CN115124992A - Ratio fluorescence sensor based on smart phone and preparation method and application thereof - Google Patents
Ratio fluorescence sensor based on smart phone and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a ratio fluorescence sensor based on a smart phone and a preparation method and application thereof, wherein the preparation method comprises the following steps: mixing chloroauric acid, bovine serum albumin and sodium hydroxide to obtain gold cluster reaction liquid; mixing sodium citrate, 3-aminopropyl triethoxy and glycerol for reaction to obtain a silicon quantum dot solution; reacting the silicon quantum dot solution, lipopolysaccharide aptamer, EDC and NHS in ultrapure water to obtain aptamer-modified silicon quantum dots; and (3) mixing and reacting the silicon quantum dots modified by the lipopolysaccharide aptamer and the gold cluster to obtain the ratiometric fluorescence sensor. The ratio fluorescence sensor based on the smart phone, which is prepared by the invention, has good stability and strong specificity, can eliminate fluorescence interference in an environmental matrix, is used for lipopolysaccharide analysis and detection by combining colorimetric analysis of a color picker of the smart phone, can realize rapid quantitative detection of lipopolysaccharide visible to the naked eye in vitro, and meets the requirement of field monitoring.
Description
Technical Field
The invention belongs to the technical field of analysis and detection, and particularly relates to a ratio fluorescence sensor based on a smart phone, a preparation method of the ratio fluorescence sensor and application of the ratio fluorescence sensor in detection of lipopolysaccharide.
Background
Liver cancer, i.e. malignant tumor of liver, is one of the most common clinical malignant tumors, and the morbidity is still in a high-grade state, and has received close attention. There is increasing evidence that the level of lipopolysaccharide is closely related to the stage of liver cancer development. Lipopolysaccharide, also called bacterial endotoxin, is a main component of gram-negative bacterial cell walls, and trace lipopolysaccharide enters a body to cause infection shock of a target organ and organ failure. Therefore, the rapid detection of lipopolysaccharide is urgently needed, and the method has important significance for health care and quality control of food and medicine. Clinically, lipopolysaccharide detection mainly depends on a limulus reagent, but the reagent is expensive and is greatly influenced by environmental factors such as temperature, pH and the like, so that the detection is insensitive, and the application of the reagent in the biomedical field is greatly limited. Compared with the traditional detection method, the method which is simple, efficient and sensitive to develop is required to be used for rapidly detecting the lipopolysaccharide.
The development of the fluorescence sensing technology makes it possible to develop a sensor independent of limulus reagent, and the quantum dots are considered as a novel fluorescent nano material, and have the advantages of adjustability, good chemical stability and high response speed due to narrow emission spectrum, thus gradually becoming a novel fluorescence sensor. However, since many quantum dot materials contain heavy metal ions which seriously affect the environment, there is a great interest in developing fluorescent probes with good biocompatibility and strong stability. The silicon quantum dots, the gold nanoclusters and the like have low toxicity, stable optical properties have potential application value in the fields of optical detection and biomarkers, and in addition, the research adopts a double-emission ratiometric fluorescent probe, so that the self-calibration effect is achieved, and the detection specificity can be effectively improved. In addition, the ratio fluorescence is combined with a detection device of a smart phone, a simple, quick and convenient reading device is introduced for signal output, and the on-site quick analysis of lipopolysaccharide is easy.
Disclosure of Invention
The invention aims to: aiming at the problems in the prior art, the invention provides a ratio fluorescence sensor based on a smart phone, which improves the detection sensitivity through a ratio fluorescence technology and plays a self-calibration role; meanwhile, the fluorescent sensor has good biocompatibility and light stability, has the characteristic of lipopolysaccharide concentration dependency 'on-off', can be used as a specific lipopolysaccharide concentration indicator, is simple in preparation process, can quickly detect the concentration of lipopolysaccharide by combining a color picker of a smart phone, and particularly realizes specific identification detection and on-site quick detection of lipopolysaccharide.
The technical scheme is as follows: in order to achieve the above object, the present invention provides a method for preparing a ratiometric fluorescence sensor based on a smartphone, comprising the following steps:
(1) mixing chloroauric acid, bovine serum albumin and sodium hydroxide to obtain gold cluster reaction liquid;
(2) mixing sodium citrate, 3-aminopropyl triethoxy and glycerol for reaction to obtain a silicon quantum dot solution;
(3) reacting silicon quantum dot solution, lipopolysaccharide aptamer, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride (EDC & HCl) and N-hydroxysuccinimide (NHS) in ultrapure water to obtain aptamer-modified silicon quantum dots;
(3) adding the silicon quantum dot solution modified by the lipopolysaccharide aptamer into the gold cluster solution to obtain a ratiometric fluorescent probe solution;
and (2) adding the chloroauric acid aqueous solution into the bovine serum albumin aqueous solution in the step (1), uniformly mixing, adding sodium hydroxide, and continuously stirring at 37-40 ℃ for 10-12 hours to obtain a gold cluster reaction solution.
Preferably, in step (1), the chloroauric acid aqueous solution is added into the bovine serum albumin aqueous solution and mixed evenly, and the sodium hydroxide solution is added under vigorous stirring at 37 ℃ for 12 hours, and the solution is observed to turn from bright yellow to red.
Wherein, the mol ratio of chloroauric acid, bovine serum albumin and sodium hydroxide in the step (1) is 5: 4: 8-5: 6: 12.
preferably, the molar ratio of chloroauric acid, bovine serum albumin and sodium hydroxide in the step (1) is 5: 5: 10.
wherein, in the step (2), the sodium citrate solution is added into the glycerol mixed solution, stirred for 15-20 minutes under argon, then the 3-aminopropyl triethoxy solution is added, stirred for 10-15 minutes, and reacted for 1-1.5 hours at 185-200 ℃, and the solution is changed from colorless to yellow to obtain the reaction solution.
Preferably, in the step (2), the sodium citrate solution is added into the glycerol mixed solution, stirred for 15-20 minutes under argon, then the 3-aminopropyl triethoxy solution is added, stirred for 10 minutes gently, and reacted for 1.5 hours at 185 ℃, and the solution is changed from colorless to yellow to obtain a reaction solution.
Further, the above reaction solution should be used immediately upon successful preparation.
Wherein, the mol ratio of the sodium citrate, the glycerol and the 3-aminopropyl triethoxy solution in the step (2) is 1: 84: 6-1: 88: 8.
preferably, the molar ratio of the sodium citrate, the glycerol and the 3-aminopropyltriethoxy solution in the step (2) is 1: 85.7: 6.9.
wherein the sequence of the lipopolysaccharide aptamer in the step (3) is COOH- (CH) 2 ) 6 -TTTTTCTTCTGCCCGCCTCTCTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT。
Wherein, the molar ratio of the lipopolysaccharide aptamer, EDC, NHS and silicon quantum dots in the step (3) is 1: 3.4: 3.4: 1-1: 3.4: 4.3: 2.
preferably, in the step (3), the molar ratio of the lipopolysaccharide aptamer, EDC, NHS and silicon quantum dot is 1: 3.4: 3.4: 1.
and (3) adding the lipopolysaccharide aptamer into the silicon quantum dot obtained in the step (2) and the gold cluster mixed solution obtained in the step (1) and continuously oscillating for 2-3 hours to generate a modified grafted reaction solution at room temperature.
Preferably, the shaking table is continued for 2 hours in the mixed solution of the lipopolysaccharide aptamer modified silicon quantum dot solution and the gold cluster solution in the step (4), and a ratiometric fluorescent probe solution is generated at room temperature.
Preferably, the volume ratio of the silicon quantum dot solution to the gold cluster solution in the step (4) is 1: 1.
the ratio fluorescence sensor based on the smart phone prepared by the preparation method is disclosed by the invention.
The ratio fluorescence sensor based on the smart phone prepared by the preparation method is applied to the rapid visible detection of lipopolysaccharide.
The smartphone color picker is used for analyzing and detecting a ratio fluorescence sensor, analyzing pictures of lipopolysaccharide detection with different concentrations, and establishing a linear curve according to the lipopolysaccharide concentration and an RGB value.
The invention prepares the ratio fluorescence sensor by taking the gold cluster and the silicon dots as the ratio fluorescence sensor for the first time, and is used for detecting lipopolysaccharide for the first time. The invention takes pure water as a solvent, prepares the gold clusters and the silicon quantum dots with good water solubility at normal temperature and normal pressure, takes the gold clusters as built-in correction to eliminate environmental interference, ensures the detection accuracy and the silicon quantum dots have good response to lipopolysaccharide. Lipopolysaccharide is grafted on the surface of the lipopolysaccharide to combine with single-stranded DNA, so that the lipopolysaccharide can be specifically combined with lipopolysaccharide and the change of fluorescence intensity and color is caused, thereby realizing the accurate quantification of the lipopolysaccharide and effectively solving the problem of poor selectivity in lipopolysaccharide detection.
The smart phone color picker selected by the invention has the function of rapidly detecting lipopolysaccharide, and not only is the device simple, but also the technical problems of high cost, low detection speed and the like of the traditional detection mode can be solved. The smartphone color picker reads specific numerical values according to different changes of detection colors of lipopolysaccharides with different concentrations, so that a detector obtains an accurate result.
The ratio fluorescence sensor based on the smart phone prepared by the invention can be used for rapidly and visually detecting the concentration of lipopolysaccharide. According to the ratio fluorescence sensor based on the smart phone, the design of the ratio fluorescence sensor can realize a self-calibration function and can realize visual analysis and detection, and the smart phone color picker can effectively realize intelligent analysis of photo chromaticity under an ultraviolet lamp, so that the detection sensitivity and the analysis speed can be effectively improved. And secondly, the gold cluster is used as built-in correction to avoid the fluorescent background interference of the environment, thereby improving the identification accuracy. And finally, the preparation process of the fluorescent sensor is simple and feasible, and is easy for large-scale production. The invention discloses a preparation method of a ratio fluorescence sensor based on a smart phone, which comprises the following steps: gold clusters and silicon quantum dots with good water solubility are prepared at normal temperature and normal pressure, and the aptamer-modified silicon quantum dots and lipopolysaccharides have good response. The lipopolysaccharide aptamer is combined with the silicon quantum dot through the reaction of carboxyl and amino so as to be used for specific recognition of lipopolysaccharide. The lipopolysaccharide aptamer used in the invention can be specifically combined with lipopolysaccharide, the sensor can perform specific recognition by combining the lipopolysaccharide aptamer with lipopolysaccharide, and the existence of lipopolysaccharide can quench the fluorescence of silicon quantum dots in a probe, so that the fluorescence intensity of the ratiometric fluorescence sensor can be changed when the lipopolysaccharide is added into a probe solution, wherein the fluorescence intensity of red gold clusters is kept unchanged, and the fluorescence intensity of blue silicon quantum dots is gradually reduced. Simultaneously, with the increase of the concentration of the lipopolysaccharide, the fluorescence of the sensor can be converted from blue to red along with the increase of the concentration of the lipopolysaccharide under an ultraviolet lamp, and the chromaticity is analyzed through a smart phone color picker, so that the relation between the concentration of the lipopolysaccharide and the chromaticity is established for the visual and rapid analysis of the concentration of the lipopolysaccharide.
The invention also develops a visual lipopolysaccharide detection method, which is simple, convenient and quick and can meet the requirements of on-site monitoring; the intelligent mobile phone colorimetric analysis is used for analyzing an ultraviolet lamp photo of a ratiometric fluorescent probe, and a tool is provided for accurate and quantitative detection of lipopolysaccharide; the ratiometric fluorescence sensor adopts silicon quantum dots and gold nanoclusters which are good in biocompatibility and stable in fluorescence, and is simple in preparation method, low in price and stable in performance.
The invention firstly proposes that the lipopolysaccharide is rapidly detected on site by using the smart phone, large instruments and equipment are not needed, the cost is low, the device is simple and easy to prepare, and the specificity analysis with high sensitivity is realized. Solves the problems of time-consuming method, high price and low detection specificity in the detection of lipopolysaccharide. According to the material synthesized by the invention, different colors can be presented after lipopolysaccharide with different concentrations is added, and the intelligent mobile phone color picker is used for reading numerical values of different colors generated by reaction.
According to the invention, the RGB values read by the smart phone color pick-up device are adopted, different colors can be presented under an ultraviolet lamp after lipopolysaccharide with different concentrations is added into a sensor solution, the smart phone color pick-up device can analyze the change of chromaticity, and a linear curve can be established according to the change of concentration and chromaticity, so that the method is used for semi-quantitative detection of lipopolysaccharide. If a smart phone color picker is not adopted, the sensor can detect the fluorescence intensity by using fluorescence spectrum for quantitative detection of lipopolysaccharide. When the sensor prepared by the invention is used for detection, fluorescence quenching can occur along with the increase of the concentration of lipopolysaccharide. When the method is used for detection, firstly, the silicon dots are used as responses, the gold clusters are used as references, the interference of the environment can be eliminated, a built-in correction is carried out, secondly, the field detection can be realized more quickly than the plasma by using the smart phone, a large-scale device is not needed, only the smart phone is needed, and the analysis only needs to take pictures to obtain numerical values.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the ratio fluorescence sensor based on the smart phone, prepared by the invention, has the advantages of low background fluorescence interference and light damage, rapid detection, simple device and low cost.
2. The ratio fluorescence sensor based on the smartphone selects the gold clusters and the silicon quantum dots as the double-emission quantum dots, the gold clusters do not respond to the lipopolysaccharide, meanwhile, the silicon quantum dots respond to the lipopolysaccharide, the lipopolysaccharide can be better detected based on the principle without background interference, and the whole sensor has good biocompatibility and stability and can well detect the lipopolysaccharide.
3. The surface of the ratio fluorescence sensor based on the smart phone, which is prepared by the invention, is modified with lipopolysaccharide combined single-stranded DNA, so that the ratio fluorescence sensor can specifically capture lipopolysaccharide in a complex biological medium and generate the change of a fluorescence signal, the near-infrared fluorescence sensor has linear response to the lipopolysaccharide of 50-3000ng/mL, the detection limit is 29.3ng/mL, and the performance is excellent.
5. The preparation process of the ratio fluorescence sensor based on the smart phone is simple and easy to implement, the prepared sensor is good in stability, the interference function of background fluorescence is eliminated, the ratio fluorescence sensor can be used for in-vitro detection of lipopolysaccharide, and meanwhile, the preparation process is simple and easy to implement and easy for large-scale production.
6. The ratio fluorescence sensor based on the smart phone disclosed by the invention is a fluorescence 'turn-off' response to the concentration of lipopolysaccharide, namely, the fluorescence intensity of the fluorescence sensor is gradually reduced along with the increase of the concentration of the lipopolysaccharide, and the effect is obvious.
7. The invention provides possibility for further application of the sensor, and the specificity of detecting lipopolysaccharide by the sensor can be effectively improved through modification of single-stranded DNA specificity recognition.
Drawings
FIG. 1 is a fluorescence emission spectrum of silicon quantum dots (Si QDs) (a), gold clusters (Au NCs) (b) and a ratiometric fluorescence sensor (Au NCs-Si QDs-Apt) prepared according to the present invention;
FIG. 2 is an infrared spectrum of silicon quantum dots (Si QDs) (a), gold clusters (Au NCs) (b) and ratiometric fluorescence sensors (Au NCs-Si QDs-Apt) prepared according to the present invention;
FIG. 3 is a graph showing the temperature stability of ratiometric fluorescence sensors (Au NCs-Si QDs-Apt) prepared in accordance with the present invention;
FIG. 4 is a graph showing the temporal stability of ratiometric fluorescence sensors (Au NCs-Si QDs-Apt) prepared in accordance with the present invention;
FIG. 5 is a graph of the fluorescence emission spectra of ratiometric fluorescence sensors prepared in accordance with the present invention in response to different concentrations of lipopolysaccharide;
FIG. 6 is a linear fit curve of the response of ratiometric fluorescent sensors prepared in accordance with the present invention to different concentrations of lipopolysaccharide;
fig. 7 is a schematic diagram (a) of the detection of the color picker device of the smart phone according to the present invention; a photo (b) of lipopolysaccharide with different concentrations under the wavelength of 365nm by an ultraviolet lamp after being added into the ratio fluorescence sensor; a linear fitting curve of the R/B value and the lipopolysaccharide concentration is obtained through analysis of a smart phone color picker;
FIG. 8 is a graph of the fluorescence response of ratiometric fluorescence sensors prepared in accordance with the present invention with other interferents.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
The chloroauric acid used in the examples was obtained from Shanghai Michelin, Inc.
Lipopolysaccharide aptamer single-stranded DNA synthesis was purchased from Shanghai Biotech Ltd, and has the sequence 5' -COOH- (CH) 2 )6-TTTTTCTTCTGCCCGCCTCTCTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3'。
The smart phone can be any commercially available smart phone, and the smart phone color picker app can read RGB.
Example 1
Preparation of smartphone-based ratiometric fluorescent sensors
1. Preparation of gold Cluster solution
5mL of bovine serum albumin (0.5mM) and 5mL of an aqueous chloroauric acid solution (0.5mM) were mixed well, and 500. mu.L of an aqueous sodium hydroxide solution (0.01M) was added under vigorous stirring to react at 37 ℃ for 12 hours, whereupon it was observed that the reaction liquid turned from bright yellow to red.
2. Preparation of silicon quantum dots
0.3180g of sodium citrate is added into a round-bottom flask, then 8mL of glycerol is added, argon is introduced for protection, stirring is carried out for 20 minutes, 1.89g of 3-aminopropyl triethoxy is added, stirring is carried out for 10 minutes, then the mixture is transferred into an oil bath pot, the mixture is vigorously stirred at 185 ℃ for reaction for 1.5 hours, cooling is carried out to room temperature, colorless change is observed to be yellow solution, the concentration of the silicon quantum dot solution is 0.107mol/L (calculated according to the amount of 3-aminopropyl triethoxy), and the solution is diluted into 0.01mol/L silicon quantum dot solution by secondary water for standby.
3. Preparation of lipopolysaccharide aptamer modified silicon quantum dot
And (3) adding 5mL of the silicon quantum dot solution (0.01mol/L) obtained in the step (2) into EDC & HCl (32mg) and NHS (20mg), ultrasonically dissolving, stirring for reacting for 30min, adding 0.1mL of lipopolysaccharide aptamer solution of 0.5mol/L, and continuing to react at room temperature for 3h to obtain the surface aptamer modified silicon quantum dot solution.
4. Preparation of ratiometric fluorescence sensors
And (4) mixing the reaction liquid obtained in the step (1) and the step (3) according to a volume ratio of 1: 1 and shaking the table at room temperature for 2 hours to obtain a ratio fluorescence sensor solution.
5. Establishment of smart phone color picker device
Preparing lipopolysaccharide with different concentrations, adding into a ratiometric fluorescent probe solution, reading specific color values by using a smart phone color pick-up device under the irradiation of an ultraviolet lamp with the wavelength of 365nm, and calculating the ratio of R/B.
Example 2
Fluorescence emission spectrum of the silicon quantum dot prepared in example 1.
1mL of the pure silicon quantum dot solution prepared in example 1 was weighed.
Fluorescence emission plot testing: the above solutions were tested for fluorescence emission spectra. The fluorescence emission spectrogram scans spectrograms within the wavelength range of 400nm to 750nm by using an excitation wavelength of 380nm and a slit width for excitation and emission of 5nm/5 nm. The obtained fluorescence emission spectrum is shown in figure 1(a), and the fluorescence intensity value indicates that the silicon quantum dots are successfully synthesized.
Example 3
Fluorescence emission spectrum of gold cluster prepared in example 1.
1mL of the pure gold cluster solution prepared in example 1 was weighed.
Fluorescence emission plot testing: the above solutions were tested for fluorescence emission spectra. The fluorescence emission spectrum was scanned at an excitation wavelength of 480nm for a spectrum in the wavelength range of 500nm to 750 nm. The obtained fluorescence emission spectrum is shown in FIG. 1(b), and the maximum emission wavelength position and intensity of the fluorescence spectrum indicate that the gold cluster is successfully synthesized.
Example 4
The aptamer-modified silicon quantum dot solution prepared in example 1, and the gold cluster were mixed in a volume ratio of 1: 1, reacting for 2 hours in a shaking table at room temperature after mixing, scanning a spectrogram in a wavelength range of 400 nm-750 nm by using a fluorescence emission spectrogram with an excitation wavelength of 380nm and excitation and emission slit widths of 5nm/5nm, and indicating that the ratiometric fluorescence sensor (Au NCs-Si QDs-Apt) is successfully prepared as shown in a figure 1 (c).
Example 5
The ratiometric fluorescent sensor prepared in example 1 was characterized by infrared spectroscopy, and the silicon quantum dot solution, the gold nanocluster solution, and the aptamer-modified ratiometric fluorescent sensor solution were lyophilized and then characterized by infrared spectroscopy. The obtained infrared spectrum is shown in FIG. 2, in FIG. 2(a), 3407cm -1 O-H group stretching vibration peak belonging to gold cluster, 1674cm -1 C ═ O carboxyl group stretching vibration peak. The presence of these characteristic peaks demonstrates the successful synthesis of gold nanoclusters. FIG. 2(b)1048cm -1 The peak belongs to Si-O bond stretching vibration peak, 1652cm -1 And 3211cm -1 Belongs to N-H bending vibration and stretching vibration, and proves the synthesis of the silicon quantum dots. In FIG. 2(c), 1241cm -1 The appearance of the phosphate peak proves that the aptamer is successfully grafted to the surface of the silicon quantum dot, and the synthesis of the sensor is structurally proved to be successful.
Example 6
The ratiometric fluorescent sensor solutions prepared in example 1 were respectively placed in solutions at 15 to 45 ℃ for 10min, and changes in fluorescence intensity were detected by fluorescence spectroscopy, as shown in fig. 3, the fluorescence intensity of the ratiometric fluorescent probe was substantially unchanged within the range of 15 to 45 ℃, demonstrating that it has better temperature stability.
Example 7
The ratiometric fluorescence sensor solutions prepared in example 1 were respectively stored in a refrigerator at 4 deg.c, and the change in fluorescence intensity with time was measured, as shown in fig. 4, and the fluorescence intensity of the ratiometric fluorescence probe was substantially constant within 7 days, demonstrating that they had better fluorescence stability.
Example 8
Fluorescence emission spectra of ratiometric fluorescence sensors in response to different concentrations of lipopolysaccharide were prepared in example 1.
0.1mg of lipopolysaccharide and 10mL of pure water are weighed to prepare a mother solution with the concentration of 10ug/mL, the mother solution is used to prepare lipopolysaccharide aqueous solutions with the concentrations of 50, 100, 200, 500, 1000, 2000 and 3000ng/mL respectively, and the ratio fluorescence sensor prepared in the example 1 and the lipopolysaccharides with different concentrations are taken according to the volume ratio of 1: 1, incubation at room temperature for 5 minutes to test the fluorescence emission spectrum. Fluorescence emission spectrometry was performed with excitation at 380nm, with excitation and emission slit widths of 5nm/5 nm. The obtained fluorescence emission spectrum is shown in FIG. 5, FIG. 5 shows that the fluorescence intensity of the silicon quantum dots in the ratiometric fluorescence sensor gradually decreases with the increase of the concentration of lipopolysaccharide, the fluorescence intensity of the gold clusters is basically unchanged, and the fluorescence intensity of the ratiometric fluorescence sensor is linearly related with the fluorescence intensity of the ratiometric fluorescence sensor at the concentration of lipopolysaccharide of 50-3000ng/mL, the fitting curve is shown in FIG. 6, and the fitting curve is that y is 3.929-7.398x (R is R, R is the ratio of y to y, and R is the ratio of y to R, R is the ratio of 3.929-7.398x 2 0.9915), detection limit was 29.3 ng/mL. The ratiometric fluorescent sensor prepared by the invention has the capability of detecting lipopolysaccharide, and meanwhile, fig. 5 can show that the ratiometric fluorescent sensor prepared based on the smartphone has low background fluorescence interference and low light damage.
Example 9
After the solutions of lipopolysaccharide at different concentrations obtained in example 8 were added to the ratiometric fluorescent probe sensor solution, photographs were taken with a smartphone under 365nm uv light, as shown in fig. 7. Then, through RGB (RED, GREEN, BLUE) chromaticity analysis of the color picker app, a linear relation is established according to the lipopolysaccharide concentration and RGB, and a linear curve is used for building the smart phone color pickerThe app device performs color extraction, as shown in fig. 7, the app generates different RGB values, and then establishes a linear curve according to the relationship between the read RGB values and the concentration of lipopolysaccharide, where the linear equation is y-2.46 × 10 -4 x+1.54×10 -5 (R 2 0.9769) to show that a linear relationship is established between RGB obtained by smartphone analysis and lipopolysaccharide concentration, so that the method can be used for analysis of lipopolysaccharide in a sample with unknown concentration.
Example 10
Respectively preparing interference substance (interference) Na with lipopolysaccharide concentration of 1 μ g/mL + The concentration is 0.9mg/mL, and other interference substances are Ca 2+ ，Mg 2+ Bovine Serum Albumin (BSA), glucose (glucose), Adenosine Monophosphate (AMP), Adenosine Diphosphate (ADP), Adenosine Triphosphate (ATP), and citric acid (citrate) were all 50. mu.g/mL. In addition, preparing two-by-two mixed solution of lipopolysaccharide and interference substance, wherein the concentration of lipopolysaccharide in the mixed solution is 1 microgrammer/mL, and the concentration of the interference substance (interference) Na + The concentration is 0.9Mg/mL, and other interference substances Mg 2+ ，Ca 2+ Bovine Serum Albumin (BSA), glucose (glucose), and citric acid (citrate) were all 50. mu.g/mL. Taking the ratio of the fluorescent sensor to lipopolysaccharide based on the smartphone prepared in the embodiment 1, the interfering substance and the mixed solution of the two solutions according to the volume ratio of 1: 1 incubation at room temperature for 5 minutes to test the fluorescence emission spectrum. Fluorescence emission spectrometry was performed with excitation at 380nm, with excitation and emission slit widths of 5nm/5 nm. The obtained fluorescence response condition is shown in fig. 8, the lipopolysaccharide of the fluorescence sensor in fig. 8 has a strong fluorescence enhancement effect, and in addition, under the condition that 50 times of interferents exist, the fluorescence sensor can still realize good fluorescence response without interference of the interferents. The ratio fluorescence sensor based on the smart phone prepared by the invention has good specificity.
Example 11
Example 11 was prepared identically to example 1, except that: adding the chloroauric acid aqueous solution into the bovine serum albumin aqueous solution, uniformly mixing, adding sodium hydroxide, and continuously stirring for 10 hours at 40 ℃ to obtain a gold cluster reaction solution; the mol ratio of chloroauric acid, bovine serum albumin and sodium hydroxide is 5: 4: 8. adding the sodium citrate solution into the glycerol mixed solution in the step (2), stirring for 20 minutes under argon, then adding the 3-aminopropyltriethoxy solution, slightly stirring for 10 minutes, reacting for 1 hour at 200 ℃, and obtaining a reaction solution after the solution turns from colorless to yellow, wherein the molar ratio of the sodium citrate to the glycerol to the 3-aminopropyltriethoxy solution in the step (2) is 1: 84: 6.
example 12
Example 12 was prepared identically to example 1, except that: in the step (1), the molar ratio of the chloroauric acid to the bovine serum albumin to the sodium hydroxide is 5: 6: 12; in the step (2), the molar ratio of the sodium citrate to the glycerol to the 3-aminopropyltriethoxy solution is 1: 88: 8.
sequence listing
<110> university of Nanjing university
<120> ratio fluorescence sensor based on smart phone and preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 91
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tttttcttct gcccgcctct ctcctagccg gatcgcgctg gccagatgat ataaagggtc 60
agccccccag gagacgagat aggcggacac t 91
Claims (10)
1. A preparation method of a ratio fluorescence sensor based on a smart phone is characterized by comprising the following steps:
(1) mixing chloroauric acid, bovine serum albumin and sodium hydroxide to obtain gold cluster reaction liquid;
(2) mixing sodium citrate, 3-aminopropyl triethoxy and glycerol for reaction to obtain a silicon quantum dot solution;
(3) reacting silicon quantum dot solution, lipopolysaccharide aptamer, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in ultrapure water to obtain aptamer-modified silicon quantum dot;
(4) and (3) mixing and reacting the silicon quantum dots modified by the lipopolysaccharide aptamer and the gold cluster to obtain the ratiometric fluorescence sensor.
2. The preparation method according to claim 1, wherein the aqueous solution of chloroauric acid is added into the aqueous solution of bovine serum albumin and mixed evenly in the step (1), then sodium hydroxide is added, and the mixture is continuously stirred for 10 to 12 hours at 37 to 40 ℃ to obtain gold cluster reaction liquid; the mol ratio of the chloroauric acid to the bovine serum albumin to the sodium hydroxide is 5: 4: 8-5: 6: 12.
3. the method according to claim 1, wherein in the step (2), the sodium citrate solution is added into the glycerol mixed solution, stirring is carried out for 15-20 minutes under argon, then the 3-aminopropyl triethoxy solution is added, stirring is carried out for 10-15 minutes, reaction is carried out for 1-1.5 hours at 185-200 ℃, and the solution is changed from colorless to yellow to obtain the reaction solution.
4. The method according to claim 1, wherein the molar ratio of the sodium citrate, the glycerol and the 3-aminopropyltriethoxy solution in the step (2) is 1: 84: 6-1: 88: 8.
5. the preparation method of claim 1, wherein the silicon quantum dots are added into ultrapure water in the step (3), then EDC and NHS are added, the ultrapure water is completely dissolved by ultrasonic, the lipopolysaccharide aptamer solution is added after stirring for 25-30min, and the reaction is continued to obtain the aptamer-modified silicon quantum dot solution.
6. The method according to claim 1, wherein the sequence of the lipopolysaccharide aptamer in step (3) is preferably 5' -COOH- (CH) 2 ) 6 -TTTTTCTTCTGCCCGCCTCTCTCCTAGCCGGATCGCGCTGGCCAGATGATATAAAGGGTCAGCCCCCCAGGAGACGAGATAGGCGGACACT-3′。
7. The method according to claim 1, wherein the lipopolysaccharide aptamer modified silicon quantum dot in the step (4) is added to the gold cluster solution obtained in the step (1) and continuously oscillated for 2 to 3 hours to generate a ratiometric fluorescent probe solution at room temperature.
8. A smartphone-based ratiometric fluorescence sensor made by the method of claim 1.
9. The application of the ratio fluorescence sensor based on the smart phone prepared by the preparation method of claim 1 in rapid visual detection of lipopolysaccharide.
10. The application of claim 9, wherein the smartphone color picker is used for analytical detection of a ratiometric fluorescence sensor, photographs of lipopolysaccharide detection at different concentrations are analyzed, a linear curve is established according to the lipopolysaccharide concentration and the RGB values, and the lipopolysaccharide concentration in the sample is obtained according to the RGB values of the sample.
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