solution into the gold cluster solution to obtain a ratio fluorescent probe solution;

[0010] In step (1), the aqueous solution of chloroauric acid is added to the aqueous solution of bovine serum albumin and mixed, then sodium hydroxide solution is added, and the mixture is continuously stirred at 37-40℃ for 10-12 hours to obtain the gold cluster reaction solution.

[0011] As a preferred, in step (1), the aqueous solution of chloroauric acid is added to the aqueous solution of bovine serum albumin and mixed, and the sodium hydroxide solution is added under vigorous stirring, and the solution is observed to change from bright yellow to red after continuously stirring at 37℃ for 12 hours.

[0012] In step (1), the molar ratio of chloroauric acid, bovine serum albumin and sodium hydroxide is 5:4:8-5:6:12.

[0013] As a preferred, in step (1), the molar ratio of chloroauric acid, bovine serum albumin and sodium hydroxide is 5:5:10.

[0014] Wherein, in step (2), the sodium citrate solution is added to the glycerol mixed solution, stirred for 15-20 minutes under argon, then the 3-aminopropyl triethoxysilane solution is added and stirred for 10-15 minutes, the solution is changed from colorless to yellow after reaction at 185-200℃ for 1-1.5h to obtain the reaction solution.

[0015] As a preferred, in step (2), the sodium citrate solution is added to the glycerol mixed solution, stirred for 15-20 minutes under argon, then the 3-aminopropyl triethoxysilane solution is added and stirred for 10 minutes, the solution is changed from colorless to yellow after reaction at 185℃ for 1.5h to obtain the reaction solution.

[0016] Further, the above reaction solution should be used immediately after successful preparation.

[0017] Wherein, in step (2), the molar ratio of sodium citrate, glycerol and 3-aminopropyl triethoxysilane solution is 1:84:6-1:88:8.

[0018] As a preferred, in step (2), the molar ratio of sodium citrate, glycerol and 3-aminopropyl triethoxysilane solution is 1:85.7:6.9.

[0019] Wherein, in step (3), the lipopolysaccharide aptamer sequence is COOH–(CH2)6-TTTTTCTTCTGCCCGCCTCTCTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT.

[0020] Wherein, in step (3), the molar ratio of lipopolysaccharide aptamer, EDC, NHS and silicon quantum dots is 1:3.4:3.4:1-1:3.4:4.3:2.

[0021] As a preferred, in step (3), the molar ratio of lipopolysaccharide aptamer, EDC, NHS and silicon quantum dots is 1:3.4:3.4:1.

[0022] Wherein, in step (3), the lipopolysaccharide aptamer is added to the mixture of silicon quantum dots of step (2) and gold cluster solution, and the mixture is continuously oscillated for 2-3 hours to generate the modified grafted reaction solution at room temperature.

[0023] As a preferred, in step (4), the mixture of lipopolysaccharide aptamer modified silicon quantum dot solution and gold cluster solution is continuously shaken for 2 hours to generate the ratio fluorescent probe solution at room temperature.

[0024] As a preferred, in step (4), the volume ratio of silicon quantum dot solution and gold cluster solution is 1:1.

[0025] The preparation method of the application prepares a smartphone-based ratio fluorescent sensor.

[0026] The application of the smartphone-based ratio fluorescent sensor prepared by the preparation method of the application in rapid visible detection of lipopolysaccharide.

[0027] The smartphone color picker is used for analysis and detection of the ratio fluorescent sensor, photos of detection of different concentrations of lipopolysaccharide are analyzed, and a linear curve is established according to the concentration of lipopolysaccharide and the RGB value.

[0028] The application first uses gold clusters and silicon quantum dots as preparation of a ratio fluorescent sensor, and is first used for detection of lipopolysaccharide. The application uses pure water as a solvent to prepare gold clusters and silicon quantum dots with good water solubility at normal temperature and pressure. The gold clusters are used as built-in correction to exclude environmental interference and ensure the accuracy of detection, and the silicon quantum dots respond well to lipopolysaccharide. A single-stranded DNA is connected to the surface of the lipopolysaccharide to enable specific binding with lipopolysaccharide and cause changes in fluorescence intensity and color, thereby realizing accurate quantification of lipopolysaccharide and effectively solving the problem of poor selectivity in detection of lipopolysaccharide.

[0029] The selected smartphone color picker has the function of rapidly detecting lipopolysaccharide, is simple in device, and can solve the technical problems of high cost and slow detection speed in traditional detection methods. The smartphone color picker reads specific numerical values according to the color changes of different concentrations of lipopolysaccharide, so that the detector can obtain accurate results.

[0030] The ratio fluorescent sensor based on the smart phone prepared by the application can be used for rapid visual detection of the concentration of lipopolysaccharide. The ratio fluorescent sensor based on the smart phone can realize self-calibration and visual analysis and detection, the smart phone color picker can effectively realize the intelligent analysis of the colorimetry of the photo under the ultraviolet lamp, so that the sensitivity and analysis speed of detection can be effectively improved. Secondly, the gold cluster as the built-in correction can avoid the fluorescent background interference of the environment, so as to improve the accuracy of identification. Finally, the preparation process of the fluorescent sensor is simple and easy to scale production. The preparation method of the ratio fluorescent sensor based on the smart phone of the application is as follows: gold clusters and silicon quantum dots with good water solubility are prepared at normal temperature and pressure, and the aptamer modified silicon quantum dots and lipopolysaccharide have good response. The lipopolysaccharide aptamer is combined with the silicon quantum dots by the reaction of carboxyl and amino, so as to be used for specific identification of lipopolysaccharide. The lipopolysaccharide aptamer used in the application can specifically bind with lipopolysaccharide, and the sensor can specifically identify lipopolysaccharide by combining the lipopolysaccharide aptamer with lipopolysaccharide. Since the presence of lipopolysaccharide can quench the fluorescence of the silicon quantum dots in the probe, when the lipopolysaccharide is added to the probe solution, the fluorescence intensity of the ratio fluorescent sensor will change, wherein the fluorescence intensity of the red gold cluster remains unchanged, and the fluorescence intensity of the blue silicon quantum dots gradually decreases. At the same time, with the increase of the concentration of lipopolysaccharide, the fluorescence of the sensor under the ultraviolet lamp will change from blue to red with the increase of the concentration of lipopolysaccharide, and the colorimetry is analyzed by the smart phone color picker, so as to establish the relationship between the concentration of lipopolysaccharide and the colorimetry, and to realize the visual rapid analysis of the concentration of lipopolysaccharide.

[0031] The application also develops a visual lipopolysaccharide detection method, which is simple, fast and can meet the needs of on-site monitoring; the colorimetry of the smart phone is used for the analysis of the ultraviolet lamp photo of the ratio fluorescent probe, which provides a tool for accurate quantitative detection of lipopolysaccharide; the ratio fluorescent sensor adopts silicon quantum dots and gold nanoclusters with good biocompatibility and stable fluorescence, and has the advantages of simple preparation method, low price and stable performance.

[0032] The application first proposes to use a smart phone to realize rapid on-site detection of lipopolysaccharide, which does not require large instruments and equipment, has low cost, simple device and easy preparation, and has high sensitivity and specificity. The problems of time-consuming, high cost and low detection specificity in the detection of lipopolysaccharide are solved. The material synthesized in the application will show different colors after different concentrations of lipopolysaccharide are added, and the different colors generated in the reaction are read by using the smart phone color picker.

[0033] The present application adopts the RGB value read by the smart phone color picker, after different concentrations of lipopolysaccharide are added to the sensor solution, different colors will be presented under the ultraviolet lamp, the smart phone color picker can analyze the chroma change, and a linear curve can be established according to the concentration and the chroma change, so as to be used for the semi-quantitative detection of lipopolysaccharide. If the smart phone color picker is not used, the sensor of the present application can be used for the quantitative detection of lipopolysaccharide by detecting the fluorescence intensity by fluorescence spectrum. When the sensor prepared in the present application is detected, with the increase of the concentration of lipopolysaccharide, fluorescence quenching will occur. When the present application is detected, the silicon point is used as the response, and the gold cluster is used as the reference, so that the environmental interference can be eliminated, and an internal correction is made, and then the smart phone is used, so that the on-site detection can be realized more quickly than the plasma, and a large device is not needed, and only the smart phone is needed, and the analysis only needs to take a photo to obtain the value.

[0034] Advantages: Compared with the prior art, the present application has the following advantages:

[0035] 1. The ratio fluorescent sensor based on a smart phone prepared in the present application has the advantages of low background fluorescence interference, low light damage, rapid detection, simple device and low cost.

[0036] 2. The ratio fluorescent sensor based on a smart phone prepared in the present application selects gold clusters and silicon quantum dots as double-emitting quantum dots, the gold clusters have no response to lipopolysaccharide, and the silicon quantum dots have a response to lipopolysaccharide, based on this principle, the lipopolysaccharide can be better detected without background interference, the whole sensor has good biocompatibility and stability, and can well detect lipopolysaccharide.

[0037] 3. The ratio fluorescent sensor based on a smart phone prepared in the present application is modified with lipopolysaccharide binding single-stranded DNA, so that it can specifically capture lipopolysaccharide in a complex biological medium and produce a change in fluorescence signal. The near-infrared fluorescent sensor has a linear response to 50-3000 ng / mL of lipopolysaccharide, and the detection limit is 29.3 ng / mL, and the performance is excellent.

[0038] 5. The preparation process of the ratio fluorescent sensor based on a smart phone in the present application is simple and easy to implement, and the prepared sensor has good stability and background fluorescence interference elimination function, and can be used for in vitro detection of lipopolysaccharide. At the same time, the preparation process is simple and easy to implement, and easy to scale production.

[0039] 6. The ratio fluorescent sensor based on a smart phone in the present application has a fluorescence 'off' response to the concentration of lipopolysaccharide, that is, with the increase of the concentration of lipopolysaccharide, the fluorescence intensity of the fluorescent sensor gradually decreases, and the effect is obvious.

[0040] 7. The present application provides the possibility for further application, and the modification of specific recognition of single-stranded DNA can effectively improve the specificity of the sensor in detecting lipopolysaccharide. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Fluorescence emission spectra of silicon quantum dots (Si QDs) (a), gold nanoclusters (Au NCs) (b) and the ratiometric fluorescent sensor (Au NCs-Si QDs-Apt) prepared in the present application;

[0042] Figure 2 Infrared spectra of silicon quantum dots (Si QDs) (a), gold nanoclusters (Au NCs) (b) and the ratiometric fluorescent sensor (Au NCs-Si QDs-Apt) prepared in the present application;

[0043] Figure 3 Temperature stability of the ratiometric fluorescent sensor (Au NCs-Si QDs-Apt) prepared in the present application;

[0044] Figure 4 Time stability of the ratiometric fluorescent sensor (Au NCs-Si QDs-Apt) prepared in the present application;

[0045] Figure 5 Fluorescence emission spectra of the ratiometric fluorescent sensor prepared in the present application in response to different concentrations of lipopolysaccharide;

[0046] Figure 6 Linear fitting curve of the ratiometric fluorescent sensor prepared in the present application in response to different concentrations of lipopolysaccharide;

[0047] Figure 7 Detection principle diagram of the color picker device of the smart phone installed in the present application (a); photograph under the wavelength of 365 nm of the ultraviolet lamp after different concentrations of lipopolysaccharide were added into the ratiometric fluorescent sensor (b); linear fitting curve of the R / B value obtained by the color picker analysis of the smart phone and the concentration of lipopolysaccharide;

[0048] Figure 8 Fluorescence response of the ratiometric fluorescent sensor prepared in the present application to other interferents. DETAILED DESCRIPTION

[0049] The present application can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the content described in the examples is only for illustrating the present application, and should not and will not limit the present application described in detail in the claims.

[0050] The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0051] Among them, the chloroauric acid used in the examples is purchased from Shanghai Maikelin Co., Ltd.

[0052] Lipopolysaccharide aptamer single-stranded DNA synthesis was purchased from Shanghai Biotech Co., Ltd., and the sequence was 5'-COOH-(CH2)6-TTTTTCTTCTGCCCGCCTCTCTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3'.

[0053] Smartphone Any commercially available smartphone can be used, and the smartphone color picker app can read RGB.

[0054] Example 1

[0055] Preparation of a smartphone-based ratiometric fluorescent sensor

[0056] 1. Preparation of gold cluster solution

[0057] Mix 5 mL of bovine serum albumin (0.5 mM) with 5 mL of chloroauric acid aqueous solution (0.5 mM), and under vigorous stirring, add 500 μL of sodium hydroxide aqueous solution (0.01 M) and react at 37°C for 12 hours. Observe that the reaction solution changes from bright yellow to red.

[0058] 2. Preparation of silicon quantum dots

[0059] Add 0.3180 g of sodium citrate to a round-bottom flask, then add 8 mL of glycerol, and protect with argon gas. Stir for 20 minutes, then add 3-aminopropyl triethoxyl 1.89 g, and stir for 10 minutes. Then transfer to an oil bath, and react at 185°C under vigorous stirring for 1.5 hours. Cool to room temperature, and observe that the colorless solution turns yellow. The concentration of the obtained silicon quantum dot solution is 0.107 mol / L (calculated based on the amount of 3-aminopropyl triethoxyl). Dilute with water to obtain a 0.01 mol / L silicon quantum dot solution for later use.

[0060] 3. Preparation of lipopolysaccharide aptamer-modified silicon quantum dots

[0061] Add 5 mL of the silicon quantum dot solution (0.01 mol / L) obtained in step (2), EDC-HCl (32 mg), and NHS (20 mg). After ultrasonic dissolution, stir for 30 min, then add 0.1 mL of 0.5 mol / L lipopolysaccharide aptamer solution, and continue to react at room temperature for 3 h to obtain the surface aptamer-modified silicon quantum dot solution.

[0062] 4. Preparation of ratiometric fluorescent sensor

[0063] Mix the reaction solutions obtained in steps (1) and (3) in a volume ratio of 1:1, and shake on a shaker at room temperature for 2 hours to obtain the ratiometric fluorescent sensor solution.

[0064] 5. Building of smartphone color picker device

[0065] Different concentrations of lipopolysaccharide were prepared and added to the ratiometric fluorescent probe solution. Under the irradiation of a wavelength of 365 nm ultraviolet light, the color-specific numerical values were read by the smartphone color picker, and the R / B ratio was calculated.

[0066] Example 2

[0067] Fluorescence emission spectrum of the silicon quantum dots prepared in Example 1.

[0068] 1 mL of the pure silicon quantum dot solution prepared in Example 1 was weighed.

[0069] Fluorescence emission spectrum test: The fluorescence emission spectrum of the above solution was tested. The fluorescence emission spectrum was scanned in the wavelength range of 400 nm-750 nm with an excitation wavelength of 380 nm and an excitation and emission slit width of 5 nm / 5 nm. The obtained fluorescence emission spectrum is shown in FIG. 1(a), and the fluorescence intensity values indicate the successful synthesis of silicon quantum dots. Figure 1

[0070] Example 3

[0071] Fluorescence emission spectrum of the gold clusters prepared in Example 1.

[0072] 1 mL of the pure gold cluster solution prepared in Example 1 was weighed.

[0073] Fluorescence emission spectrum test: The fluorescence emission spectrum of the above solution was tested. The fluorescence emission spectrum was scanned in the wavelength range of 500 nm-750 nm with an excitation wavelength of 480 nm. The obtained fluorescence emission spectrum is shown in FIG. 2(b), and the fluorescence spectrum maximum emission wavelength position and intensity indicate the successful synthesis of gold clusters. Figure 1

[0074] Example 4

[0075] The aptamer-modified silicon quantum dot solution prepared in Example 1 and the gold clusters were mixed in a volume ratio of 1:1 and reacted on a shaking table at room temperature for 2 hours. The fluorescence emission spectrum was scanned in the wavelength range of 400 nm-750 nm with an excitation wavelength of 380 nm and an excitation and emission slit width of 5 nm / 5 nm. The obtained fluorescence emission spectrum is shown in FIG. 3(c), indicating the successful preparation of the ratiometric fluorescent sensor (Au NCs-Si QDs-Apt). Figure 1

[0076] Example 5

[0077] ​​​The ratio fluorescent sensor prepared in Example 1 was characterized by infrared spectrum. The silicon quantum dot solution, the gold nanocluster solution and the aptamer modified ratio fluorescent sensor solution were freeze-dried respectively, and then characterized by infrared spectrum. The obtained infrared spectrum is shown in Figure 2 Figure 2 (a) 3407 cm -1 belongs to the O-H group stretching vibration peak of gold cluster, 1674 cm -1 is the C=O carboxyl stretching vibration peak. The presence of these characteristic peaks proves the successful synthesis of gold nanoclusters. Figure 2 (b) 1048 cm -1 peak belongs to Si-O stretching vibration peak, 1652 cm -1 and 3211 cm -1 belong to N-H bending vibration and stretching vibration, which proves the synthesis of silicon quantum dots. Figure 2 (c) 1241 cm -1 The appearance of the phosphate peak proves that the aptamer is successfully grafted to the surface of the silicon quantum dot, which proves the successful synthesis of the sensor from the structure.

[0078] Example 6

[0079] The ratio fluorescent sensor solution prepared in Example 1 was placed in a 15-45℃ solution for 10 min respectively, and the change of fluorescence intensity was detected by fluorescence spectrum, as shown in Figure 3 The fluorescence intensity of the ratio fluorescent probe is basically unchanged in the range of 15-45℃, which proves that it has good temperature stability.

[0080] Example 7

[0081] The ratio fluorescent sensor solution prepared in Example 1 was placed in a 4℃ refrigerator for storage, and the change of fluorescence intensity with time was measured, as shown in Figure 4 The fluorescence intensity of the ratio fluorescent probe is basically unchanged in the range of 7 days, which proves that it has good fluorescence stability.

[0082] Example 8

[0083] The ratio fluorescent sensor prepared in Example 1 was placed in a 4℃ refrigerator for storage, and the change of fluorescence intensity with time was measured, as shown in

[0084] ​Take 0.1 mg of lipopolysaccharide and add 10 mL of pure water to prepare a mother liquor with a concentration of 10 ug / mL. Use the mother liquor to prepare lipopolysaccharide aqueous solutions with concentrations of 50, 100, 200, 500, 1000, 2000, and 3000 ng / mL, respectively. Take the ratio fluorescent sensor prepared in Example 1 and different concentrations of lipopolysaccharide according to the volume ratio of 1:1, incubate at room temperature for 5 minutes, and test the fluorescence emission spectrum. The fluorescence emission spectrum is determined by excitation at 380 nm, and the excitation and emission slit width is 5 nm / 5 nm. The obtained fluorescence emission spectrum is shown in Figure 5 , Figure 5 It is shown that as the concentration of lipopolysaccharide increases, the fluorescence intensity of silicon quantum dots in the ratio fluorescent sensor gradually decreases, while the fluorescence intensity of gold clusters remains basically unchanged. When the concentration of lipopolysaccharide is in the range of 50-3000 ng / mL, the fluorescence intensity of the ratio fluorescent sensor is linearly related, and the fitting curve is shown in Figure 6 The fitting curve is y = 3.929-7.398x (R 2 = 0.9915), and the detection limit is 29.3 ng / mL. It is shown that the ratio fluorescent sensor prepared by the present application has the ability to detect lipopolysaccharide, and at the same time Figure 5 It can be shown that the ratio fluorescent sensor based on the smart phone prepared has low background fluorescence interference and low light damage.

[0085] Example 9

[0086] According to the different concentrations of lipopolysaccharide solutions obtained in Example 8, after adding the ratio fluorescent probe sensor solution, the smart phone is photographed under the irradiation of 365 nm ultraviolet lamp, as shown in Figure 7 Then through the RGB (RED, GREEN, BLUE) colorimetric analysis of the color picker app, a linear relationship between the concentration of lipopolysaccharide and RGB is established, and the linear curve is shown in Figure 7 The app generates different RGB values, and then a linear curve is established between the read RGB values and the concentration of lipopolysaccharide, and the linear equation is y = 2.46 x 10 -4 x + 1.54 x 10 -5 (R 2 = 0.9769), which shows that a linear relationship between the RGB obtained by the smart phone analysis and the concentration of lipopolysaccharide is established, so it can be used for the analysis of lipopolysaccharide in unknown concentration samples.

[0087] Example 10

[0088] Respectively configure the concentration of lipopolysaccharide to be 1 ug / mL, the concentration of interference Na + is 0.9 mg / mL, and other interference substances Ca 2+ , Mg 2+, bovine serum albumin (BSA), gluocose, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), citrate were all 50 μg / mL. In addition, two two mixed solutions of lipopolysaccharide and interference were prepared, and the concentration of lipopolysaccharide in the mixed solution was 1 μg / mL, and the interference Na + , the concentration of the other interference substance Mg 2+ , Ca 2+ , bovine serum albumin (BSA), gluocose, citrate were all 50 μg / mL. Take the preparation of the smart phone based on the ratio of fluorescence sensor and lipopolysaccharide, interference and two two mixed solution in example 1 respectively according to the volume ratio of 1:1 at room temperature for 5 minutes to test the fluorescence emission spectrum. The fluorescence emission spectrum was measured with 380nm excitation, and the excitation and emission slit width was 5nm / 5nm. The obtained fluorescence response is shown in Figure 8 , Figure 8 The fluorescence sensor has strong fluorescence enhancement effect, and in the presence of 50 times of interference, the fluorescence sensor can still realize good fluorescence response without the interference of the interference. It shows that the smart phone based on the ratio of fluorescence sensor prepared by the application has good specificity.

[0089] Example 11

[0090] Example 11 and example 1 preparation method is same, the difference is: in step (1) the chloroauric acid aqueous solution is added to the bovine serum protein aqueous solution, and then sodium hydroxide is added, and the stirring is continued at 40℃ for 10 hours to obtain the gold cluster reaction solution; the molar ratio of chloroauric acid, bovine serum protein and sodium hydroxide is 5:4:8. In step (2), the sodium citrate solution is added to the glycerol mixed solution, and stirred under argon for 20 minutes, then 3-aminopropyl triethoxy solution is added and stirred for 10 minutes, and the solution is changed from colorless to yellow after reaction at 200℃ for 1h to obtain the reaction solution. The molar ratio of sodium citrate, glycerol and 3-aminopropyl triethoxy solution in step (2) is 1:84:6.

[0091] Example 12

[0092] Example 12 and example 1 preparation method is same, the difference is: in step (1) the molar ratio of chloroauric acid, bovine serum protein and sodium hydroxide is 5:6:12; in step (2) the molar ratio of sodium citrate, glycerol and 3-aminopropyl triethoxy solution is 1:88:8.

Claims

1. The application of a smartphone-based ratio fluorescence sensor in the preparation of a reagent for rapid and visual detection of lipopolysaccharide, wherein the detection involves incubating the ratio fluorescence sensor and lipopolysaccharide solution at a volume ratio of 1:1 at room temperature for 5 minutes and then testing the fluorescence emission spectrum. The fluorescence emission spectrum is measured with excitation at 380 nm and the slit width between excitation and emission is 5 nm / 5 nm. The method for fabricating the smartphone-based ratio fluorescence sensor includes the following steps: (1) Chloroauric acid, bovine serum albumin and sodium hydroxide were mixed to obtain a gold cluster reaction solution; (2) Sodium citrate, 3-aminopropyltriethoxysilane, and glycerol were mixed and reacted to obtain a silicon quantum dot solution; (3) Silicon quantum dot solution, lipopolysaccharide aptamer, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were reacted in ultrapure water to obtain aptamer-modified silicon quantum dots. (4) A ratiometric fluorescence sensor was obtained by mixing and reacting lipopolysaccharide aptamer-modified silicon quantum dots with gold clusters; The lipopolysaccharide aptamer sequence is 5'-COOH-(CH2)6-TTTTTCTTCTGCCCGCCTCTCTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3'; In step (1), chloroauric acid aqueous solution is added to bovine serum albumin aqueous solution and mixed well. Then sodium hydroxide is added and stirred continuously at 37-40℃ for 10-12 hours to obtain gold cluster reaction solution. The molar ratio of chloroauric acid, bovine serum albumin and sodium hydroxide is 5:(4-6):(8-12). In step (2), sodium citrate solution is added to glycerol mixture and stirred under argon for 15-20 minutes. Then, 3-aminopropyltriethoxysilane solution is added and stirred gently for 10-15 minutes. The reaction is carried out at 185-200℃ for 1-1.5 h. The solution changes from colorless to yellow to obtain the reaction solution. In step (2), the molar ratio of sodium citrate, glycerol, and 3-aminopropyltriethoxy solution is 1:(84-88):(6-8). In step (3), silicon quantum dots are added to ultrapure water, followed by EDC and NHS. The mixture is completely dissolved in the ultrapure water by sonication. After stirring for 25-30 min, a lipopolysaccharide aptamer solution is added to the mixture, and the reaction continues to obtain an aptamer-modified silicon quantum dot solution. In step (4), the lipopolysaccharide aptamer-modified silicon quantum dots are added to the gold cluster solution obtained in step (1) and continuously oscillated for 2-3 hours to generate a ratio fluorescent probe solution at room temperature.

2. The application according to claim 1, characterized in that, The smartphone color picker was used for analysis and detection by a ratiometric fluorescence sensor. Photos of lipopolysaccharide (LPS) at different concentrations were analyzed. A linear curve was established based on the LPS concentration and RGB values. The LPS concentration in the sample was obtained from the RGB values ​​of the sample.

Images