CN118745453B - A single bacteria electrical impedance imaging method based on surface plasmon resonance and its application - Google Patents
A single bacteria electrical impedance imaging method based on surface plasmon resonance and its application Download PDFInfo
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- CN118745453B CN118745453B CN202411227112.0A CN202411227112A CN118745453B CN 118745453 B CN118745453 B CN 118745453B CN 202411227112 A CN202411227112 A CN 202411227112A CN 118745453 B CN118745453 B CN 118745453B
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Abstract
本发明涉及一种基于表面等离子共振的单细菌电阻抗成像方法及应用，属于细菌药敏检测技术领域。本发明的药敏芯片通过优化噬菌体修饰试剂及参数，能够结合更多的噬菌体，提高细菌捕获效率，从而方便后续进行单细菌的高通量检测。利用本发明的方法，基于表面等离子体共振能够获得单细菌水平的电阻抗图像，可用于检测单细菌水平的细胞结构变化，进一步用于药敏检测。本发明具有高通量分析单细菌的优势，能够进行细菌耐药机制及异质性耐药研究。
The present invention relates to a single bacteria electrical impedance imaging method and application based on surface plasmon resonance, and belongs to the technical field of bacterial drug sensitivity detection. The drug sensitivity chip of the present invention can combine more bacteriophages and improve the efficiency of bacterial capture by optimizing phage modification reagents and parameters, thereby facilitating subsequent high-throughput detection of single bacteria. Using the method of the present invention, electrical impedance images at the level of single bacteria can be obtained based on surface plasmon resonance, which can be used to detect cell structure changes at the level of single bacteria, and further used for drug sensitivity detection. The present invention has the advantage of high-throughput analysis of single bacteria, and can conduct research on bacterial resistance mechanisms and heterogeneous resistance.
Description
Technical Field
The invention belongs to the technical field of bacterial drug sensitivity detection, and particularly relates to a single-bacterium electrical impedance imaging method based on surface plasmon resonance and application thereof.
Background
The current primary methods for bacterial susceptibility testing remain conventional antibiotic susceptibility testing (Antibiotic Susceptibility Testing, AST), such as paper spread, agar dilution, broth microdilution, and the like. The technology needs to culture, separate and enrich bacteria in clinical samples, and then obtains bacterial drug-sensitive results by observing the inhibition effect of antibiotics on bacterial growth, but the process is complicated and takes a long time (2-4 days).
In recent years, various rapid drug sensitivity detection techniques have been developed. Wherein a "phenotype" based method directly detects bacterial phenotypic characteristics, such as bacterial size, morphology, number, electrical characteristics, etc., which is a gold standard AST detection method. A rapid drug sensitivity detection method based on bacterial impedance is an important research direction. The response of bacteria to an electric field is an inherent feature related to the size and shape of the bacteria, the internal structure of the bacteria, and the conductivity and dielectric constant of the different bacterial components. Any differences and changes in the bacterial composition will affect their electrical signals, making impedance analysis a good method for rapid drug sensitive detection of bacteria and for antibiotic mechanism of action research.
However, conventional impedance detection methods are mainly based on spectroscopic techniques to measure the average impedance of the entire sensor area, such as Electrochemical Impedance Spectroscopy (EIS). Therefore, the heterogeneity research of single bacteria is difficult to complete due to the lack of imaging capability, and a brand-new bacterial electrical impedance imaging and drug sensitivity detection method is urgently needed.
Disclosure of Invention
In order to solve at least one of the technical problems, the invention adopts the following technical scheme:
the first aspect of the invention provides a phage-modified coated substrate prepared by the steps of:
plating 3-6 nm of chromium on the transparent substrate by using a magnetron sputtering method, and then plating 33-60 nm of gold to obtain a coated substrate;
Sulfhydrylation of phage with 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide and cysteamine;
and placing the coated substrate in a sulfhydrylation phage solution for phage self-assembly monolayer modification to obtain the phage modified coated substrate.
In some embodiments of the invention, the step of sulfhydrylating the phage is:
20mg of 1-ethyl- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 15mg of cysteamine were added to 1mL of 10 5~107 PFU/mL phage solution and mixed, and incubated for 8-18 hours with shaking.
In some embodiments of the present invention, the transparent substrate is made of a material selected from one of glass, polycarbonate, polymethyl methacrylate, and fluorosilicone.
Among these, polycarbonate is a transparent, high strength thermoplastic material. It has excellent impact resistance and heat resistance while being lighter than glass.
Polymethyl methacrylate (PMMA), also known as acrylic or plexiglass, has excellent optical transparency and weatherability. It has a lower density than glass and is easy to process and shape.
The fluorosilicone resin (Fluorosilicone Resin) increases chemical resistance and transparency while maintaining good flexibility of the silicone. It is useful as a substitute material for glass in optical applications, and is excellent in weather resistance and heat resistance in particular.
According to a second aspect of the present invention, there is provided a chip obtained by bonding an organosilicon film to the phage-modified coated substrate according to the first aspect of the present invention, wherein the organosilicon film has holes penetrating therethrough from top to bottom, the holes allow the organosilicon film to form a detection chamber after bonding with the coated substrate, and the phage is exposed in the detection chamber.
In some embodiments of the invention, the silicone is selected from one of polydimethylsiloxane, polymethylphenylsiloxane, methylsiloxane, vinylsiloxane, fluorosilicone, polyphenylsiloxane.
Among them, polydimethylsiloxane (PDMS) is a very common organosilicon compound, and the chemical structure of PDMS is (-Si (CH 3)2-O-)n), in which dimethylsiloxane units (-Si (CH 3)2 -O-) are linked by a siloxane bond (Si-O)) to form a long chain polymer.
Polymethylphenylsiloxane (PMDS) has a structure similar to PDMS but with a portion of the methyl groups replaced with phenyl groups.
Polymethylsiloxane (PMS) is a variant of PDMS, containing only methyl groups in the molecular chain.
Vinyl siloxane introduces vinyl side groups on the basis of PDMS, can form a higher-strength elastomer through a crosslinking reaction, and has higher weather resistance and chemical stability.
The fluorosilicone introduces fluorine groups on the basis of PDMS, and has excellent chemical corrosion resistance, hydrophobicity and high temperature resistance
The polyphenyl siloxane is obtained by substituting part of methyl of PDMS with phenyl, and has good high temperature resistance.
The third aspect of the invention provides a single-bacterium electrical impedance imaging method based on surface plasmon resonance, which comprises the following steps:
Adding a bacterial solution to the detection chamber of any of the chips of the second aspect of the invention such that bacteria bind to the phage and unbound bacteria are washed away;
Applying potential modulation to bacteria in a detection cavity, and acquiring images of the other surface of the chip relative to the detection cavity by using an objective type total internal reflection microscope to obtain surface plasma resonance images under different modulation voltages;
And carrying out Fourier transform on the surface plasma resonance image to obtain an electrical impedance image of the single bacteria.
In the invention, the image acquisition frequency is more than 2 times of the potential modulation frequency so as to meet the image acquisition frequency requirement of electrical impedance imaging.
The fourth aspect of the invention provides a method for detecting single bacterial drug sensitivity, comprising the following steps:
Exposing bacteria to be tested to different concentrations of antibiotics in the detection chamber of any of the chips of the second aspect of the invention,
Obtaining electrical impedance images of bacteria to be detected before and after exposure in antibiotics with different concentrations by using the method of any one of the third aspects of the invention, and obtaining amplitude changes of the electrical impedance images of each single bacteria before and after exposure;
If the amplitude change of the electrical impedance image of the single bacteria with a certain proportion before and after exposure in the antibiotics with a certain concentration is smaller than a preset threshold value and the amplitude change of the electrical impedance image of the single bacteria with a certain proportion before and after exposure in the antibiotics with a certain concentration is larger than the preset threshold value, the concentration is the minimum antibacterial concentration of the antibiotics to the single bacteria,
Wherein the certain proportion is 60% -90%;
In some embodiments of the invention, the certain proportion is 70% -90%.
In some embodiments of the invention, the certain proportion is 80% -90%.
In the present invention, the antibiotic is at least one antibiotic selected from the group consisting of β -lactams, aminoglycosides, macrolides, tetracyclines, quinolones, sulfonamides, glycopeptides, lincomamides, nitroimidazoles, and polypeptides.
Among them, β -lactams include penicillins including but not limited to penicillin G (Penicillin G), ampicillin (AMPICILLIN), and piperacillin (PIPERACILLIN), cephalosporins including but not limited to cefazolin (Cefazolin), ceftriaxone (Ceftriaxone), ceftazidime (Ceftazidime), and Cefotaxime (ceftaxime), and carbapenems including but not limited to imipenem (Imipenem), meropenem (Meropenem), and ertapenem (ERTAPENEM).
Aminoglycosides include, but are not limited to, gentamicin (GENTAMICIN), tobramycin (Tobramycin), and amikacin (Amikacin).
Macrolides include, but are not limited to, erythromycin (Erythromycin), azithromycin (Azithromycin), and clarithromycin (Clarithromycin).
Tetracyclines include, but are not limited to, tetracycline (TETRACYCLINE), doxycycline (Doxycycline), and minocycline (Minocycline).
Quinolones include, but are not limited to, ciprofloxacin (Ciprofloxacin), levofloxacin (Levofloxacin), and moxifloxacin (Moxifloxacin).
Sulfonamides include, but are not limited to, sulfamethoxazole/trimethoprim (Sulfamethoxazole/Trimethoprim, SMZ-TMP).
Glycopeptides include, but are not limited to, vancomycin (Vancomycin) and teicoplanin (Teicoplanin).
Lincomamides include, but are not limited to, clindamycin (CLINDAMYCIN) and lincomycin (Lincomycin).
Nitroimidazoles include, but are not limited to, metronidazole (Metronidazole).
Polypeptides include, but are not limited to, polymyxin B (Polymyxin B) and Colistin.
Of course, the person skilled in the art can also apply the invention to the drug sensitive detection of other antibiotics such as Linezolid (Linezolid), daptomycin (Daptomycin), tigecycline (TIGECYCLINE) and fusidic acid (Fusidic acid).
In the invention, according to the action mechanisms of different antibiotics (such as blocking cell wall synthesis, damaging cell membranes to affect permeability, blocking ribosomal protein synthesis, affecting folic acid metabolism or blocking RNA and DNA synthesis, etc.), the potential modulation frequency needs to be adjusted so as to detect the electrical impedance change of the relevant cell structure of the bacteria acting on the potential modulation frequency. For example, polymyxin B in one embodiment of the invention affects permeability by damaging the bacterial membrane, thereby altering the single cell electrical impedance, and experimentally verifies that the modulation frequency is selected to be 10Hz.
Similarly, for different antibiotics/bacteria, the corresponding preset threshold value needs to be adjusted accordingly, which can be obtained using the population sample.
The beneficial effects of the invention are that
Compared with the prior art, the invention has the following beneficial effects:
the drug sensitive chip can combine more phages by optimizing phage modification reagents and parameters, and improves the bacterial capturing efficiency, thereby facilitating the subsequent high-flux detection of single bacteria.
By utilizing the method, the electrical impedance image of the single bacteria level can be obtained based on the potential modulation surface plasmon resonance, and the method can be used for detecting the cell structure change of the single bacteria level and further used for detecting the drug sensitivity.
The method has the advantage of high throughput analysis of single bacteria, and can be used for research on bacterial drug resistance mechanism and heterogeneous drug resistance.
Drawings
FIG. 1 shows a schematic diagram of a drug sensitive detection chip;
fig. 2 shows a schematic diagram of an objective type surface plasmon resonance imaging system.
FIG. 3 shows SPR images of E.coli based on surface plasmon resonance.
Fig. 4 shows an electrical impedance image of e.coli based on surface plasmon resonance.
FIG. 5 shows the electrical impedance amplitude as a function of time after E.coli is exposed to 4. Mu.g/mL polymyxin B.
FIG. 6 shows the variation of the electrical impedance amplitude as a function of concentration for 60 minutes after E.coli is exposed to a gradient of polymyxin B.
Detailed Description
Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited in this application are incorporated by reference, particularly as if they were set forth in the relevant terms of art. If the definition of a particular term disclosed in the prior art is inconsistent with any definition provided in the present application, the definition of the term provided in the present application controls.
The numerical ranges in the present application are approximations, so that it may include the numerical values outside the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.
The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or process, and are not related to whether or not such other component, step or process is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising", "including" or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant or compound. Rather, the term "consisting essentially of" excludes any other component, step, or process from the scope of any of the terms recited below, except as necessary for operability. The term "consisting of" does not include any components, steps, or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.
In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
The experimental methods in the following examples are conventional methods unless otherwise specified. The apparatus used in the examples described below, unless otherwise specified, were all laboratory conventional apparatus, and the test materials used in the examples described below, unless otherwise specified, were all purchased from conventional biochemical reagent stores.
Example 1 preparation of drug sensitive detection chip #1
1. Preparation and treatment of film-coated glass slide
And plating 3-6 nm of chromium (Cr) and then 33-60 nm of gold (Au) on the BK7 optical slide by using a magnetron sputtering method to obtain the 36-66 nm thick film plating slide. The coated glass slide is washed by a piranha solution (the volume ratio of H 2SO4 to H 2O2 is 7:3), and is dried by nitrogen, and then is treated by hydrogen flame to improve the flatness of the coated glass slide and remove impurity particles on the surface.
2. Phage modification
1MLM phage solution (10 6 PFU/mL), 20mg 1-ethyl- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 15mg cysteamine were mixed and incubated with shaking for 12 hours. The thiolated phage was filtered using an ultrafilter and resuspended in ultrapure water to give a thiolated phage solution, which was stored at 4 ℃.
And (3) washing the surface of the coated glass slide by using absolute ethyl alcohol, immersing the coated glass slide in a sulfhydrylation phage solution, incubating for 1.5-6 hours to carry out phage self-assembly monolayer (SAM) modification, and washing by using deionized water.
3. Preparation of drug sensitive detection chip #1
In the embodiment, polydimethylsiloxane (PDMS) is adopted to prepare a detection cavity, PDMS monomer and cross-linking agent (DOW CORNING) are mixed according to the mass ratio of 10:1, the mixture is stirred uniformly and then is kept stand for removing bubbles, then the bubble-removed PDMS glue is poured into a plastic culture dish with the diameter of 9cm and the surface of which is flat, the plastic culture dish is placed on a heating plate for baking for 1.5 hours at the temperature of 75 ℃, the PDMS film is removed and then cut into square blocks with the diameter of 10mm multiplied by 10mm, the thickness of the PDMS film is about 2mm, and a puncher is used for punching the cut PDMS film to prepare the detection cavity, wherein the detection cavity is cylindrical and the diameter of the detection cavity is 2.5mm.
And (3) cleaning the coated glass slide in a surface plasma treatment instrument for 30 seconds, and then placing the prepared PDMS on the coated glass slide to combine the coated glass slide with the PDMS for 1 hour to obtain the drug sensitive detection chip #1 for containing a bacterial solution and for drug sensitive detection. The obtained drug sensitive detection chip #1 can be stored in a refrigerator at 4 ℃ for standby.
Example 2 preparation of drug sensitive detection chip #2
In this example, phage modification was performed by referring to the method disclosed in chinese patent publication No. CN101251541a, which specifically comprises the following steps:
Preparation and treatment of the coated slide the same as in example 1 was followed by immersing the coated slide in 50mM mercaptoundecanoic acid ethanol solution for 24 hours, immersing the coated slide in a mixed aqueous solution of EDC and N-hydroxysuccinimide (NHS) having a molar concentration of 200mM and 50mM respectively for 30 minutes after washing and blow-drying, preparing the phage solution of example 1, injecting the phage solution into a container containing the coated slide at a flow rate of 2. Mu.L/min for 30 minutes, and then washing the nonspecific adsorption with 0.02% PBS-T solution.
The preparation of the detection chamber and the combination method of the detection chamber and the coated glass were the same as in example 1, to obtain a drug sensitive detection chip #2.
Example 3 preparation of drug sensitive detection chip #3
The difference from example 1 is that, when phage was modified, cysteamine was replaced with an equimolar amount of N-hydroxysuccinimide, and the rest was the same as in example 1, to obtain a drug sensitive detection chip #3.
Example 4 preparation of drug sensitive detection chip #4
The difference from example 1 is that cysteamine was replaced with equimolar amount of N-hydroxysulfosuccinimide (Sulfo-NHS) at the time of phage modification, and the remainder was the same as example 1, to obtain a drug sensitive detection chip #4.
Example 5 preparation of drug sensitive detection chip #5
The difference from example 1 is that when phage were modified, cysteamine was replaced with equimolar quantity of N-ethylmaleimide (NEM), and the remainder was the same as in example 1, to obtain a drug sensitive detection chip #5.
Example 6 preparation of drug sensitive detection chip #6
The difference from example 1 is that cysteamine was replaced with equimolar amount of N-iodosuccinimide at the time of phage modification, and the remainder was the same as example 1, to obtain a drug sensitive detection chip #6.
Example 7 preparation of drug sensitive detection chip #7
The difference from example 1 is that, when phage was modified, cysteamine was replaced with an equimolar amount of N-aminophthalimide, and the remainder was the same as in example 1, to obtain a drug sensitive detection chip #7.
Example 8 preparation of drug sensitive detection chip #8
The difference from example 1 is that, when phage was modified, cysteamine was replaced with an equimolar amount of N-ethoxyformylphthalimide, and the rest was the same as in example 1, to obtain a drug sensitive detection chip #8.
Example 9 phage monolayer detection
The charge density wave of a metal Surface is described in solid physics as being quantized as "Surface plasmon". The physical phenomenon in which surface plasmons are excited is called "surface plasmon resonance" (surface plasmon resonance, SPR).
SPR is a physical optical phenomenon that results from the interaction of electromagnetic waves of incident light with free electrons on the surface of a metallic conductor. When a beam of light is incident into air from glass or quartz matrix, its refraction angle is greater than incident angle, and when the incident angle is greater than a specific angle (critical angle), total reflection phenomenon can be produced, so as to produce evanescent wave, and the free electrons on the surface of metal film plated on the matrix can be induced to produce surface plasma. When the incident angle is a certain value (namely resonance angle), the surface plasma resonates with the evanescent wave, the energy of the incident light is partially transferred to the surface plasma, the energy of the reflected light is rapidly reduced, a formant (dark band) appears on the reflection spectrum, and the position of the formant changes along with the change of the refractive index of the surface of the metal film. SPR is very sensitive to the refractive index of the medium attached to the surface of a metal film, and the resonance angle will be different when the properties of the surface medium are changed or the amount of attachment is changed. And directly measuring the change of the resonance angle of the surface of the drug sensitive detection chip by using an image sensor to obtain an SPR signal.
In the present invention, the inventors have combined SPR with imaging techniques using a total internal reflection microscope (IX 73, olympus, japan) system. Specifically, the laser source uses Super Light Emitting Diode (SLED), p polarized light with power of 15mW and wavelength of 660nm, total internal reflection microscope uses 60 times of oil mirror (NA=1.49), and camera uses CCD camera (Allied Vision Pike F-032). The CCD camera can detect the light and shade changes in the field of view of the chip surface, namely SPR images, calculate the changes of refractive indexes of different areas, and the gray value of the SPR images is the SPR signal value.
In the invention, phage are modified on the surface of the coated glass, and the refractive index is influenced, so that the change of the overall SPR signal value is influenced, and therefore, the quantity of phage on different drug sensitive detection chips can be evaluated by utilizing the change of the overall SPR signal value (average gray scale of SPR images) of the coated glass before and after phage modification. The results are shown in Table 1.
TABLE 1 variation of SPR Signal values for different drug sensitive detection chips
As can be seen from Table 1, the signal value variation of the drug sensitive detection chip #1 SPR is significantly higher than that of other drug sensitive detection chips, which indicates that the number of phages bound on the drug sensitive detection chip is the greatest, and the method of example 1 is proved to have the best phage modification effect on the coated glass, so that more phages can be bound, and the bacterial capturing efficiency is further improved, thereby facilitating the subsequent high-throughput detection of single bacteria.
Example 10 SPR-based Single cell Electrical impedance imaging
1. Phage capture
After adding 10 6 CFU/mL of escherichia coli solution (CaMHB broth culture) to the PDMS detection chamber, the bacteria were incubated at 37 ℃ for 30 minutes to allow the bacteria to be captured by phage on the chip surface (as shown in fig. 1), and then unbound bacteria were washed away using CaMHB broth.
2. Single cell electrical impedance model
When an electric field is applied to bacterial cells, the resistive component, the ion flow through the conductive bacterial cell wall and cytoplasm, and the capacitive component (i.e., plasma membrane charging) all affect the electrical impedance of the cell.
The electrical impedance model of a single bacterium includes one capacitive region, a cell membrane, and three resistive regions (cell wall, cytoplasm, and cell membrane). When potential modulation is applied, the total currentIt can be calculated as:
wherein R S is medium resistance, R wall is cell wall resistance, R cyt is cytoplasmic resistance, R m is cell membrane resistance, Is a film capacitance, V is an applied potential modulation, j isW is the angular frequency of the potential modulation.
To simplify the formula, the cell membrane resistance is not considered, and thus, the membrane charge densityThe calculation formula is as follows:
where f is the modulation frequency.
3. Electrical impedance imaging method based on SPR
In the present invention, the inventors found that the refractive index of the interface of the coated slide is linear with the film charge density of the bacterial surface to which the surface is bound under applied potential modulation, so that the SPR signal value is also linear with the film charge density of the bacterial surface under applied potential modulation.
Therefore, the calculation formula of the SPR signal intensity S is as follows:
where α is a linear coefficient.
When the membrane charge density of the bacterial surface is changed slightly, the resonance angle is changed obviously, and the SPR signal value is changed very sensitively. The SPR image may be converted to an electrical impedance image by performing a Fast Fourier Transform (FFT).
4. Electrical impedance imaging system based on SPR
As shown in FIG. 2, after capturing E.coli with phage, when potential modulation is applied, the film charge density on the E.coli surface will change with the modulation, resulting in a change in the optical refractive index of the coated slide, resulting in a periodic change in the SPR signal value.
The potential modulation technique is to control the potential of bacteria by using a standard three-electrode electrochemical measurement configuration, a potentiostat (PINE RESEARCH instruments, ARFDE 5), a coated glass slide as a working electrode, ag/AgCl as a reference electrode, a platinum (Pt) coil as a counter electrode, apply potential modulation of different frequencies and amplitudes to a sample by using an external function generator (Agilent, 33220A), and realize synchronization between electrochemical measurement and a camera by using a data acquisition card (DAQ, USB-6250,National Instruments) while recording the applied potential and a trigger signal of the camera.
Setting the voltage amplitude range of potential modulation as 500 mV, the frequency as 10 Hz, the modulation time length as 5 seconds, setting the camera acquisition frame rate as 106 fps, the imaging view field as 640 x 480 pixels, the image acquisition time length as 5 seconds, carrying out surface plasma resonance image acquisition on coliform bacteria before adding polymyxin, and converting into an electrical impedance image.
Example 11 drug sensitivity detection Using SPR-based single bacterial Electrical impedance imaging
The antibiotic and bacteria can destroy the structures such as bacterial cell walls, and the like, so that the membrane charge density on the surface of the bacteria is changed, the refractive index is changed, and the drug-resistant bacteria is not obviously changed, so that the sterilization and bacteriostasis effects of the antibiotic can be evaluated through the change of SPR signal values.
Polymyxin B was diluted to 0.125, 0.25, 0.5, 1,2, 4, 8 μg/mL using CaMHB broth, respectively, and added to the different detection chambers, and SPR images of bacteria were acquired every 5 minutes (as shown in fig. 3). The method comprises the steps of observing the morphological change of the escherichia coli through an optical image, processing an SPR image by using Fast Fourier Transform (FFT) to obtain an electrical impedance image (shown in figure 4), further obtaining the electrical impedance amplitude of the escherichia coli, and normalizing the electrical impedance amplitude after exposure to the polymyxin B based on the electrical impedance amplitude before exposure to the polymyxin B.
Taking the example of adding 4 mug/mL polymyxin B, after FFT, a curve of normalized electrical impedance amplitude of the escherichia coli with time is obtained, as shown in figure 5. The increase in the electrical impedance amplitude is due to the fact that polymyxin B changes the permeability of cell membranes, and increases the membrane charge density caused by the increase in bacterial membrane capacitance, so that the electrical impedance amplitude is increased. As can be seen from FIG. 5, the variation of the electrical impedance amplitude of the E.coli 1 was somewhat different from that of E.coli, the variation of the electrical impedance amplitude of E.coli 2 was slightly lower than that of E.coli 2, but was about 0.39, and the variation of the electrical impedance amplitude of E.coli 3 was only 0.044 after 60 minutes of exposure to polymyxin B, indicating that there was very large heterogeneity between individual cells.
The change of the electrical impedance amplitude of the coliform bacteria after 60 minutes can be calculated by the change curve of the normalized electrical impedance amplitude of the coliform bacteria along with time. Coli was exposed to 0.125, 0.25, 0.5, 1, 2, 4, 8 μg/mL polymyxin B, respectively, and observed for changes in electrical impedance magnitude before and after exposure, and 5 single bacteria were randomly selected for each antibiotic concentration, and the results are shown in fig. 6.
As can be seen from FIG. 6, the overall response of E.coli to antibiotic concentrations was relatively consistent, but individual single bacteria were different from other bacteria, such as in 1. Mu.g/mL polymyxin B and in 4. Mu.g/mL polymyxin B, with individual single cells having a greater variation in electrical impedance magnitude than other single bacteria, and exhibited significantly lower electrical impedance magnitude than other single bacteria at the same concentrations, indicating that these single bacteria were relatively resistant to the drug. The inventor eliminates the electrical impedance amplitude variation of the individual single bacteria and calculates the average value of the electrical impedance amplitude variation of other single bacteria.
It is worth noting that these isolated single bacteria indicate drug resistance variation in the population, and that a sample needs to be monitored with emphasis as the drug resistance bacteria increase in proportion.
As is further evident from FIG. 6, the average electrical impedance amplitude of E.coli to be tested was less over time at a polymyxin B concentration of 0.5. Mu.g/mL or less and more over time at a polymyxin B concentration of 1. Mu.g/mL or more, so that the minimum inhibitory concentration of E.coli to polymyxin B was determined to be 1. Mu.g/mL.
The inventor further detects the minimum inhibitory concentration of the escherichia coli by a micro-dilution method as a gold standard control, and the minimum inhibitory concentration of the escherichia coli on the polymyxin B is 1 mug/mL. It can be seen that the minimum inhibitory concentration result detected by the drug sensitivity detection method of single-bacteria electrical impedance imaging is consistent with the result of the gold standard method microdilution method.
In addition, the method is based on single-bacterium electrical impedance imaging, has the advantage of analyzing single bacterium at high flux, and can be used for researching a bacterial drug resistance mechanism and heterogeneous drug resistance.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
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