Rapid bacterium detection and counting method based on thiohydracrylic acid quantum dot fluorescence labeling

Abstract

The invention discloses a rapid bacterium detection and counting method based on thiohydracrylic acid quantum dot fluorescence labeling, and the labeling method comprises the following steps: carrying out water-soluble modification on oil-phase quantum dots to obtain water-soluble quantum dot solids, uniformly dispersing the water-soluble quantum dot solids in water, and adjusting the pH value to 12-13 to obtain a water-phase quantum dot solution, the method for water-soluble modification of the oil-phase quantum dots comprises the following steps: dissolving solid oil-phase quantum dots in toluene, uniformly dispersing to obtain a quantum dot solution, adding thiohydracrylic acid into the quantum dot solution, carrying out oscillation reaction, standing, and drying precipitates obtained by centrifugation after precipitates are generated to obtain water-soluble quantum dot solids; and adding the aqueous-phase quantum dot solution into the bacterial liquid, and standing for reaction to finish marking. The bacteria marked by the solid oil phase quantum dots can be quickly imaged through a fluorescence microscope, and counting and morphological analysis of the bacteria can be quickly completed in combination with image analysis software capable of automatically identifying and counting the bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a rapid detection and counting method for bacteria based on mercaptopropionic acid quantum dot fluorescent labeling. Background Technology

[0002] Bacteria are a major cause of many infectious diseases. They are widely distributed in the natural environment and readily multiply on surfaces such as food, water, and medical equipment, posing a potential threat to public health. Therefore, rapid and accurate detection and counting of bacteria are crucial for disease diagnosis, food safety, environmental monitoring, and clinical treatment.

[0003] Most existing bacterial detection methods have limitations and cannot meet the demand for rapid detection. For example, both culture and biochemical identification methods require inoculating samples onto culture media and observing bacterial growth, which typically takes at least 24 hours—unacceptable for clinical scenarios requiring rapid diagnosis. Furthermore, culture methods have stringent requirements for the operating environment, demanding strict aseptic techniques, and some bacteria are difficult to grow on standard culture media, resulting in low detection sensitivity. While biochemical identification methods have some specificity, they require high sample purity; the presence of multiple microorganisms in the sample may interfere with the results. Molecular biology methods, such as polymerase chain reaction (PCR) and gene sequencing, while offering high sensitivity and specificity, are cumbersome to operate, require sophisticated sample processing, and are costly, making them unsuitable for rapid bacterial detection. Therefore, with the development of modern medicine and public health, the need for rapid bacterial detection technologies is becoming increasingly urgent. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a labeling method based on mercaptopropionic acid quantum dot fluorescence. This labeling method uses mercaptopropionic acid and oil-phase quantum dot fluorescence to label bacteria, enabling rapid detection and counting. Through the specific labeling of oil-phase quantum dots and fluorescence microscopy imaging technology, combined with image analysis software, it achieves rapid and accurate detection and counting of bacteria, overcoming the limitations of traditional detection methods in terms of detection speed, sensitivity, and specificity. This provides an efficient and reliable technical means for fields such as clinical diagnosis, food safety, and environmental monitoring.

[0005] The objective of this invention is achieved through the following technical solutions.

[0006] A labeling method based on mercaptopropionic acid quantum dot fluorescence includes the following steps:

[0007] S1, the oil-phase quantum dots are modified to be water-soluble to obtain water-soluble quantum dot solids. The water-soluble quantum dot solids are uniformly dispersed in deionized water, and the pH value is adjusted to 12-13 to obtain an aqueous quantum dot solution. The method of modifying the oil-phase quantum dots to be water-soluble includes: dissolving solid oil-phase quantum dots in toluene, sonicating for at least 10 min to make them uniformly dispersed to obtain a quantum dot solution, adding mercaptopropionic acid to the quantum dot solution, shaking for at least 5 min, letting it stand for 10-15 min until precipitation occurs, centrifuging to remove the supernatant, and vacuum drying the precipitate to obtain water-soluble quantum dot solids. The volume fraction of mercaptopropionic acid to the mass fraction of solid oil-phase quantum dots in the quantum dot solution is (25-30):1, where the volume fraction is in μL and the mass fraction is in mg.

[0008] In S1, the concentration of water-soluble quantum dot solid in the aqueous quantum dot solution is 0.1–0.3 mg / mL.

[0009] In S1, the pH is adjusted using tetramethylammonium hydroxide.

[0010] In S1, the concentration of solid oil phase quantum dots in the quantum dot solution is 0.5–0.8 mg / mL.

[0011] In S1, the ultrasound duration is 10-15 minutes and the ultrasound power is 200-300W.

[0012] In S1, the oscillation frequency is 300-500 rpm and the oscillation time is 5-10 min.

[0013] In S1, the centrifugation speed is 8000-10000 rpm, and the centrifugation time is 3-5 min.

[0014] In S1, the vacuum drying temperature is 40-50°C, and the vacuum drying time is 1-2 hours.

[0015] In S1, the method for obtaining solid oil-phase quantum dots includes: rotary evaporating the oil-phase quantum dot solution to remove the solvent and obtain solid oil-phase quantum dots.

[0016] In the above technical solution, the temperature of rotary evaporation is 40-50℃, and the pressure of rotary evaporation is 0.01-0.02MPa.

[0017] In S1, the solid oil phase quantum dot is CdSe.

[0018] In S1, the mercaptopropionic acid is 3-mercaptopropionic acid.

[0019] S2, add aqueous quantum dot solution to the bacterial culture, mix well and let stand for 10-15 minutes to obtain the labeled bacterial culture, thus completing the labeling process.

[0020] In S2, the ratio of bacterial culture to aqueous quantum dot solution by volume is (80-120):1.

[0021] A rapid detection method for bacteria based on mercaptopropionic acid quantum dot fluorescent labeling includes: dropping the labeled bacterial suspension onto a glass slide, covering it with a coverslip to obtain an imaging sample, placing the imaging sample under a fluorescence microscope, and imaging it under excitation wavelength to obtain a fluorescence image.

[0022] In the above technical solution, the excitation wavelength is 460-480nm.

[0023] In the above technical solution, bacteria are counted based on the fluorescence image.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The solid oily quantum dots modified with mercaptopropionic acid of the present invention have high fluorescence intensity, high detection sensitivity, and strong specificity. They can bind to specific molecules on the surface of bacteria, achieving specific labeling of bacteria in low-concentration bacterial samples and reducing interference from non-specific binding.

[0026] 2. The labeling method of the present invention is simple to operate and does not require complex equipment or professional technicians. Bacteria labeled with solid oil phase quantum dots can be rapidly imaged using a fluorescence microscope. Combined with image analysis software that can automatically identify and count bacteria, bacterial counting and morphological analysis can be completed quickly, reducing human error and improving the accuracy and repeatability of detection. It is suitable for application in a variety of detection scenarios and provides an efficient and accurate technical means for clinical diagnosis and public health safety. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the water-soluble modification of oil-phase quantum dots in Example 1 of the present invention;

[0028] Figure 2 This is a flowchart illustrating the marking method in Embodiment 1 of the present invention;

[0029] Figure 3 The above is a bacterial counting and analysis diagram of quantum dot fluorescent labeling in Example 1 of the present invention, wherein a is a fluorescence image, b is a fluorescence image of picking up bacterial boundaries, c is a bacterial area distribution diagram processed by ImageJ, and d is a fluorescent labeling light intensity distribution diagram processed by ImageJ.

[0030] Figure 4The image shows the counting and analysis of quantum dot fluorescently labeled bacteria in Comparative Example 1, where a is an image of the quantum dot fluorescently labeled bacteria and b is a fluorescence intensity distribution image processed by ImageJ. Detailed Implementation

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] The method for obtaining solid oil-phase quantum dots in the following embodiments includes: in a rotary evaporator, the oil-phase quantum dot solution is rotary evaporated at 40°C (the rotary evaporation pressure is 0.01 MPa) to remove n-hexane and obtain solid oil-phase quantum dots.

[0033] The oil phase quantum dot solution was purchased from Wuhan Zhunling Nano Technology Co., Ltd., and its trade name is oil-soluble cadmium selenide quantum dots. It is a mixture of solid oil phase quantum dots (the solid oil phase quantum dots are CdSe) and n-hexane. The concentration of solid oil phase quantum dots in the oil phase quantum dot solution is 3 mg / mL.

[0034] Toluene: Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0035] 3-Mercaptopropionic acid: Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0036] Tetramethylammonium hydroxide: Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0037] Bacteria: Escherichia coli (ATCC25922).

[0038] The equipment models and manufacturers used in the following embodiments are as follows:

[0039] Rotary evaporator, model: N-1210BV-WB, manufacturer: Tianjin Sipuning Technology Co., Ltd.

[0040] Inverted fluorescence microscope, model: TE2000-U, manufacturer: Nikon Optical Instruments Co., Ltd., the inverted fluorescence microscope is equipped with a 525nm single-pass filter.

[0041] Example 1

[0042] like Figure 2 As shown, a labeling method based on mercaptopropionic acid quantum dot fluorescence includes the following steps:

[0043] S1. The oil-phase quantum dots were modified to be water-soluble to obtain water-soluble quantum dot solids. The water-soluble quantum dot solids were mixed with deionized water and magnetically stirred at 300 rpm for 10 min to uniformly disperse the water-soluble quantum dot solids in the deionized water. The pH value was adjusted to 13 using tetramethylammonium hydroxide, and stirring was continued for 5 min to obtain a stable aqueous quantum dot solution. The concentration of water-soluble quantum dot solids in the aqueous quantum dot solution was 0.3 mg / mL. The method of water-soluble modification of the oil-phase quantum dots included: dissolving 0.30 mg of solid oil-phase quantum dots in toluene and sonicating for 10 min to uniformly disperse them (ultrasonic power was 200 W) to obtain a quantum dot solution. The concentration of solid oil-phase quantum dots in the quantum dot solution was 0.6 mg / mL. Mercaptopropionic acid (3-mercaptopropionic acid) was added to the quantum dot solution, and the mixture was shaken at 300 rpm for 5 min, then allowed to stand for 10 min. (Through ligand exchange reaction, mercaptopropionic acid was modified onto the surface of the quantum dots, achieving water-soluble modification of the quantum dots.) Figure 1 As shown), after precipitation occurs, the supernatant is removed by centrifugation at 8000 rpm for 3 min. The precipitate obtained by centrifugation is then vacuum dried at 40℃ for 2 h (vacuum degree of vacuum drying is -0.1 MPa) to obtain water-soluble quantum dot solids. The volume fraction of mercaptopropionic acid to the mass fraction of solid oil phase quantum dots in the quantum dot solution is 29:1. The volume fraction is in μL and the mass fraction is in mg.

[0044] S2, add aqueous quantum dot solution to 1 mL of bacterial suspension with an OD value of 0.8 (the bacterial suspension is a mixture of liquid LB medium and bacteria, purchased from Sangon Biotech Co., Ltd.), shake for 30 seconds to mix, and then let it stand at room temperature for 10 minutes to obtain the labeled bacterial suspension, thus completing the labeling process. The ratio of bacterial suspension to aqueous quantum dot solution by volume is 100:1. During the reaction, quantum dots will specifically or non-specifically bind to the bacterial surface, thereby achieving bacterial labeling.

[0045] A rapid detection method for bacteria based on mercaptopropionic acid quantum dot fluorescent labeling includes: adding 1 μL of labeled bacterial suspension onto a glass slide, covering it with a coverslip (taking care to avoid air bubbles), obtaining an imaging sample, placing the imaging sample under a fluorescence microscope, and imaging at an excitation wavelength of 488 nm to obtain a fluorescence image, such as... Figure 3 As shown in a, bacterial counting is based on fluorescence images: the fluorescence images are imported into ImageJ software, which can pick out bacterial boundaries, such as... Figure 3As shown in b, background correction and contrast adjustment are then performed. The software's automatic counting function is used to count the bacteria in the image, generating a counting report. Area and light intensity distribution analysis is then performed on the bacteria in the fluorescence image, generating area and light intensity distribution maps for the bacteria, as shown below. Figure 3 As shown in diagrams c to d. By analyzing the area of ​​the bacteria, we can understand their size distribution; by analyzing the light intensity of the bacteria, we can understand the uniformity and intensity of the quantum dot labeling.

[0046] Comparative Example 1

[0047] A rapid detection and counting method for bacteria based on quantum dot fluorescent labeling includes: adding the quantum dot solution from Example 1 to 1 mL of bacterial suspension with an OD value of 0.8 (the bacterial suspension is the same as that in Example 1), shaking for 30 seconds to mix, and then allowing it to stand at room temperature for 10 minutes to obtain the labeled bacterial suspension. The ratio of bacterial suspension to quantum dot solution by volume is 100:1.

[0048] 1 μL of labeled bacterial suspension was dropped onto a glass slide, covered with a coverslip, and an imaging sample was prepared. The imaging sample was placed under a fluorescence microscope, and imaging was performed at an excitation wavelength of 488 nm to acquire fluorescence images. Figure 4 As shown in a;

[0049] The fluorescence images were imported into ImageJ software for background correction and contrast adjustment. The software's automatic counting function was then used to analyze the light intensity distribution of bacteria in the images, generating a light intensity distribution map of the bacteria. Figure 4 As shown in b.

[0050] As can be seen from Comparative Example 1 and Example 1, the light intensity of Example 1 is stronger than that of Comparative Example 1. The fluorescence intensity of Example 1 is mainly distributed between 75 and 85, while the fluorescence intensity of Comparative Example 1 is mainly distributed between 0 and 1. It can be considered that the unmodified quantum dots basically did not bind to bacteria. This proves that in the labeling method of the present invention, the quantum dots modified with mercaptopropionic acid have high fluorescence intensity, high detection sensitivity, and strong specificity. They can bind to specific molecules on the surface of bacteria, providing an efficient and accurate technical means for clinical diagnosis and public health safety.

[0051] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A labeling method based on mercaptopropionic acid quantum dot fluorescence, characterized in that, Includes the following steps: S1, the oil-phase quantum dots are modified to be water-soluble to obtain water-soluble quantum dot solids. The water-soluble quantum dot solids are uniformly dispersed in water, and the pH value is adjusted to 12-13 to obtain an aqueous quantum dot solution. The method of modifying the oil-phase quantum dots to be water-soluble includes: dissolving solid oil-phase quantum dots in toluene, sonicating for at least 10 min to make them uniformly dispersed to obtain a quantum dot solution, adding mercaptopropionic acid to the quantum dot solution, shaking for at least 5 min, letting stand for 10-15 min, centrifuging to remove the supernatant, and vacuum drying the precipitate obtained by centrifugation to obtain water-soluble quantum dot solids. The volume fraction of mercaptopropionic acid to the mass fraction of solid oil-phase quantum dots in the quantum dot solution is (25-30):1, where the volume fraction is in μL and the mass fraction is in mg. S2, add aqueous quantum dot solution to the bacterial culture, mix well and let stand for at least 10 minutes to obtain the labeled bacterial culture, thus completing the labeling process.

2. The marking method according to claim 1, characterized in that, In S1, the concentration of water-soluble quantum dot solid in the aqueous quantum dot solution is 0.1–0.3 mg / mL.

3. The marking method according to claim 1, characterized in that, In S1, the concentration of solid oil phase quantum dots in the quantum dot solution is 0.5–0.8 mg / mL.

4. The marking method according to claim 1, characterized in that, In S1, the method for obtaining solid oil-phase quantum dots includes: rotary evaporating the oil-phase quantum dot solution to remove the solvent and obtain solid oil-phase quantum dots.

5. The marking method according to claim 4, characterized in that, The temperature of rotary evaporation is 40–50℃, and the pressure is 0.01–0.02 MPa.

6. The marking method according to claim 1, characterized in that, In S1, the solid oil phase quantum dot is CdSe.

7. The marking method according to claim 1, characterized in that, In S1, the mercaptopropionic acid is 3-mercaptopropionic acid.

8. The marking method according to claim 1, characterized in that, In S2, the ratio of bacterial culture to aqueous quantum dot solution by volume is (80-120):

1.

9. A rapid detection method for bacteria based on mercaptopropionic acid quantum dot fluorescent labeling, characterized in that, include: The labeled bacterial suspension as described in claims 1 to 8 is dropped onto a glass slide, covered with a coverslip to obtain an imaging sample. The imaging sample is placed under a fluorescence microscope and imaged under excitation wavelength to obtain a fluorescence image.

10. The rapid detection method according to claim 9, characterized in that, Bacterial counting is performed based on the fluorescence images.

Images