阅读说明：本技术 一种基于aie荧光探针检测细菌抗药性的方法 (Method for detecting bacterial drug resistance based on AIE fluorescent probe ) 是由 付文金 邓任堂 赖丽莎 谢岭平 张露 于 2019-08-30 设计创作，主要内容包括：本发明涉及微量检测技术领域,具体涉及一种基于AIE荧光探针检测细菌抗药性的方法。包括如下步骤：将待测菌株用生理盐水配置至麦氏浊度为0.5的菌悬液；吸取6PD-DPAN荧光探针和上述菌悬液稀释至MOPS缓冲液中,制得混合液；向无菌96孔板中的每个孔均加入上述的混合液,向除第一孔外的每个孔中均加入不同浓度的抗菌药物溶液,制得若干个待测样,检测并记录初始荧光强度值；将上述带有待测样的无菌96孔板进行孵育,每孵育一段时间,检测并记录待测样的荧光强度值。本发明的检测方法简单,易于实现,利用6PD-DPAN荧光探针的聚集诱导发光特性和高光稳定性,可实时监测6PD-DPAN荧光探针与菌株结合的动力学过程。(The invention relates to the technical field of micro detection, in particular to a method for detecting bacterial drug resistance based on an AIE fluorescent probe. The method comprises the following steps: preparing a bacterial strain to be detected into a bacterial suspension with the turbidity of 0.5 in McLeod with physiological saline; absorbing the 6PD-DPAN fluorescent probe and the bacterial suspension to dilute into MOPS buffer solution to prepare mixed solution; adding the mixed solution into each hole of a sterile 96-hole plate, adding antibacterial drug solutions with different concentrations into each hole except the first hole to prepare a plurality of samples to be detected, and detecting and recording the initial fluorescence intensity value; and (3) incubating the sterile 96-well plate with the sample to be detected, and detecting and recording the fluorescence intensity value of the sample to be detected every time the incubation is carried out for a period of time. The detection method is simple and easy to realize, and the dynamic process of the combination of the 6PD-DPAN fluorescent probe and the strain can be monitored in real time by utilizing the aggregation-induced emission characteristic and the high light stability of the 6PD-DPAN fluorescent probe.)

1. A method for detecting bacterial drug resistance based on an AIE fluorescent probe is characterized in that: the method comprises the following steps:

step (1): preparing a bacterial strain to be detected into a bacterial suspension with the turbidity of 0.5 in McLeod with physiological saline;

step (2): sucking 1mL of 6PD-DPAN fluorescent probe and 10 mu L of the bacterial suspension obtained in the step (1) to dilute the probe into 10mL of MOPS buffer solution, and shaking up to obtain a mixed solution;

and (3): adding 50 mu L of the mixed solution prepared in the step (2) into each hole in a disposable sterile 96-hole plate, then adding 50 mu L of MOPS buffer solution into a first hole in the disposable sterile 96-hole plate to prepare a control sample, adding 50 mu L of antibacterial drug solutions with different concentrations into each hole except the first hole to prepare a plurality of samples to be detected, and detecting and recording the initial fluorescence intensity value of the 6PD-DPAN fluorescent probe combined with the strain in the mixed solution by using a transparent film closing plate;

and (4): and (3) placing the disposable sterile 96-well plate with the sample to be detected in incubation equipment with the constant temperature of 35 ℃ for incubation, taking the disposable sterile 96-well plate out of the incubation equipment and placing the disposable sterile 96-well plate in fluorescence detection equipment every 15min for incubation, and detecting and recording the fluorescence intensity value of the sample to be detected.

2. The method for detecting bacterial resistance based on AIE fluorescent probe as claimed in claim 1, wherein: in the step (1), the mass fraction of the physiological saline is 0.85%.

3. The method for detecting bacterial resistance based on AIE fluorescent probe as claimed in claim 1, wherein: in the step (3), the molecular formula of the 6PD-DPAN fluorescent probe is as follows:

4. the method for detecting bacterial resistance based on AIE fluorescent probe as claimed in claim 3, wherein: in the step (3), the Stokes shift value of the 6PD-DPAN fluorescent probe is 134nm, the Zeta potential is 35.9mV, and the particle size is 81.55 nm.

5. The method for detecting bacterial resistance based on AIE fluorescent probe as claimed in claim 1, wherein: in the step (4), the specific steps of detecting the fluorescence intensity value are as follows: the fluorescence detection equipment is adjusted to be in an automatic real-time continuous fluorescence detection mode, and the fluorescence intensity value is tested every 15min of incubation.

6. The method for detecting bacterial resistance based on AIE fluorescent probe as claimed in claim 5, wherein: the detection parameters of the fluorescence detection equipment are as follows: the temperature is constant at 35 ℃, the excitation wave band is 457-477nm, and the fluorescence intensity value with the wave band of 490-610nm is detected and recorded.

7. A detection system for use in the method for detecting bacterial resistance based on the AIE fluorescent probe according to any one of claims 1 to 6, wherein: the device comprises a sample adding system, an incubation and detection system and a data processing system which are connected in sequence according to the using sequence, wherein the sample adding system, the incubation and detection system and the data processing system are all electrically connected with a power supply;

the incubation and detection system comprises incubation equipment for incubating strains to be detected, fluorescence detection equipment for detecting fluorescence intensity values in the strains to be detected, a mechanical arm for transporting a container filled with samples to be detected to the fluorescence detection equipment from the incubation equipment, an air cylinder for driving the mechanical arm, a temperature regulator for monitoring and regulating the temperature and a controller for controlling the operation of the incubation equipment, the fluorescence detection equipment, the mechanical arm, the temperature regulator and the data processing system.

8. The detection system of claim 7, applied to a method for detecting bacterial resistance based on AIE fluorescent probes, characterized in that: the sample adding system comprises a disposable sterile 96-well plate and a plurality of quantitative sample adding devices for quantitatively adding sample liquid to the disposable sterile 96-well plate; the incubation device is a slat rack.

9. The detection system of claim 8, applied to a method for detecting bacterial resistance based on AIE fluorescent probes, wherein: the fluorescence detection equipment comprises a light source emitter for emitting a detection light source, an excitation light filter for filtering stray light in an excitation light source emitted by the light source emitter, a fluorescence filter for filtering the stray light in a fluorescence light source emitted by a sample to be detected, and a detector for detecting fluorescence intensity; the mechanical arm is used for clamping the disposable sterile 96-pore plate added with the sample liquid from the incubation equipment and transporting the disposable sterile 96-pore plate to the position below the light source emitter; the light source emitter and the detector are electrically connected with the controller.

10. The detection system of claim 9, applied to a method for detecting bacterial resistance based on AIE fluorescent probes, wherein: the data processing system comprises a data processing unit and a display, wherein the signal output end of the detector is transmitted to the data processing unit through a signal amplifier, and the signal output end of the data processing unit is connected with the signal input end of the display; the signal amplifier, the data processing unit and the display are electrically connected with the controller.

Technical Field

The invention relates to the technical field of micro detection, in particular to a method for detecting bacterial drug resistance based on an AIE fluorescent probe.

Background

The bacterial drug sensitivity test (AST) is an important means for providing information of degree of sensitivity of clinical pathogenic bacteria to different antibacterial drugs, and has important values for guiding clinical medication, monitoring treatment effect, finding drug-resistant strains and researching, developing and screening novel antibacterial drugs. However, the conventional bacterial susceptibility test (AST) is a growth-based susceptibility test method, which relies on the growth of microorganisms, and thus, it takes 24 to 48 hours or more to achieve a degree of visual or instrumental discrimination. The strain is difficult to determine the growth key point accurately and repeatedly, and has the defects of strong subjectivity in result judgment, incapability of measuring an MIC (paper diffusion method), limited applicable drugs and strains and the like, and the strain can not bring timely result feedback for clinic, thereby influencing the accurate medication of critically ill patients and also influencing the clinical value of drug sensitivity test to a great extent.

To avoid these problems, several other methods have been reported, including the fluorescence-labeled phage method, electrochemical detection method, real-time PCR method, and nanoparticle-based method, but these methods also have disadvantages: expensive instruments, high cost, complex operation, low sensitivity, high repeatability and the like, and the application of the method is limited. Therefore, the development of a rapid detection method for a novel bacterial drug sensitive test which can be rapid, simple and accurate has urgent and important clinical value.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a method for detecting bacterial drug resistance based on an AIE fluorescent probe, which is simple and easy to implement, can monitor the dynamic process of the combination of the 6PD-DPAN fluorescent probe and a bacterial strain in real time by utilizing a real-time dynamic tracking mode, induces the luminescence characteristic through the aggregation of the 6PD-DPAN fluorescent probe, has high light stability, and can realize the real-time observation of the action process of the 6PD-DPAN fluorescent probe on the bacterial strain through the change of the fluorescence intensity.

The invention aims to provide a detection system applied to a method for detecting bacterial drug resistance based on an AIE fluorescent probe, the detection system can dynamically track and monitor the dynamic process of the combination of a 6PD-DPAN fluorescent probe and a bacterial strain in real time, realizes rapid and accurate bacterial drug detection, and has high practicability.

The purpose of the invention is realized by the following technical scheme: a method for detecting bacterial drug resistance based on an AIE fluorescent probe comprises the following steps:

step (1): preparing a bacterial strain to be detected into a bacterial suspension with the turbidity of 0.5 in McLeod with physiological saline with the mass fraction of 0.85%;

step (2): sucking 1mL of 6PD-DPAN fluorescent probe and 10 mu L of the bacterial suspension obtained in the step (1) to dilute the probe into 10mL of MOPS buffer solution, and shaking up to obtain a mixed solution;

and (3): adding 50 mu L of the mixed solution prepared in the step (2) into each hole in a disposable sterile 96-hole plate, then adding 50 mu L of MOPS buffer solution into the first hole in the disposable sterile 96-hole plate to prepare a reference sample, adding 50 mu L of antibacterial drug solutions with different concentrations into each hole except the first hole to prepare a plurality of samples to be detected, and detecting and recording the initial fluorescence intensity value of the 6PD-DPAN fluorescent probe combined with the strain in the mixed solution by using a transparent film closing plate;

and (4): and (3) placing the disposable sterile 96-well plate with the sample to be detected in incubation equipment with the constant temperature of 35 ℃, taking out the disposable sterile 96-well plate from the incubation equipment and placing the disposable sterile 96-well plate in fluorescence detection equipment every 15min of incubation, and detecting and recording the fluorescence intensity value of the sample to be detected.

The method for detecting the concentration of the bacterial drug is simple, easy to implement, rapid and accurate, and utilizes a real-time dynamic tracking mode, the dynamic process of the combination of the 6PD-DPAN fluorescent probe and the bacterial strain can be monitored in real time through the change of the fluorescence intensity, so that the problems that the bacteria are dyed by adopting the traditional dye and self-quenching occurs when the high-density dye is dyed are solved; the 6PD-DPAN fluorescent probe can be combined with various bacteria to cause aggregation-induced luminescence, has sensitive fluorescent response to the bacteria, has high aggregation state luminescence efficiency and high light stability, has the fluorescence intensity in direct proportion to the bacterial quantity, and shows and detects the drug resistance of the strain to the antibacterial drugs through the change of the fluorescence intensity.

The invention is provided with a control sample as a positive control sample to prove the normal growth kinetics of bacteria without the action of antibacterial drugs.

When the antibacterial agent is added, the proliferation of bacteria is inhibited by the antibacterial agent, and the fluorescence intensity of the 6PD-DPAN fluorescent probe is weakened; under the condition that no antibacterial drug is added, the bacteria grow exponentially, the 6PD-DPAN fluorescent probe emits stronger fluorescent reaction, and the fluorescence intensity is higher; the 6PD-DPAN fluorescent probe adopted by the invention does not influence the growth of bacteria, and the sensitivity of the bacteria to antibacterial drugs, namely drug resistance (drug resistance), is tested through the change of the fluorescence intensity.

In the invention, the relative change of the fluorescence intensity is obtained by detecting the initial fluorescence intensity value of the combination of the 6PD-DPAN fluorescent probe and the bacteria just after the antibacterial agent is added and the final fluorescence intensity value after the incubation is finished, and the relative change value of the fluorescence intensity is calculated by specifically adopting the following equation:

RC_FRET＝(FRET_max/FRET₀)-(FRET₁/FRET₀)；

wherein, RC_FRET: a relative change value representing the fluorescence intensity;

FRET_max: the maximum fluorescence intensity value of the combined 6PD-DPAN fluorescent probe and the strain in the mixed solution;

FRET₀: initial fluorescence intensity value of the 6PD-DPAN fluorescent probe after being completely combined with the strain in the mixed solution;

FRET₁: after adding the antibacterial agent, after incubating for 15min, the fluorescence intensity value of the 6PD-DPAN fluorescent probe combined with the strain in the mixed solution; detecting once after each incubation for 15min to obtain multiple fluorescence intensity values FRET at different times₁。

The larger the final fluorescence intensity value, or the FRET of multiple fluorescence intensity values at different times₁Gradually increase, then RC_FRETThe smaller or gradually decreased value proves that the more strains combined with the 6PD-DPAN fluorescent probe, the more the number of the strains is increased, and the situation that the proliferation of the strains is not inhibited by antibacterial drugs, the sensitivity is lower and the strains are resistant to the drugs is shown. On the contrary, the final fluorescence intensity value or a plurality of fluorescence intensities at different timesValue FRET₁The strain has no obvious change and no large change, proves that the proliferation of the strain is inhibited by the antibacterial drug, and shows that the strain has higher sensitivity to the corresponding antibacterial drug and is non-drug resistant.

Preferably, in the step (3), the molecular formula of the 6PD-DPAN fluorescent probe is as follows:

preferably, in the step (3), the Stokes shift value of the 6PD-DPAN fluorescent probe is 134nm, the Zeta potential is 35.9mV, and the particle size is 81.55 nm.

The 6PD-DPAN fluorescent probe adopted by the invention has aggregation-induced emission characteristics, sensitive fluorescent response to bacteria, high aggregation state luminous efficiency, high light stability, high fluorescence intensity and bacteria quantity in direct proportion, can realize real-time observation of the action process of the 6PD-DPAN fluorescent probe on strains through fluorescence intensity change, and has high sensitivity and high accuracy.

Preferably, in the step (4), the specific steps of detecting the fluorescence intensity value are as follows: adjusting the fluorescence detection equipment to be in an automatic real-time continuous fluorescence detection mode, and testing the fluorescence intensity value of the fluorescence detection equipment every 15min of incubation; the detection parameters of the fluorescence detection equipment are as follows: the temperature is constant at 35 ℃, the excitation wave band is 457-477nm, and the fluorescence intensity value with the wave band of 490-610nm is detected and recorded.

The other purpose of the invention is realized by the following technical scheme: the detection system applied to the method for detecting the drug resistance of the bacteria based on the AIE fluorescent probe comprises a sample adding system, an incubation and detection system and a data processing system which are sequentially connected according to a using sequence, wherein the sample adding system, the incubation and detection system and the data processing system are electrically connected with a power supply;

the incubation and detection system comprises incubation equipment for incubating strains to be detected, fluorescence detection equipment for detecting fluorescence intensity values in the strains to be detected, a mechanical arm for transporting a container filled with samples to be detected to the fluorescence detection equipment from the incubation equipment, an air cylinder for driving the mechanical arm, a temperature regulator for monitoring and regulating the temperature and a controller for controlling the operation of the incubation equipment, the fluorescence detection equipment, the mechanical arm, the temperature regulator and the data processing system.

By adopting the detection system, the dynamic process of combining the 6PD-DPAN fluorescent probe and the strain can be dynamically tracked and monitored in real time, the rapid and accurate detection of the bacterial drugs is realized, and the practicability is high; wherein, the container filled with the sample to be detected is transported from the incubation system to the fluorescence system through the mechanical arm, so that the dynamic process of combining the 6PD-DPAN fluorescent probe and the strain can be dynamically tracked and monitored in real time, and the rapidity and the real-time performance of detection are improved. The mechanical arm is driven to operate by an air cylinder, and a container filled with a sample to be detected is transported from the incubation equipment to the fluorescence detection equipment for fluorescence intensity value detection; the temperature controller can monitor the temperature change in the incubation and detection system in time and transmit the temperature change to the controller, and if the temperature is too high or too low, the controller sends a temperature control instruction to the temperature controller for temperature control, so that the temperature of the incubation and detection system is kept at a constant temperature of 35 ℃.

Preferably, the sample adding system comprises a disposable sterile 96-well plate and a plurality of quantitative sample applicators for quantitatively adding the sample liquid to the disposable sterile 96-well plate; the incubation device is a slat rack.

According to the invention, the disposable sterile 96-well plate is adopted, a plurality of strains and strains with different concentrations can be incubated at the same time, the detection rapidity and high efficiency are improved, the adopted quantitative sample injector can be regulated and controlled according to the actual sample injection amount, and materials are accurately added, for example, 50 mu L of mixed liquid is accurately added into each hole in the disposable sterile 96-well plate in the step (3), 50 mu L of MOPS buffer solution is accurately added into the first hole in the disposable sterile 96-well plate, 50 mu L of antibacterial drug solution with different concentrations is accurately added into each hole except the first hole in the disposable sterile 96-well plate, and the detection accuracy is improved. And the disposable sterile 96-well plate added with the sample liquid can be placed in a plate rack for incubation through manual operation.

Preferably, the fluorescence detection device comprises a light source emitter for emitting a detection light source, an excitation light filter for filtering stray light in an excitation light source emitted by the light source emitter, a fluorescence filter for filtering stray light in a fluorescence light source emitted by a sample to be detected, and a detector for detecting fluorescence intensity; the mechanical arm is used for clamping the disposable sterile 96-pore plate added with the sample liquid from the incubation equipment and transporting the disposable sterile 96-pore plate to the position below the light source emitter; the light source emitter and the detector are electrically connected with the controller. Specifically, the excitation light filter is arranged at a light source emitting end of the light source emitter, and the fluorescence filter is arranged at a light source receiving end of the detector.

Preferably, the data processing system comprises a data processing unit and a display, the signal output end of the detector is transmitted to the data processing unit through a signal amplifier, and the signal output end of the data processing unit is connected with the signal input end of the display; the signal amplifier, the data processing unit and the display are electrically connected with the controller.

The incubation and detection system and the data processing system provided by the invention have the working processes that: a plurality of disposable sterile 96-hole plates for loading samples to be tested are placed in the plate rack, the controller sends an instruction to the incubation and detection system, the mechanical arm is controlled to transport the disposable sterile 96-hole plates to the sample placement plate to be tested, then the controller regulates and controls the detection condition, sends out an exciting light instruction to the light source emitter, starts the light source emitter to emit a laser light source, the laser light source enters a sample to be detected containing the 6PD-DPAN fluorescent probe after passing through the exciting light filter, under the irradiation of exciting light, the 6PD-DPAN fluorescent probe emits a fluorescent light source, and the emitted fluorescent light source filters stray light in the fluorescent light source through a fluorescent filter, then the fluorescence light source enters a detector, the detector detects the intensity of the fluorescence light source, the fluorescence intensity value signal is output and is input to a data processing unit through a signal amplifier for data processing, and the processed data displays the detection result through a display. The whole detection process is convenient and quick, the detection sensitivity is high, the accuracy is high, and the dynamic process of the combination of the 6PD-DPAN fluorescent probe and the strain and the action process on the strain can be monitored in real time through the change of the fluorescence intensity.

The invention has the beneficial effects that: the method for detecting the bacterial drug resistance based on the AIE fluorescent probe is simple to operate and easy to realize, utilizes a real-time dynamic tracking mode, can monitor the dynamic process of the combination of the 6PD-DPAN fluorescent probe and the bacterial strains in real time through the change of the fluorescence intensity, has sensitive fluorescence response to the bacteria through the characteristic that the 6PD-DPAN fluorescent probe is combined with various bacteria to cause aggregation induced luminescence, has high aggregation state luminescence efficiency and high light stability, and presents and detects the drug resistance of the bacterial strains to antibacterial drugs through the change of the fluorescence intensity, wherein the fluorescence intensity is in a positive proportion to the bacterial amount.

The invention is applied to the detection system of the method for detecting the drug resistance of bacteria based on the AIE fluorescent probe, can dynamically track and monitor the dynamic process of the combination of the 6PD-DPAN fluorescent probe and the bacterial strain in real time, realizes the rapid and accurate detection of the bacterial drugs and has high practicability.

Drawings

FIG. 1 is a flow chart of the detection steps of the present invention;

FIG. 2 is a block diagram of the detection system of the present invention;

FIG. 3 is a control principle framework diagram of the present invention;

FIG. 4 is a fluoroscope of 6PD-DPAN fluorescent probe on Escherichia coli in example 1 of the present invention;

FIG. 5 is a graph showing the change in fluorescence intensity of the 6PD-DPAN fluorescent probe against Escherichia coli strain (control sample A) in example 1 of the present invention;

FIG. 6 is a graph showing the relationship between the concentration of the antibacterial agent and the amount of Escherichia coli strain measured by fluorescence intensity when a 6PD-DPAN fluorescent probe is applied to Escherichia coli strain in example 1 of the present invention;

FIG. 7 is a fluoroscope of the 6PD-DPAN fluorescent probe on Klebsiella pneumoniae strain in example 2 of the present invention;

FIG. 8 is a fluoroscope of the 6PD-DPAN fluorescent probe against Staphylococcus aureus strain in example 3 of the present invention.

The reference signs are: 1-sample adding system, 11-disposable sterile 96-pore plate, 12-quantitative sample adding device, 2-incubation and detection system, 21-incubation device, 22-fluorescence detection device, 221-light source emitter, 222-excitation light filter, 223-fluorescence filter, 224-detector, 23-mechanical arm, 24-cylinder, 25-temperature controller, 26-controller, 3-data processing system, 31-signal amplifier, 32-data processing unit, 33-display.

Detailed Description

For the understanding of those skilled in the art, the present invention will be further described with reference to the following examples and accompanying drawings 1 to 8, and the content of the embodiments is not intended to limit the present invention.