A single bacteria electrical impedance imaging method based on surface plasmon resonance and its application

Abstract

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

[0001] The invention belongs to the technical field of bacterial drug sensitivity detection, and in particular relates to a single bacteria electrical impedance imaging method based on surface plasmon resonance and an application thereof. Background Art

[0002] At present, the main method for bacterial drug sensitivity testing is still the traditional antibiotic susceptibility testing (AST), such as paper diffusion, agar dilution, broth microdilution, etc. These technologies require the cultivation, separation and enrichment of bacteria in clinical samples, and then obtain bacterial drug sensitivity results by observing the inhibitory effect of antibiotics on bacterial growth. Although accurate, the process is complicated and time-consuming (2 to 4 days).

[0003] In recent years, various rapid drug sensitivity detection technologies have made progress. Among them, the "phenotype"-based method directly detects bacterial phenotypic characteristics, such as bacterial size, morphology, quantity, electrical characteristics, etc. This is the gold standard AST detection method. Rapid drug sensitivity detection methods based on bacterial impedance are an important research direction. The response of bacteria to electric fields is an inherent characteristic related to the size and shape of bacteria, the internal structure of bacteria, and the conductivity and dielectric constant of different bacterial components. Any differences and changes in bacterial components will affect their electrical signals, making impedance analysis a good method for rapid bacterial drug sensitivity detection and research on the mechanism of action of antibiotics.

[0004] However, traditional impedance detection methods are mainly based on spectroscopy technology, measuring the average impedance of the entire sensor area, such as electrochemical impedance spectroscopy (EIS). Therefore, it lacks imaging capabilities and is difficult to complete the heterogeneity study of single bacteria. A new bacterial electrical impedance imaging and drug sensitivity detection method is urgently needed. Summary of the invention

[0005] In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a phage-modified coated substrate, which is prepared by the following steps:

[0007] A transparent substrate is first plated with 3-6 nm chromium and then with 33-60 nm gold by magnetron sputtering to obtain a coated substrate;

[0008] The phage was thiolated using 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide and cysteamine;

[0009] The coated substrate is placed in a thiol-modified phage solution to be modified by a phage self-assembled monolayer to obtain the phage-modified coated substrate.

[0010] In some specific embodiments of the present invention, the step of thiolating the bacteriophage is:

[0011] 1mL 10 5 ~10 7 PFU / mL phage solution, add 20 mg 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 15 mg cysteamine, mix, and incubate with shaking for 8-18 hours.

[0012] In some embodiments of the present invention, the material of the transparent substrate is selected from one of glass, polycarbonate, polymethyl methacrylate, and fluorosilicone resin.

[0013] Among them, polycarbonate is a transparent, high-strength thermoplastic material. It has excellent impact resistance and heat resistance, and is lighter than glass.

[0014] Polymethyl methacrylate (PMMA), also known as acrylic or plexiglass, has excellent optical clarity and weather resistance. It has a lower density than glass and is easy to process and shape.

[0015] Fluorosilicone Resin maintains the good flexibility of siloxane while increasing chemical resistance and transparency. It can be used as a substitute for glass in optical applications, especially in terms of weather resistance and heat resistance.

[0016] The second aspect of the present invention provides a chip obtained by combining an organic silicon film on the phage-modified coated substrate described in the first aspect of the present invention, wherein the organic silicon film has holes running through it from top to bottom, and the holes enable the organic silicon film to form a detection cavity after combining with the coated substrate, and the phage is exposed in the detection cavity.

[0017] In some embodiments of the present invention, the silicone is selected from one of polydimethylsiloxane, polymethylphenylsiloxane, methylsiloxane, vinylsiloxane, fluorosilicone and polyphenylsiloxane.

[0018] Among them, polydimethylsiloxane (PDMS) is a very common organosilicon compound. The chemical structure of PDMS is (-Si(CH3)2-O-) n , in which dimethylsiloxane units (-Si(CH3)2-O-) are connected by silicon-oxygen bonds (Si-O) to form long-chain polymers. Due to the presence of silicon-oxygen bonds, PDMS has unique flexibility, thermal stability and low surface energy.

[0019] The structure of polymethylphenylsiloxane (PMDS) is similar to that of PDMS, but some of the methyl groups are replaced by phenyl groups.

[0020] Polymethylsiloxane (PMS) is a variant of PDMS that contains only methyl groups in the molecular chain.

[0021] Vinyl siloxane introduces vinyl side groups on the basis of PDMS, which can form a higher strength elastomer through cross-linking reaction, and has higher weather resistance and chemical stability.

[0022] Fluorosilicone introduces fluorine groups on the basis of PDMS, which has excellent chemical corrosion resistance, hydrophobicity and high temperature resistance.

[0023] Polyphenylsiloxane is obtained by replacing some of the methyl groups of PDMS with phenyl groups, and has good high temperature resistance.

[0024] The third aspect of the present invention provides a single bacteria electrical impedance imaging method based on surface plasmon resonance, comprising the following steps:

[0025] Adding a bacterial solution into the detection chamber of any chip according to the second aspect of the present invention, so that the bacteria bind to the bacteriophage, and washing away unbound bacteria;

[0026] Applying potential modulation to the bacteria in the detection cavity, and using an objective lens type total internal reflection microscope to collect images of the other side of the chip relative to the detection cavity, to obtain surface plasmon resonance images under different modulation voltages;

[0027] The surface plasmon resonance image is subjected to Fourier transformation to obtain an electrical impedance image of a single bacterium.

[0028] In the present invention, the image acquisition frequency is greater than twice the potential modulation frequency to meet the image acquisition frequency requirement of electrical impedance imaging.

[0029] The fourth aspect of the present invention provides a single bacterial drug sensitivity detection method, comprising the following steps:

[0030] The bacteria to be tested are exposed to different concentrations of antibiotics in the detection chamber of any chip described in the second aspect of the present invention,

[0031] Obtaining electrical impedance images of the bacteria to be tested before and after exposure to different concentrations of antibiotics for a period of time using any of the methods described in the third aspect of the present invention, and obtaining amplitude changes of the electrical impedance images of each single bacterium before and after exposure for a period of time;

[0032] If the amplitude change of the electrical impedance image of more than a certain proportion of single bacteria before exposure to antibiotics below a certain concentration and after exposure for a period of time is less than a preset threshold, and the amplitude change of the electrical impedance image of more than a certain proportion of single bacteria before exposure to antibiotics above the concentration and after exposure for a period of time is greater than the preset threshold, then the concentration is the minimum inhibitory concentration of the antibiotic for the single bacteria,

[0033] Wherein, the certain proportion is 60%~90%;

[0034] In some embodiments of the present invention, the certain proportion is 70% to 90%.

[0035] In some embodiments of the present invention, the certain proportion is 80% to 90%.

[0036] In the present invention, the antibiotic is selected from at least one of β-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, sulfonamides, glycopeptides, lincosamides, nitroimidazoles and polypeptide antibiotics.

[0037] Among them, β-lactams include penicillins, cephalosporins and carbapenems. Penicillins include but are not limited to penicillin G, ampicillin and piperacillin; cephalosporins include but are not limited to cefazolin, ceftriaxone, ceftazidime and cefotaxime; carbapenems include but are not limited to imipenem, meropenem and ertapenem.

[0038] Aminoglycosides include, but are not limited to, gentamicin, tobramycin, and amikacin.

[0039] Macrolides include, but are not limited to, erythromycin, azithromycin, and clarithromycin.

[0040] Tetracyclines include, but are not limited to, tetracycline, doxycycline, and minocycline.

[0041] Quinolones include, but are not limited to, Ciprofloxacin, Levofloxacin, and Moxifloxacin.

[0042] Sulfonamides include but are not limited to Sulfamethoxazole / Trimethoprim (SMZ-TMP).

[0043] Glycopeptides include, but are not limited to, vancomycin and teicoplanin.

[0044] Lincosamides include, but are not limited to, clindamycin and lincomycin.

[0045] Nitroimidazoles include but are not limited to metronidazole.

[0046] Peptides include but are not limited to Polymyxin B and Colistin.

[0047] Of course, those skilled in the art can also apply the present invention to the drug sensitivity detection of other antibiotics such as Linezolid, Daptomycin, Tigecycline and Fusidic acid.

[0048] In the present invention, according to the action mechanism of different antibiotics (such as blocking cell wall synthesis, damaging cell membrane to affect permeability, blocking ribosomal protein synthesis, affecting folic acid metabolism or blocking RNA and DNA synthesis, etc.), it is necessary to adjust the potential modulation frequency to detect the change in the electrical impedance of the relevant cell structure of the bacteria. For example, polymyxin B in a specific embodiment of the present invention affects the permeability by damaging the bacterial membrane and thus changes the single cell electrical impedance. The modulation frequency is selected to be 10Hz through experimental verification.

[0049] Similarly, for different antibiotics / bacteria, the corresponding preset thresholds need to be adjusted accordingly, and the preset thresholds can be obtained using population samples.

[0050] Beneficial effects of the present invention

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The drug sensitivity chip of the present invention can bind more bacteriophages and improve the bacteria capture efficiency by optimizing the bacteriophage modification reagents and parameters, thereby facilitating subsequent high-throughput detection of single bacteria.

[0053] By using the method of the present invention, an electrical impedance image at the single bacteria level can be obtained based on potential-modulated surface plasmon resonance, which can be used to detect cell structure changes at the single bacteria level and further used for drug sensitivity detection.

[0054] The method of the present invention has the advantage of high-throughput analysis of single bacteria and can be used to study bacterial resistance mechanisms and heterogeneous resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 A schematic diagram of a drug sensitivity detection chip is shown;

[0056] Figure 2 A schematic diagram of an objective lens type surface plasmon resonance imaging system is shown.

[0057] Figure 3 Shown is a surface plasmon resonance-based SPR image of Escherichia coli.

[0058] Figure 4 Shown is an electrical impedance image of Escherichia coli based on surface plasmon resonance.

[0059] Figure 5 The curve showing the change of electrical impedance amplitude over time after Escherichia coli was exposed to 4 μg / mL polymyxin B.

[0060] Figure 6 The curve showing the change of electrical impedance amplitude as a function of concentration after Escherichia coli was exposed to gradient concentrations of polymyxin B for 60 minutes. DETAILED DESCRIPTION

[0061] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0062] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally regarded as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0063] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0064] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0065] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0066] 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, and the disclosure and materials cited therein are hereby incorporated by reference.

[0067] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0068] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0069] Example 1 Preparation of drug sensitivity detection chip #1

[0070] 1. Preparation and treatment of coated slides

[0071] The BK7 optical glass slide was first coated with 3~6nm chromium (Cr) and then 33~60nm gold (Au) by magnetron sputtering to obtain a 36~66nm thick coated glass slide. The coated glass slide was rinsed with piranha solution (H2SO4 and H2O2 volume ratio 7:3), dried with nitrogen, and then treated with hydrogen flame to improve the flatness of the coated glass slide and remove impurities on the surface.

[0072] 2. Phage modification

[0073] 1 mL of M13 phage solution (10 6 PFU / mL), 20 mg 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 15 mg cysteamine were mixed and incubated with shaking for 12 hours. The thiolated phages were filtered using an ultrafilter and resuspended in ultrapure water to obtain a thiolated phage solution, which was stored at 4°C.

[0074] The surface of the coated glass slide was cleaned with anhydrous ethanol, and then the coated glass slide was immersed in a thiolated phage solution, incubated for 1.5 to 6 hours for phage self-assembled monolayer (SAM) modification, and then washed with deionized water.

[0075] 3. Preparation of drug sensitivity detection chip #1

[0076] In this embodiment, polydimethylsiloxane (PDMS) is used to prepare the detection cavity. First, the PDMS monomer and the cross-linking agent (DOWCORNING) are mixed in a mass ratio of 10:1, stirred evenly and allowed to stand to remove bubbles. The PDMS glue with all bubbles removed is then poured into a plastic culture dish with a flat surface and a diameter of 9 cm, and baked on a heating plate at 75°C for 1.5 hours. The PDMS film is peeled off and cut into 10 mm×10 mm square blocks. The thickness of the PDMS film is about 2 mm. A hole puncher is used to punch holes in the cut PDMS film to make the detection cavity. The detection cavity is cylindrical and has a diameter of 2.5 mm.

[0077] The coated glass slide was cleaned in a surface plasma treatment apparatus for 30 seconds, and then the prepared PDMS was placed on the coated glass slide to allow the two to combine for 1 hour, and the drug sensitivity detection chip #1 was obtained to hold the bacterial solution and be used for drug sensitivity detection. The obtained drug sensitivity detection chip #1 can be stored in a 4°C refrigerator for future use.

[0078] Example 2 Preparation of drug sensitivity detection chip #2

[0079] This example refers to the method disclosed in Chinese invention patent publication number CN101251541A to perform phage modification, and the specific steps are as follows:

[0080] The preparation and treatment of the coated glass slide were the same as in Example 1, and then the coated glass slide was immersed in a 50 mM mercaptoundecanoic acid ethanol solution for 24 hours; after cleaning and drying, the coated glass slide was immersed in a mixed aqueous solution of EDC and N-hydroxysuccinimide (NHS) with molar concentrations of 200 mM and 50 mM, respectively, for 30 minutes; the phage solution was prepared as in Example 1, and injected into the container containing the coated glass slide at a flow rate of 2 μL / min for 30 minutes; then nonspecific adsorption was washed with 0.02% PBS-T solution.

[0081] The preparation of the detection cavity and the method of combining it with the coated glass slide are the same as in Example 1, and a drug sensitivity detection chip #2 is obtained.

[0082] Example 3 Preparation of drug sensitivity detection chip #3

[0083] The difference from Example 1 is that when modifying the phage, cysteamine is replaced by an equimolar amount of N-hydroxysuccinimide, and the rest is the same as Example 1, to obtain a drug sensitivity detection chip #3.

[0084] Example 4 Preparation of drug sensitivity detection chip #4

[0085] The difference from Example 1 is that when modifying the phage, cysteamine is replaced by an equimolar amount of N-hydroxysulfosuccinimide (Sulfo-NHS), and the rest is the same as Example 1, to obtain a drug sensitivity detection chip #4.

[0086] Example 5 Preparation of drug sensitivity detection chip #5

[0087] The difference from Example 1 is that when modifying the phage, cysteamine is replaced by an equimolar amount of N-ethylmaleimide (NEM), and the rest is the same as Example 1, to obtain a drug sensitivity detection chip #5.

[0088] Example 6 Preparation of drug sensitivity detection chip #6

[0089] The difference from Example 1 is that when modifying the phage, cysteamine is replaced by an equimolar amount of N-iodosuccinimide, and the rest is the same as Example 1, to obtain a drug sensitivity detection chip #6.

[0090] Example 7 Preparation of drug sensitivity detection chip #7

[0091] The difference from Example 1 is that when modifying the phage, cysteamine is replaced by an equimolar amount of N-aminophthalimide, and the rest is the same as Example 1, to obtain a drug sensitivity detection chip #7.

[0092] Example 8 Preparation of drug sensitivity detection chip #8

[0093] The difference from Example 1 is that when modifying the phage, cysteamine is replaced by an equimolar amount of N-ethoxycarbonylphthalimide, and the rest is the same as Example 1, to obtain a drug sensitivity detection chip #8.

[0094] Example 9 Phage Monolayer Detection

[0095] The charge density wave on the metal surface is quantized and described as "surface plasmon" in solid-state physics. The physical phenomenon of surface plasmon being excited is called "surface plasmon resonance" (SPR).

[0096] SPR is a physical optical phenomenon, which is generated by the interaction between the electromagnetic wave of the incident light and the free electrons on the surface of the metal conductor. When a beam of light is incident from a glass or quartz substrate into the air, its refraction angle will be greater than the incident angle; and when the incident angle is greater than a certain angle (critical angle), total reflection will occur, generating an evanescent wave, which triggers the free electrons on the surface of the metal film coated on the substrate to generate surface plasma. When the incident angle is a certain value (i.e., the resonance angle), the surface plasma resonates with the evanescent wave, and the incident light energy is partially transferred to the surface plasma, causing the reflected light energy to drop sharply, and a resonance peak (dark band) appears in the reflection spectrum; the position of the resonance peak changes with the change of the refractive index of the metal film surface. SPR is very sensitive to the refractive index of the medium attached to the surface of the metal film. When the properties of the surface medium change or the amount of attachment changes, the resonance angle will be different. The SPR signal is obtained by directly measuring the change of the resonance angle on the surface of the drug sensitivity detection chip using an image sensor.

[0097] In the present invention, the inventors combined SPR with imaging technology using a total internal reflection microscope (IX73, Olympus, Japan) system. Specifically, the laser light source uses a superluminescent diode (SLED), uses p-polarized light, has a power of 15mW, and a wavelength of 660nm; the total internal reflection microscope uses a 60x oil objective (NA=1.49); and the camera uses a CCD camera (Allied VisionPike F-032). The CCD camera can detect the light and dark changes in the field of view on the chip surface, that is, the SPR image, and calculate the changes in the refractive index of different regions. The grayscale value of the SPR image is the SPR signal value.

[0098] In the present invention, modifying the phage on the surface of the coated glass slide also affects its refractive index, thereby affecting the change in the overall SPR signal value. Therefore, the change in the overall SPR signal value (average grayscale of the SPR image) of the coated glass slide before and after the modification of the phage can be used to evaluate the number of phages on different drug sensitivity detection chips. The results are shown in Table 1.

[0099] Table 1 SPR signal value changes of different drug sensitivity detection chips

[0100]

[0101] As can be seen from Table 1, the change in the SPR signal value of drug sensitivity detection chip #1 is significantly higher than that of other drug sensitivity detection chips, indicating that the number of phages bound thereto is the largest, proving that the phage modification effect of the coated glass slide using the method of Example 1 is optimal, and more phages can be bound, further improving the bacterial capture efficiency, thereby facilitating subsequent high-throughput detection of single bacteria.

[0102] Example 10 Single cell electrical impedance imaging based on SPR

[0103] 1. Phage capture

[0104] 10 6 After adding CFU / mL of Escherichia coli solution (CaMHB broth culture) into the PDMS detection cavity, it was cultured at 37°C for 30 minutes to allow the bacteria to be captured by the phages on the chip surface (e.g. Figure 1 ), and unbound bacteria were washed away using CaMHB broth.

[0105] 2. Single Cell Electrical Impedance Model

[0106] When an electric field is applied to a bacterial cell, the resistive component, the flow of ions through the conductive bacterial cell wall and cytoplasm, and the capacitive component, i.e., the charging of the plasma membrane, affect the electrical impedance of the cell.

[0107] The electrical impedance model of a single bacterium consists of a capacitive region, the cell membrane, and three resistive regions (cell wall, cytoplasm, and cell membrane). When potential modulation is applied, the total current It can be calculated as:

[0108]

[0109] in, R S is the dielectric resistance, R wall is the cell wall resistance, R cyt is the cytoplasmic resistance, R m is the cell membrane resistance, is the membrane capacitance, V is the applied potential modulation, j for , w is the angular frequency of the potential modulation.

[0110] In order to simplify the formula, the cell membrane resistance is not considered, so the membrane charge density The calculation formula is:

[0111]

[0112] in, f is the modulation frequency.

[0113] 3. SPR-based electrical impedance imaging method

[0114] In the present invention, the inventors found that the refractive index of the coated glass slide interface is linearly related to the membrane charge density of the bacterial surface bound to it under applied potential modulation, so that the SPR signal value is also linearly related to the membrane charge density of the bacterial surface under applied potential modulation.

[0115] Therefore, the calculation formula of SPR signal intensity S is as follows:

[0116]

[0117] in, α is the linear coefficient.

[0118] When the membrane charge density on the bacterial surface changes slightly, the resonance angle changes significantly, and the SPR signal value changes very sensitively. The SPR image can be converted into an electrical impedance image by performing a fast Fourier transform (FFT).

[0119] 4. SPR-based electrical impedance imaging system

[0120] like Figure 2As shown, after the coated glass slide uses bacteriophage to capture E. coli, when potential modulation is applied, the membrane charge density on the surface of E. coli changes with the modulation, causing the optical refractive index of the coated glass slide to change, resulting in periodic changes in the SPR signal value.

[0121] The potential modulation technique was developed by using a standard three-electrode electrochemical measurement configuration. The potential of the bacteria was controlled by a potentiostat (Pine Research Instrumentation, ARFDE5), with a coated glass slide as the working electrode, Ag / AgCl as the reference electrode, and a platinum (Pt) coil as the counter electrode. An external function generator (Agilent, 33220A) was used to apply potential modulation of different frequencies and amplitudes to the sample. A data acquisition card (DAQ, USB-6250, National Instruments) was used to synchronize the electrochemical measurement with the camera, and simultaneously record the applied potential and the trigger signal of the camera.

[0122] The voltage amplitude range of potential modulation was set to 500 mV, the frequency was set to 10 Hz, and the duration of each modulation was set to 5 seconds. The camera acquisition frame rate was set to 106 fps, the imaging field of view was set to 640×480 pixels, and the image acquisition duration was set to 5 seconds. Surface plasmon resonance images of Escherichia coli before the addition of polymyxin were acquired and converted into electrical impedance images.

[0123] Example 11 Drug sensitivity detection using single bacterial electrical impedance imaging based on SPR

[0124] The interaction between antibiotics and bacteria can destroy structures such as bacterial cell walls, causing changes in the membrane charge density on the bacterial surface, and then causing changes in its refractive index, while there is no obvious change in drug-resistant bacteria. Therefore, the bactericidal and antibacterial effects of antibiotics can be evaluated by changes in SPR signal values.

[0125] Polymyxin B was diluted to 0.125, 0.25, 0.5, 1, 2, 4, and 8 μg / mL using CaMHB broth and added to different detection chambers. SPR images of bacteria were collected every 5 minutes (e.g. Figure 3 The morphological changes of E. coli can be observed through optical images; the SPR images are processed using fast Fourier transform (FFT) to obtain electrical impedance images (as shown in Figure 4 As shown in FIG. 1 ), the electrical impedance amplitude of Escherichia coli can be obtained, and the electrical impedance amplitude after exposure to polymyxin B is normalized based on the electrical impedance amplitude before exposure to polymyxin B.

[0126] Taking the addition of 4 μg / mL polymyxin B as an example, after FFT, the normalized impedance amplitude of Escherichia coli is obtained as a function of time, as shown in Figure 5The increase in the impedance amplitude is because polymyxin B changes the permeability of the cell membrane, increases the bacterial membrane capacitance, and causes the membrane charge density to increase, which in turn increases the impedance amplitude. Figure 5 It can also be seen that the changes in the impedance amplitudes of different Escherichia coli are different to a certain extent. The impedance amplitude change of Escherichia coli 1 after being exposed to polymyxin B for 60 minutes reaches 0.46, and the impedance amplitude change of Escherichia coli 2 is slightly lower, but also reaches about 0.39, while the impedance amplitude change of Escherichia coli 3 after being exposed to polymyxin B for 60 minutes is only 0.044, indicating that there is a very large heterogeneity between single cells.

[0127] The change in the impedance amplitude of E. coli caused by different concentrations of polymyxin B after 60 minutes can be calculated through the curve of the change in the normalized impedance amplitude of E. coli. E. coli was exposed to 0.125, 0.25, 0.5, 1, 2, 4, and 8 μg / mL polymyxin B, and the change in the impedance amplitude before and after was observed. For each antibiotic concentration, 5 single bacteria were randomly selected to observe the change in the impedance amplitude. The results are shown in Figure 6 shown.

[0128] from Figure 6 It can be seen that the overall response of Escherichia coli to antibiotic concentration is relatively consistent, but some individual bacteria are different from other bacteria. For example, in 1μg / mL of polymyxin B and in 4μg / mL of polymyxin B, the impedance amplitude change of individual single cells is quite different from that of other single bacteria, which is manifested in that the impedance amplitude change is significantly lower than that of other single bacteria at the same concentration, indicating that the drug resistance of these single bacteria is relatively strong. After eliminating the impedance amplitude change of these individual single bacteria, the inventor calculated the average of the impedance amplitude change of other single bacteria.

[0129] It is worth noting that these outlier single bacteria indicate the emergence of drug-resistant mutations in the population. When the proportion of drug-resistant bacteria increases, the samples need to be monitored closely.

[0130] from Figure 6 It can be further seen that for the tested Escherichia coli, its average impedance amplitude changes less with time when the polymyxin B concentration is 0.5 μg / mL and below, and changes more with time when the polymyxin B concentration is 1 μg / mL and above, so it can be determined that the minimum inhibitory concentration of Escherichia coli for polymyxin B is 1 μg / mL.

[0131] The inventors further tested the minimum inhibitory concentration of Escherichia coli by microdilution method as a gold standard control, and found that the minimum inhibitory concentration of Escherichia coli for polymyxin B was 1 μg / mL. It can be seen that the minimum inhibitory concentration results obtained by the single bacteria electrical impedance imaging drug sensitivity detection method are consistent with the results of the gold standard method microdilution method.

[0132] In addition, since the method of the present invention is based on single-bacteria electrical impedance imaging, it has the advantage of high-throughput analysis of single bacteria and can be used to study bacterial resistance mechanisms and heterogeneous resistance.

[0133] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Application of a phage-modified coated substrate in the preparation of a kit for single-bacteria drug sensitivity detection based on surface plasmon resonance single-bacteria electrical impedance imaging, characterized in that: The phage-modified coated substrate is prepared by the following steps: A transparent substrate is first plated with 3-6 nm chromium and then with 33-60 nm gold by magnetron sputtering to obtain a coated substrate; The phage was thiolated using 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide and cysteamine; placing the coated substrate in a thiol-modified phage solution to perform phage self-assembled monolayer modification to obtain the phage-modified coated substrate; The step of thiolating the bacteriophage is: 1mL 10 5 ~10 7 PFU / mL phage solution, add 20 mg 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 15 mg cysteamine, mix, shake and incubate for 8-18 hours, The material of the transparent substrate is glass, The application includes the steps of preparing the phage-modified coated substrate into a chip: An organic silicon film is combined on the coating substrate modified by the bacteriophage, and the organic silicon film has holes penetrating from top to bottom, and the holes enable the organic silicon film to form a detection cavity after combining with the coating substrate, and the bacteriophage is exposed in the detection cavity. The single bacterial drug sensitivity test comprises the following steps: Exposing the bacteria to be tested to different concentrations of antibiotics in the detection chamber of the chip; Obtaining electrical impedance images of the bacteria to be tested before and after exposure to antibiotics of different concentrations for a period of time, and obtaining amplitude changes of the electrical impedance images of each single bacterium before and after exposure for a period of time; If the amplitude change of the electrical impedance image of more than a certain proportion of single bacteria before exposure to antibiotics below a certain concentration and after exposure for a period of time is less than a preset threshold, and the amplitude change of the electrical impedance image of more than a certain proportion of single bacteria before exposure to antibiotics above the concentration and after exposure for a period of time is greater than the preset threshold, then the concentration is the minimum inhibitory concentration of the antibiotic to the bacteria, Wherein, the certain proportion is 60% to 90%. The electrical impedance image is obtained by the following steps: Applying potential modulation to the bacteria in the detection cavity, and using an objective lens type total internal reflection microscope to collect images of the other side of the chip relative to the detection cavity, to obtain surface plasmon resonance images under different modulation voltages; The surface plasmon resonance image is subjected to Fourier transformation to obtain an electrical impedance image of a single bacterium.

2. The use according to claim 1, characterized in that: The organic silicon is selected from one of polydimethylsiloxane, polymethylphenylsiloxane, methylsiloxane, vinylsiloxane, fluorosilicone and polyphenylsiloxane.

3. The use according to claim 1, characterized in that: The certain proportion is 70%~90%.

4. The use according to claim 3, characterized in that: The certain proportion is 80%~90%.

5. The use according to any one of claims 1 to 4, characterized in that: The antibiotic is selected from at least one of β-lactams, aminoglycosides, macrolides, tetracyclines, quinolones, sulfonamides, glycopeptides, lincosamides, nitroimidazoles and polypeptide antibiotics.

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