Detection of Residual Amoxicillin in Breast Milk by Combining Aptamer Chromogenic Assay Based on Gold Nanoparticles (AuNPs) with a Smartphone

Abstract

The present patent invents a method for detecting amoxicillin in breast milk based on the colorimetric method of aptamer combined with gold nanoparticles (AuNPs) and a smartphone. It is characterized in that the aptamer conjugated with gold nanoparticles specifically binds to amoxicillin in breast milk. In the presence of a salt solution, different concentrations of amoxicillin can cause different degrees of aggregation of gold nanoparticles, resulting in different colors of the solution. The color of the solution is collected by an image acquisition device and transmitted to a smartphone, and the smartphone analyzes the change in the RGB value of the image to obtain a quantitative result, thereby realizing the detection of residual amoxicillin in real breast milk samples. This method can obtain quantitative detection results in a short time, and has the advantages of high sensitivity, good specificity, low cost, simple operation, etc., and is easy to promote and apply.

Description

Technical Field

[0001] The present invention relates to a method for detecting residual amoxicillin in breast milk by combining the colorimetric method of aptamer with gold nanoparticles (AuNPs) and a smart phone. Background Art

[0002] Breast milk is an important natural food required by infants. Breast milk contains rich carbohydrates, proteins, fats, various trace elements and natural antibodies, which play a crucial role in the growth and development of infants. Breast milk can not only provide nutrients and energy for infants, but also the various effective substances in breast milk can provide a natural "protective cover" for infants who leave the mother's body. Since the 21st century, the abuse of antibiotics has become a worldwide problem, and the consequences have seriously endangered people's lives and health. Antibiotic resistance has attracted great attention in clinical practice. With the increase in the types and amounts of antibiotics used in recent years, the types of antibiotics that produce resistance and the resistance have also been gradually increasing. Due to the particularity of the infant group, the presence of antibiotics in breast milk due to lactating mothers receiving antibiotic treatment deserves special attention. Antibiotics in breast milk will affect the breast milk microbiota and the normal flora of the infant's intestine, and there have been experiments proving that the use of antibiotics in the early life of mammals will increase their susceptibility to allergic asthma. In order to avoid infants being exposed to antibiotics prematurely and increasing the possibility of developing antibiotic resistance as much as possible, it is urgent to popularize a detection method for the content of antibiotics in breast milk. In recent years, some methods for detecting antibiotic residues have emerged, such as fluorescence method, enzyme-linked immunosorbent assay, electrochemical analysis, high performance liquid chromatography and other methods. Although these methods have high sensitivity, most of them have disadvantages such as complex operation and high price. Amoxicillin is one of the most commonly used antibiotic drugs at present. There is still little research on the content of amoxicillin in breast milk and there is no convenient and fast detection method.

[0003] Gold nanoparticles (AuNPs) have attracted much attention due to their chemical and biological properties. When gold nanoparticles are in a dispersed state in solution, they are dark purple-red, and when they aggregate, they are blue-purple. This property makes them an important tool for research. Aptamer (APT) is a specific single-stranded oligonucleotide selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. Compared with traditional recognition molecules such as antibodies, aptamers have the characteristics of simple synthesis, easy modification, good chemical stability, high flexibility, etc. The use of AuNPs to fabricate colorimetric aptasensors has gradually become popular because of its easy preparation and simple operation. When the target drug exists, under the action of the colorimetric sensor, the color of the solution will change accordingly.

[0004] Inspired by the above work, in this study, the color of the solution was collected by an image acquisition device, and the collected image was transmitted to a smartphone and the RGB values of the image were analyzed, so as to achieve the goal of quantitative detection of drug concentration. Summary of the Invention

[0005] The purpose is to establish a method for detecting residual amoxicillin in breast milk based on the colorimetric method of gold nanoparticles (AuNPs) aptamers combined with a smartphone.

[0006] The specific technical solution is as follows:

[0007] A method for detecting residual amoxicillin in breast milk based on the colorimetric method of gold nanoparticles (AuNPs) aptamers combined with a smartphone, and its preparation process includes the following steps:

[0008] (1) Preparation of gold nanoparticles (AuNPs) Soak all the utensils needed in aqua regia for about 1 hour or more, then pour the aqua regia into a recovery flask, and rinse the utensils with a large amount of ultrapure water (18.2 MΩcm, 25 °C) and dry them. Weigh 0.0824 g of chloroauric acid powder, dissolve it in 200 ml of ultrapure water, and mix evenly to form a 1 mmol / L chloroauric acid solution. Weigh 0.2282 g of trisodium citrate powder, dissolve it in 20 ml of ultrapure water, and mix evenly to form a 38.8 mmol / L trisodium citrate solution. Place the conical flask containing the chloroauric acid solution on an electromagnetic stirring heater, turn on the electromagnetic heating stirrer to heat and stir, and heat until the solution boils. While keeping the chloroauric acid solution boiling violently and stirring evenly, quickly add 20 ml of the trisodium citrate solution to the conical flask. The reaction solution quickly changes from light yellow to black, and then turns into dark wine red. Continue to stir and heat until the solution boils for at least 20 minutes, then turn off the heating power supply, and continue to stir and cool for 15 minutes. After the sample cools, transfer it to a clean dark sample bottle and store it in a 4 °C refrigerator for subsequent use.

[0009] (2) Aptamer preparation Centrifuge the EP tube containing the HPLC-purified aptamer at 4000 rpm for 5 minutes, and add ultrapure water to this EP tube to prepare a 100 μmol / L solution.

[0010] (3) Coupling of gold nanoparticles (AuNPS) and aptamer Dilute the above-prepared aptamer solution to 100 nmol / L, add an AuNPS solution with a volume ratio of 2:1 to the aptamer solution, oscillate and mix evenly for 15 seconds, and incubate at room temperature for 15 minutes.

[0011] (4) Reaction of the drug solution with the AuNPs aptamer coupling solution Add 100 μL of the AuNPs aptamer coupling solution to a 96-well plate, then add 100 μL of amoxicillin solutions of each concentration respectively, oscillate and mix for 10 minutes, and then add the salt solution.

[0012] (5), Result detection: Place the 96-well plate on the scanner. After scanning, obtain the color change picture result, transfer the image to the smartphone and analyze the image change.

[0013] (6), Detect the breast milk sample.

[0014] In step (1) above, all the utensils used for preparing AuNPs need to be soaked in aqua regia for more than 1 hour to remove all potentially interfering ions, then thoroughly rinsed with a large amount of ultrapure water and dried. It is necessary to ensure that the utensils and solutions used in the preparation process are not contaminated.

[0015] In step (1) above, the diameter of the gold nanoparticles (AuNPs) is about 18 - 25 nm, and the concentration is 17 nmol / L. When the gold nanoparticles are dispersed, there is a maximum absorption peak at about 520 nm, and when they aggregate, there is a maximum absorption peak at about 620 nm.

[0016] The aptamers in step (2) above are all purified by HPLC method.

[0017] The aptamer sequence in step (2) above is 5’-TTAGTTGGGGTTCAGTTGG-3’.

[0018] In step (2) above, the ultrapure water added to the EP tube containing the aptamer powder is 55 μL, and a 100 μmol / L solution is prepared.

[0019] In step (3) above, the aptamer solution is diluted to 100 nmol / L with ultrapure water.

[0020] In the reaction solution of step (3) above, the concentration of AuNPs is 11.3 nmol / L, and the concentration of the aptamer is 33.3 nmol / L.

[0021] The protective effect of the aptamer on the gold nanoparticles in step (3) above can be used.

[0022] In step (4) above, the drug solution is prepared with ultrapure water.

[0023] In step (4) above, the salt solution is sodium chloride solution.

[0024] In step (4) above, the concentration of the added salt solution is 500 mmol / L.

[0025] The components of the reaction solution in step (4) above include AuNPs, aptamer, amoxicillin, and sodium chloride.

[0026] In step (5) above, the brand of the scanner is Camon, and the dpi value is set to 1200.

[0027] Before detecting the breast milk sample in step (6), trichloroacetic acid needs to be added for pretreatment of the breast milk to remove proteins and lipids.

[0028] The present invention is based on the colorimetric method of aptamer conjugated with gold nanoparticles (AuNPs). Utilizing the property that aptamers specifically bind to targets, gold nanoparticles are used. When there is no aptamer present, gold nanoparticles will aggregate when encountering a high-concentration salt solution, and the color changes from dark wine red to blue-violet. When an aptamer binds to gold nanoparticles through van der Waals forces, it will protect the gold nanoparticles from aggregating in the presence of salt. When amoxicillin exists in the solution, different concentrations of amoxicillin will bind to the aptamer to different extents, resulting in different degrees of weakening of the protective effect of the aptamer on gold nanoparticles, and the gold nanoparticles will re-aggregate. Different concentrations of amoxicillin cause different degrees of aggregation of gold nanoparticles, thus producing different color changes. By collecting the color change images and transmitting them to a smartphone, and the smartphone analyzes the change in RGB values of the images, rapid detection of amoxicillin can be achieved.

[0029] The present invention uses the conjugation of aptamer and gold nanoparticles to detect amoxicillin in breast milk, causing the color of the final solution to change. The color change images are collected, and the analysis of the image results is processed using a smartphone.

[0030] Compared with the prior art, the advantages of this method are as follows:

[0031] (1) Utilize aptamers to specifically recognize amoxicillin in breast milk. Compared with antibodies, aptamers are simple to synthesize, easy to modify, have good chemical stability, high flexibility, and can bind to targets in a shorter time.

[0032] (2) Utilize the conjugation of gold nanoparticles and aptamers. The concentration of amoxicillin in breast milk is shown by the color change caused by the aggregation of gold nanoparticles, which has the advantages of high sensitivity, good specificity, and being observable by the naked eye.

[0033] (3) The color changes are collected by an image acquisition device, and a smartphone is used to process the data. Compared with traditional methods, it has the advantages of higher data inclusiveness, smaller error, and higher accuracy.

[0034] (4) The present invention can quickly and simply measure the concentration of amoxicillin in breast milk. Compared with other detection methods, it has the advantages of low cost, simple operation, and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of a method for detecting residual amoxicillin in breast milk based on the colorimetric method of aptamer conjugated with gold nanoparticles (AuNPs) combined with a smartphone;

[0036] Figure 2Spectral scanning results of the dispersion and aggregation states of gold nanoparticles

[0037] Figure 3 The protective effect of aptamers with different concentrations on gold nanoparticles

[0038] Figure 4 Color changes of the solution after adding aqueous solutions of amoxicillin with different concentrations Detailed implementation manners

[0039] The present invention will be further described below through specific examples in conjunction with the accompanying drawings

[0040] Example 1 Detection of residual amoxicillin in breast milk by combining the aptamer chromogenic method based on gold nanoparticles (AuNPs) with a smartphone

[0041] A method for detecting residual amoxicillin in breast milk by combining the aptamer chromogenic method based on gold nanoparticles (AuNPs) with a smartphone, the specific steps are as Figure 1 shown

[0042] 1. Preparation of gold nanoparticles (AuNPs):

[0043] (1) Preparation before the experiment

[0044] Multiple experiments have proved that during the preparation of gold nanoparticles, the cleanliness of the utensils used is crucial. If the utensils are not clean enough and contain other impurities, it will interfere with the synthesis of gold nanoparticles. Therefore, all the utensils to be used are soaked in aqua regia for about 1 hour or more, then the aqua regia is poured into a recovery flask, and the utensils are rinsed clean with a large amount of ultrapure water (18.2 MΩ·cm, 25 °C) and dried

[0045] (2) Specific preparation steps

[0046] Weigh 0.0824 g of chloroauric acid powder and dissolve it in 200 ml of ultrapure water, mix evenly to form a 1 mmol / L chloroauric acid solution. Weigh 0.2282 g of trisodium citrate powder and dissolve it in 20 ml of ultrapure water, mix evenly to form a 38.8 mmol / L trisodium citrate solution. Place the conical flask containing the chloroauric acid solution on an electromagnetic stirring heater, turn on the electromagnetic heating stirrer to heat and stir, and heat until the solution boils. While keeping the chloroauric acid solution boiling violently and stirring evenly, quickly add 20 ml of the trisodium citrate solution to the conical flask. The reaction solution changes from light yellow to black rapidly, and then turns into dark wine red. Continue to stir and heat until the solution boils for at least 20 minutes, then turn off the heating power supply, and continue to stir and cool for 15 minutes. After the sample cools down, transfer it to a clean dark sample bottle and store it in a 4 °C refrigerator for subsequent use. As Figure 2 shown, when the gold nanoparticles are dispersed, there is a maximum absorption peak at about 520 nm, and when aggregated, there is a maximum absorption peak at about 620 nm

[0047] 2. Preparation of Amoxicillin Aptamer:

[0048] (1) The amoxicillin aptamer sequence of the present invention is 5’-TTAGTTGGGGTTCAGTTGG-3’. The EP tube containing the HPLC-purified aptamer was centrifuged at 4000 rpm for 5 minutes, and ultrapure water was added to this EP tube to prepare a 100 μmol / L solution.

[0049] (2) The nano-gold particles have the highest absorption peak at about 520 nm during dispersion and at about 620 nm during aggregation. The protective effect of the aptamer on the nano-gold particles can be expressed as A620 / A520. As Figure 3 shown, the protective effects of aptamers at different concentrations on nano-gold are also different.

[0050] 3. Reaction of Drug Solution with AuNPs-Aptamer Conjugate Solution:

[0051] (1) Amoxicillin solutions with concentrations of 0, 100, 200, 400, 800, 1600, 3200, 6400, 12800, 25600, 51200, and 102400 nmol / L were prepared respectively.

[0052] (2) 100 μL of AuNPs-aptamer conjugate solution was added to a 96-well plate, and then 100 μL of amoxicillin solutions at each concentration were added respectively. After shaking and mixing for 10 minutes, a salt solution was added.

[0053] 4. Result Detection:

[0054] The 96-well plate was placed on a scanner, and after scanning, a color change picture result was obtained. As Figure 4 shown, the colors of the reactions of amoxicillin aqueous solutions at different concentrations are also different.

[0055] 5. Detection of Breast Milk Samples.

[0056] Before detecting breast milk samples, trichloroacetic acid needs to be added for pretreatment of breast milk to remove proteins and lipids. Then, the treated sample was added to the AuNPs-aptamer conjugate solution, and a salt solution was added, and the color change was observed.

Claims

1. A method for detecting residual amoxicillin in breast milk by combining the colorimetric method of aptamer with gold nanoparticles (AuNPs) and a smartphone, comprising the following steps: (1) Preparation of gold nanoparticles (AuNPs) Weigh 0.0824 g of chloroauric acid powder and dissolve it in 200 ml of ultrapure water, mix evenly to form a 1 mmol / L chloroauric acid solution. Weigh 0.2282 g of trisodium citrate powder and dissolve it in 20 ml of ultrapure water, mix evenly to form a 38.8 mmol / L trisodium citrate solution. While keeping the chloroauric acid solution boiling violently and stirring evenly, quickly add 20 ml of the trisodium citrate solution to the conical flask. The reaction solution rapidly changes from light yellow to black, and then turns into dark wine red. Continuously stir and heat until the solution boils for at least 20 minutes, then turn off the heating power and continue to stir and cool for 15 minutes. After the sample cools, transfer it to a clean dark sample bottle and store it in a 4°C refrigerator for subsequent use; (2) Aptamer preparation Centrifuge the centrifuge tube containing the aptamer purified by high-performance liquid chromatography (HPLC) at 4000 rpm for 5 minutes, and add ultrapure water to this centrifuge tube to prepare a 100 μmol / L aptamer solution; (3) Coupling of gold nanoparticles (AuNPs) and aptamer Dilute the above-prepared aptamer solution to 100 nmol / L with ultrapure water, add a AuNPs solution with a volume ratio of 2:1 to the 100 nmol / L aptamer solution, oscillate and mix evenly for 15 seconds, and incubate at room temperature for 15 minutes to obtain the AuNPs-aptamer coupling solution; (4) Reaction of the drug solution and the AuNPs-aptamer coupling solution Add 100 μL of the AuNPs-aptamer coupling solution prepared in step (3) to a 96-well plate, and then add 100 μL of amoxicillin solutions of different concentrations to the wells already containing the AuNPs-aptamer coupling solution, that is, only add one concentration of amoxicillin solution to each well. After oscillating and mixing evenly for 10 minutes, add a salt solution; (5) Result detection Place the 96-well plate on a scanner, obtain the color change picture result after scanning, transfer the picture to a smartphone, and analyze its RGB value to automatically calculate the concentration of residual amoxicillin in breast milk; (6) Detect breast milk samples.

2. The method for detecting residual amoxicillin in breast milk by combining the colorimetric method of aptamer with gold nanoparticles (AuNPs) and a smartphone according to claim 1, characterized in that, the diameter of the gold nanoparticles (AuNPs) is about 18 - 25 nm, the concentration is 17 nmol / L, the gold nanoparticles have the highest absorption peak at about 520 nm when dispersed and the highest absorption peak at 620 nm when aggregated.

3. The method for detecting residual amoxicillin in breast milk by combining the colorimetric method of aptamer with gold nanoparticles (AuNPs) and a smartphone according to claim 1, characterized in that, the aptamer sequence is 5’-TTAGTTGGGGTTCAGTTGG-3’.

4. The method for detecting residual amoxicillin in breast milk by combining the colorimetric method of aptamer with gold nanoparticles (AuNPs) and a smartphone according to claim 1, It is characterized in that the concentration of AuNPs is 11.3 nmol / L and the concentration of aptamer is 33.3 nmol / L.

5. The method for detecting residual amoxicillin in breast milk by combining the colorimetric method of gold nanoparticles (AuNPs) aptamer with a smartphone according to claim 1, It is characterized in that the salt solution in the preparation method is sodium chloride solution with a concentration of 500 mmol / L.

6. The method for detecting residual amoxicillin in breast milk by combining the colorimetric method of gold nanoparticles (AuNPs) aptamer with a smartphone according to claim 1, It is characterized in that the detection result is collected as an image by a scanner and then transmitted to the smartphone, and the smartphone analyzes the RGB value to calculate the concentration of residual amoxicillin in breast milk.

7. The method for detecting residual amoxicillin in breast milk by combining the colorimetric method of gold nanoparticles (AuNPs) aptamer with a smartphone according to claim 1, It is characterized in that before detecting the breast milk sample, trichloroacetic acid needs to be added for pretreatment of the breast milk to remove proteins and lipids.

Images