Intracellular pH value detection method in process of expressing heterologous protein by escherichia coli and application of intracellular pH value detection method

Abstract

The invention belongs to the technical field of single cell detection, and particularly relates to a method for detecting the intracellular pH value in the process of expressing heterologous protein by escherichia coli and application. The detection method comprises the following steps: S1, establishing a pH response curve; s2, co-incubating the fluorescent probe AF-C and the escherichia coli in the process of expressing the heterologous protein to obtain a real-time intracellular cell image of the escherichia coli; s3, processing the real-time cell image in the step S2, and calculating the fluorescence intensity of a Red channel and the fluorescence intensity of an NIR channel corresponding to a single cell; and substituting the ratio of the fluorescence intensity of the Red channel to the fluorescence intensity of the NIR channel into the pH response curve in the step S1 to obtain the intracellular pH value of the escherichia coli. The detection method provided by the invention has good permeability and retention in the process of expressing heterologous protein by escherichia coli, has fast spectral response to intracellular pH change, and is more accurate in detection result.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of single cell detection, and particularly relates to a method for detecting intracellular pH in the process of expressing heterologous proteins by Escherichia coli and application thereof. BACKGROUND

[0002] Intracellular pH refers to the acidity and alkalinity of the liquid in the cell, and usually has different pH values at different parts of the cell (such as cytoplasm, mitochondria, lysosomes, etc.). Escherichia coli is a commonly used host bacterium, which has the advantages of rapid growth and efficient protein expression, and is one of the most widely used heterologous expression hosts in the field of biological engineering at present. The core principle is to use genetic engineering technology to introduce target genes from other species (prokaryotes, eukaryotes such as humans, yeasts, plants, etc.) into Escherichia coli, and to synthesize the protein coded by the target gene (i.e. "heterologous protein") through the transcription and translation system of Escherichia coli. However, the efficient expression of heterologous proteins often accompanies the change of intracellular environment, especially the change of intracellular pH; this is because the expression of heterologous proteins may cause the reprogramming of cell metabolism, which in turn also affects the balance of intracellular pH. The change of intracellular pH not only affects the metabolic activity of the cell, but also directly affects the folding, stability and function of the protein. Therefore, accurately and real-time monitoring of intracellular pH has become an important issue in cell biology and medical research.

[0003] In the prior art, the existing intracellular pH detection technologies include: proton-selective electrode method and nuclear magnetic resonance (NMR) technology; among them, the proton-selective electrode method measures the intracellular pH value by using a proton-selective electrode; although this method has high accuracy, its operation is complex, and it is usually impossible to monitor the pH change of a single cell in real time. The nuclear magnetic resonance (NMR) technology can monitor the change of intracellular pH by analyzing the chemical shift of protons, although this method can be used for large-scale and diversified samples, but the technical requirements are high, and the resolution is low. The above defects limit the application of these methods to the determination of intracellular pH.

[0004] With the continuous development of fluorescence technology, the use of fluorescent dyes to detect intracellular pH is referred to in the patent document with publication number CN108535169A, which uses fluorescent dyes (fluorescent dyes include BCECF, SNARF, etc.) to emit different wavelengths of fluorescence at different pH values, and to monitor the change of intracellular pH in real time; although the existing fluorescence method can realize the change of intracellular pH, in the process of expressing heterologous proteins by Escherichia coli, the intracellular pH (pH i ) will change significantly with the protein homeostasis load, carbon-nitrogen metabolic redistribution and energy metabolism fluctuation, and the pH iReal-time, quantitative, non-invasive monitoring of the process control window (induction timing, temperature, feeding, buffer strength) is the key. However, most of the existing fluorescent dye probes serve mammalian or static cell environments, and have obvious shortcomings in the high expression scenario of gram-negative bacteria. The specific performance is as follows: the existing fluorescent dye probe has poor permeability, so that the fluorescent probe is difficult to quickly enter the cell in the outer membrane of Escherichia coli, resulting in slow pH response speed; and poor retention, even if it enters the cell in the expression of heterologous proteins in Escherichia coli, it is easy to leak, so it is difficult to stably obtain data for a long time, and the detection result error is large.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The purpose of the present application is to provide an intracellular pH value detection method and application in the process of Escherichia coli expressing heterologous proteins, which has good permeability and retention in the process of Escherichia coli expressing heterologous proteins, and has a faster spectral response to intracellular pH change, and the detection result is more accurate.

[0007] In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0008] An intracellular pH value detection method in the process of Escherichia coli expressing heterologous proteins, comprising the following steps:

[0009] S1, establishing a pH response curve

[0010] Prepare a solution containing fluorescent probe AF-C with different known pH values, and obtain the pH response curve between the fluorescence intensity ratio and the pH by in vitro fluorescence intensity test method;

[0011] S2, acquiring real-time cell images

[0012] Incubate the fluorescent probe AF-C with Escherichia coli in the process of expressing heterologous proteins to obtain real-time cell images in the intracellular Escherichia coli;

[0013] S3, intracellular pH test

[0014] Process the real-time cell images of step S2, calculate the fluorescence intensity of the Red channel and the fluorescence intensity of the NIR channel corresponding to a single cell; and substitute the ratio of the fluorescence intensity of the Red channel and the fluorescence intensity of the NIR channel into the pH response curve of step S1 to obtain the intracellular pH value of Escherichia coli.

[0015] Further limited, in the step S1, the solution containing the fluorescent probe AF-C is formed by the fluorescent probe AF-C powder, dimethyl sulfoxide and buffer.

[0016] Further limitation, the buffer solution is an aqueous solution containing 10 mM sodium dodecyl sulfate (SDS) and 10 mM HEPES.

[0017] Further limitation, in step S1, the specific process of obtaining the pH response curve by in vitro fluorescence intensity test method is as follows:

[0018] S1.1, at an excitation wavelength of 537 nm, the fluorescence absorption intensity I of the solution containing fluorescent probe AF-C at 630 nm is measured 630nm ;

[0019] S1.2, at an excitation wavelength of 718 nm, the fluorescence absorption intensity I of the solution containing fluorescent probe AF-C at 740 nm is measured 740nm ;

[0020] S1.3, the I 740nm / I 630nm ratio of the solution containing fluorescent probe AF-C corresponding to different known pH values is calculated;

[0021] S1.4, the pH response curve is constructed with the pH value as the abscissa and the I 740nm / I 630nm as the ordinate.

[0022] Further limitation, in step S2, the specific process of incubation is as follows:

[0023] S2.1, the E. coli is pretreated according to the streak culture method to obtain an E. coli bacterial solution;

[0024] S2.2, the E. coli bacterial solution is inoculated into LB liquid medium containing Kan resistance and cultured to OD 600 = 0.6-0.8;

[0025] S2.3, the dimethyl sulfoxide solution containing fluorescent probe AF-C is added to the system after culture, and then incubated.

[0026] Further limitation, in step S2, the incubation is carried out at 37℃, 220 rpm for 45 min-120 min.

[0027] Further limitation, in step S2, the real-time cell image of E. coli intracellular is obtained by laser confocal microscope.

[0028] Further limitation, the excitation wavelength of the laser confocal microscope is set to 561 nm and 640 nm.

[0029] Further limited, in the step S3, import Image J image processing software is adopted, the cell image is processed to reduce noise and deduct background, and then the fluorescence intensity of the Red channel and the fluorescence intensity of the NIR channel of the single cell are calculated respectively.

[0030] Application of a fluorescent probe AF-C in detecting intracellular pH in the process of expressing heterologous proteins by Escherichia coli.

[0031] Compared with the prior art, the beneficial effects of the present application are specifically manifested in that:

[0032] 1、The present application uses the fluorescent probe AF-C and different known pH standard buffers to measure the double-channel fluorescence intensity ratio at different pH values, and draw a pH response curve; then the fluorescent probe AF-C is co-incubated with Escherichia coli cells for a certain time, and the double-channel fluorescence intensity of the Escherichia coli in the heterologous expression process is observed in real time under a laser confocal microscope. Finally, the double-channel fluorescence intensity ratio is substituted into the pH response curve to calculate the real-time intracellular pH value. The fluorescent probe AF-C used in the present application has the characteristics of cell membrane permeability, retention and pH sensitivity, can quickly enter the cell in the process of expressing heterologous proteins by Escherichia coli, can stably obtain data in a long induction time, has a faster spectral response to the pH change in the cell, and the detection result is more accurate.

[0033] 2、The detection method of the present application is suitable for detecting intracellular pH in the range of pH = 6.6-8.6, and can distinguish the pH difference between 7.8 and 8.5. This pH range is relatively wide, and is consistent with the pH required for the growth of Escherichia coli, and meets the detection requirements in the process of expressing heterologous proteins by Escherichia coli.

[0034] 3、The present application selects wavelengths of 630 nm and 740 nm as the characteristic absorption wavelengths of the fluorescent probe AF-C, and constructs a pH response curve based on the fluorescence absorption intensity of the fluorescent probe AF-C at the two wavelengths; and collects cell images, realizes the detection of intracellular pH in the process of expressing heterologous proteins by Escherichia coli through the double-channel fluorescence intensity, and improves the accuracy of the detection result. The fluorescent probe AF-C used in the present application has strong anti-interference ability and higher detection sensitivity. As can be seen from the research data, the measurement accuracy of the present application can reach 0.01 pH unit, 200 nm spatial accuracy and millisecond time accuracy.

[0035] 4、The present application finds that, in the process of expressing heterologous proteins by Escherichia coli, different concentrations of the fluorescent probe AF-C are added, and the survival rate of Escherichia coli cells is more than 90%. The toxicity of the fluorescent probe AF-C is negligible, and the damage to the cells in the process of expressing heterologous proteins by Escherichia coli is small, so that the cells are in a survival state with a high probability, and the detection safety is good. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings needed to be used in the specific embodiments or prior art description will be briefly introduced.

[0037] Figure 1 Structure diagram of fluorescent probe AF-C used in the present application.

[0038] Figure 2 HPLC chromatogram of fluorescent probe AF-C used in the present application. + Ion response curve diagram.

[0039] Figure 3 Laser confocal microscope result diagram of E. coli under different expression time periods in the present application;

[0040] Figure 4 pH response curve diagram of fluorescent probe AF-C used in the present application;

[0041] Figure 5 Laser confocal microscope result diagram of E. coli under different induction expression time periods in the present application;

[0042] Figure 6 Fluorescence intensity diagram of NIR channel and Red channel of AF-C under different induction expression time periods in the present application;

[0043] Figure 7 Fluorescence intensity ratio diagram of NIR channel and Red channel of AF-C under different induction expression time periods in the present application;

[0044] Figure 8 Comparison diagram of intracellular and extracellular pH values of E. coli under different induction expression time periods in the present application;

[0045] Figure 9 Cell toxicity experiment result diagram of fluorescent probe AF-C used in the present application. DETAILED DESCRIPTION

[0046] The present application will be further described in details below in combination with the accompanying drawings and examples, but the embodiments of the present application are not limited thereto. Other methods for preparing the compounds of the present application are considered to be within the scope of the present application, with some conventional modifications according to the reaction conditions of the present application.

[0047] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those skilled in the art to which the present application belongs.

[0048] It should also be understood that the above-described specific embodiments are merely intended for the explanation of the present application, and the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and the inventive concept of the present application within the technical scope disclosed by the present application, which should be covered within the protection scope of the present application.

[0049] In the following examples, the structure of the fluorescent probe (denoted as AF-C) is shown in Figure 1 The fluorescent probe AF-C is a known compound, and the synthesis path is as follows:

[0050]

[0051] Specific reference is made to: Role of Azole Drugs in Promoting Fungal Cell Autophagy Revealed by an NIR Fluorescence-Based Theranostic Probe; Wanzhen Yang, §Yanhui Zhang, §Hao Teng, et al. Analytical Chemistry.

[0052] The fluorescent probe (denoted as AF-C) adopted in the present application has the following characteristics:

[0053] (1) AF-C is designed based on HQO skeleton, and the quinoline nitrogen N_quin and the ortho phenolic hydroxyl O-H in the HQO skeleton (HydroxyQuinoline-Based) form an O-H···N intramolecular hydrogen bond in the vicinity. The molecule can take two channels in the excited state, i.e. excited state intramolecular proton transfer (from O-H→N_quin to form keto * state, emitting NIR≈740nm) and intramolecular charge transfer / LE state radiation (red-shifted region ≈630nm).

[0054] (2) The molecular weight of AF-C is 493.25, which does not occupy ribosomes and ATP, does not change the expression resource allocation of the system to be tested, has good permeability and good retention, and can enter the cytoplasm within 75 minutes of co-incubation; its toxicity can be ignored, and can cover the induction window.

[0055] (3) The red / near-red dual-channel (630 / 740nm) avoids the strong background of protein peptone, yeast extract and flavin in LB medium at <600nm, and has high-density anti-interference ability; it does not need fluorescent protein maturation (oxygen-independent, no low-temperature maturation lag), and responds in real time at an induction temperature of 16-37℃.

[0056] The co-incubation system adopted by the present application does not contain bovine serum albumin, serum or polyvinyl alcohol, and the incubation is carried out in a container with smooth surface and not easy to adhere to cells.

[0057] The buffer adopted by the present application does not contain bovine serum albumin, serum or polyvinyl alcohol, and the composition is: NaCl 8.0 g / L, KCl 0.2 g / L, KH2PO4 1.44 g / L, Na2HPO4 0.24 g / L.

[0058] The cell culture medium adopted by the present application has the following components: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl.

[0059] It should be noted that the chemicals, reagents and the like not specially mentioned are conventional commercially available products in the art.

[0060] 1. Determining the characteristic absorption wavelength of fluorescent probe AF-C

[0061] 1.1. Pretreatment of fluorescent probe AF-C

[0062] Take 20 μM of fluorescent probe AF-C powder, add 1 mL of cell culture grade dimethyl sulfoxide (denoted as DMSO), and dissolve it under ultrasonic conditions. After dissolution, store it in a -20℃ refrigerator for standby use;

[0063] 1.2. H + ion spectrum response determination of fluorescent probe AF-C

[0064] After thawing the above DMSO solution containing fluorescent probe AF-C, inoculate it into an aqueous solution containing 10 mM sodium dodecyl sulfate (denoted as SDS) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (denoted as HEPES) at a proportion of 0.5%. After mixing, take 500 μL of the solution and adjust the pH value to 6.8; take another 500 μL of the solution and adjust the pH value to 7.4. Place the two AF-C-containing solutions with different pH values in 1 mL cuvettes, use a BioTek XS2 enzyme marker to perform ultraviolet full-wavelength scanning spectrum analysis on the solutions with pH 6.8 and pH 7.4, and use Origin 2021 software to draw the H + ion spectrum response spectrum of fluorescent probe AF-C, as shown in Figure 2 .

[0065] As can be seen from the spectrum of Figure 2 , AF-C has characteristic absorption peaks at wavelengths of 548 nm and 720 nm, so the characteristic absorption wavelength of fluorescent probe AF-C is determined to be 548 nm and 720 nm, and this wavelength is used as the excitation wavelength for subsequent pH response curve determination and laser confocal microscope imaging.

[0066] 2. Screening the optimal co-incubation time of fluorescent probe AF-C

[0067] 2.1. Pretreatment of E. coli BL21(DE3) cells

[0068] E. coli BL21(DE3) with kanamycin resistance (Kan resistance) plasmid was taken out from the cryotube in the -80℃ refrigerator, after thawing, some bacterial liquid was taken with a sterile 200 μL gun head, and then streaked on a LB plate containing kanamycin resistance according to the streaking method. After the streaking was completed, it was inverted and cultured overnight in a constant temperature incubator at 37℃ until single colonies were grown. A single colony was picked up using a sterile 10 μL gun head, and then the gun head was directly punched into 10 mL LB liquid medium containing kanamycin resistance, and cultured overnight at 37℃, 220 rpm.

[0069] 2.2. Screening of optimal co-incubation time

[0070] The powder fluorescent probe AF-C at 20 μM was dissolved in DMSO for cell culture, filtered with a 0.22 μm sterilization filter and placed in a clean bench for standby. The overnight cultured E. coli BL21(DE3) bacterial liquid was inoculated into two sterile LB liquid medium containing 50 mg / L Kan resistance at a ratio of 2%, and the shaking table parameters were set to 37℃, 220 rpm. After the culture reached the logarithmic growth phase (OD 600After adjusting the concentration (=0.6~0.8), the shaker parameters were set to 21℃ and 250rpm, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2mM for induction. After 12h of induction, 2mL of bacterial culture was taken into a sterile centrifuge tube, and 0.5% DMSO solution containing the fluorescent probe AF-C was added. The tube was then placed in a shaker (37℃, 220rpm), and the staining time was set to 45min, 60min, 75min, 90min, and 120min. After staining, the culture medium was discarded after centrifugation at 5000rpm for 3min, and the bacterial cells were washed twice with sterile PBS solution. After washing, the bacterial cells were resuspended in the same volume of PBS, and 20μL of the suspension was dropped onto a sterile pathological grade glass slide, and a coverslip was placed on top to distribute the suspension evenly on the slide. After slide preparation, open the Nikon A1plus laser confocal microscope and its accompanying computer workstation, launch the NIS-Elements software, select 60× oil immersion, and invert the slide onto the stage. In the Laser Configuration panel, select excitation wavelengths of 561nm and 640nm, click "Live" to preview, and adjust the focal plane, light intensity, and laser power. Once a suitable field of view is found, click "Capture" to acquire the image, adjust the image resolution, and finally save the image data in its original format (*.nd2 or *.ids). Figure 3 .

[0071] from Figure 3 It can be seen that under laser confocal microscopy, the fluorescence intensity of cells varies with different co-incubation times, and the co-incubation time corresponding to the group with the strongest fluorescence intensity is the optimal co-incubation time; when the incubation time is 75 min, the fluorescence intensity of the NIR channel is the highest, so the optimal incubation time is 75 min.

[0072] 3. Detection of intracellular pH during the expression of heterologous proteins in E. coli

[0073] 3.1 Determination of pH response curve of fluorescent probe AF-C

[0074] Thaw the DMSO solution containing the fluorescent probe AF-C from step 1.1, and inoculate it at a ratio of 0.5% into an aqueous solution containing 10 mM SDS and 10 mM HEPES, mixing thoroughly. After mixing, divide the solution into 11 equal portions and place them in 5 mL centrifuge tubes to prepare solutions with different pH values ​​(6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6). Turn on the Edinburgh FSL1000 steady-state fluorescence spectrometer and its accompanying computer workstation, start the Edinburgh Instruments Spectrometer Control operating system, and turn on the xenon lamp, preheating for about 15 minutes. Place the sample solutions sequentially into the matching quartz cuvettes, select Emission Spectrum mode, set the excitation wavelength to 537 nm, step size to 0.5 nm, integration time to 0.1 s, and bandwidth to 2 nm, and click "Start" to begin the measurement. The fluorescence absorption intensity of solutions containing the fluorescent probe AF-C at pH 6.6–8.6 was measured sequentially at 630 nm, and the data were recorded (I 630nm The data was saved in ASCII format; then the excitation wavelength was changed to 718 nm, and the fluorescence absorption intensity of the solutions containing the fluorescent probe AF-C at pH 6.6–8.6 was measured at 740 nm, and the data were recorded (I 740nm Save the data in ASCII format. After the test is complete, turn off the Edinburgh FSL1000 steady-state fluorescence spectrometer, import the measured data into the Organic 2021 software, and calculate I. 740nm / I 630nm After the calculation is completed, use I 740nm / I 630nm The ratio of pH to AF-C was plotted on the ordinate and pH value on the abscissa to create a pH response curve for the fluorescent probe AF-C, as shown below. Figure 4 .

[0075] from Figure 4 It can be seen that as the pH value increases, I 740nm / I 630nm The ratio initially decreases rapidly, then levels off when the pH exceeds 7.8.

[0076] right Figure 4 The equation corresponding to the pH response curve is obtained by fitting the curve using a nonlinear least squares (NLS) model of a four-parameter Henderson–Hasselbalch / 4-parameter logistic (4PL-HH) model:

[0077]

[0078] In the above formula: R is I 740nm / I 630nmThe ratios; pKa, s, B, and A are fitting constants, with pKa set to 7.218504, s to 5.521153, B to 6.294120, and A to 0.420666.

[0079] 3.2 Laser confocal imaging of E. coli at different induction time points

[0080] The overnight culture of *E. coli* BL21(DE3) from section 2.1 was inoculated at a ratio of 2% into two sterile LB broth media containing 50 mg / L Kan resistance. The culture was incubated at 37°C and 220 rpm on a shaker until the logarithmic growth phase (OD600 0.6–0.8). 2 mL of the culture was transferred to a sterile centrifuge tube, and 0.5% of a DMSO solution containing 20 μM fluorescent probe AF-C was added. The tube was incubated at 37°C and 220 rpm for the determined optimal incubation time. After incubation, the tube was centrifuged at 5000 rpm for 3 min, the culture medium was discarded, and the cells were washed three times with sterile PBS. The cells were then prepared using pathological grade slides and coverslips. Excitation wavelengths of 561 nm and 640 nm were used to observe the staining of the NIR and Red channels under a laser confocal microscope, and the image data were recorded. After induction of expression, the heterologous protein was induced by shaking at 21°C and 250 rpm. After 12 hours of induction, 2 mL of bacterial culture was taken, and the fluorescent probe AF-C was added and incubated at 21°C. After incubation, slides were prepared and the staining of NIR and Red channels was observed under a laser confocal microscope, and image data were recorded. After 24 hours of induction, 2 mL of bacterial culture was taken, and the fluorescent probe AF-C was added and incubated at 21°C. After incubation, slides were prepared and the staining of NIR and Red channels was observed under a laser confocal microscope, and image data were recorded. Figure 5 .

[0081] pass Figure 5 The images can be used to visually assess the staining of cells at different induction times, and at the same time, the fluorescence intensity of the Red channel and NIR channel of a single cell can be quantified based on these images.

[0082] 3.3 Image Data Processing

[0083] Image data of *E. coli* cells recorded by laser confocal microscopy were imported into ImageJ image processing software. After noise reduction and background subtraction, the fluorescence intensity values ​​of the Red and NIR channels of single cells at 0h, 12h, and 24h of induced expression were calculated. Figure 6 As shown.

[0084] Then, the ratio of NIR channels to Red channels at different induction times was calculated and plotted as a bar chart, as shown below. Figure 7 As shown.

[0085] Figure 7 The fluorescence intensity ratio of the NIR channel to the Red channel is displayed visually, which facilitates subsequent input into the pH response curve to determine the specific intracellular pH value of E. coli.

[0086] 3.4 Intracellular pH Measurement

[0087] Based on the pH response curve of the obtained fluorescent probe AF-C, the data obtained in step 3.3 can be substituted into the pH response curve to obtain the real-time intracellular pH of *E. coli* BL21(DE3) at different induction expression times. This can be compared with the real-time pH of the culture medium (extracellular pH). Figure 8 As shown.

[0088] pass Figure 8 A direct comparison of the difference between intracellular and extracellular pH values ​​was made, with Extracelluar representing extracellular pH and Intracelluar representing intracellular pH. The comparison revealed that the difference between intracellular and extracellular pH was less than 0.5, and this difference decreased with increasing induction time. The intracellular pH calculated by this invention was not significantly different from the real-time culture medium pH, indicating that the method of this invention is feasible and highly accurate for detecting intracellular pH during the expression of heterologous proteins in *E. coli*.

[0089] 4. Cytotoxicity assessment of the fluorescent probe AF-C

[0090] The cytotoxicity of the fluorescent probe AF-C was assessed using the CCK-8 assay.

[0091] Add 2 mL of LB liquid medium containing 50 mg / L Kan resistance to each well of a 6-well plate. Inoculate each well with 1% of the overnight cultured *E. coli* from section 2.1 as a blank control. Dissolve 5 μM, 10 μM, 20 μM, 30 μM, and 40 μM fluorescent probe AF-C powder in DMSO for cell culture as sample groups. An equal volume of DMSO without AF-C serves as a blank control. Each group is prepared in triplicate, with 0.5% of each solution added to the corresponding well. The plate is then sealed with film and incubated at 37°C and 220 rpm for 12 h in a shaker. After incubation, aspirate 1 mL of bacterial culture from each well, centrifuge at 5000 rpm for 3 min to discard the medium, and wash the cells three times with sterile PBS. After washing, add CCK-8 (10% concentration in serum-free medium) to each well and incubate for 30 min. After incubation, centrifuge at 5000 rpm for 3 min to discard the bacterial pellet. Collect the clear supernatant and measure the absorbance at 450 nm using a microplate reader. Perform five replicates for each concentration. Calculate cell viability based on the absorbance data using the following formula:

[0092]

[0093] Wherein: Ab sample wells are the absorbance values ​​corresponding to the sample group, Ab blank wells are the absorbance values ​​corresponding to the blank group, and Ab control wells are the absorbance values ​​corresponding to the blank control group.

[0094] Cell survival data such as Figure 9 As shown.

[0095] from Figure 9 It can be seen that the cell survival rate is above 90% under different concentrations of fluorescent probe AF-C, indicating that the fluorescent probe AF-C has very low cytotoxicity, which can be ignored; it does not kill cells when detecting intracellular pH, and has good detection safety.

[0096] The above are several preferred embodiments of the preparation method of the present invention, but they should not be regarded as limitations on the technical solutions protected by the present invention. Any alternative solutions obtained by those skilled in the art based on the technical ideas of the present invention without creative labor should fall within the protection scope of the present invention.

Claims

1. A method for detecting intracellular pH during the expression of heterologous proteins in *Escherichia coli*, characterized in that, Includes the following steps: S1. Establish pH response curve Solutions containing the fluorescent probe AF-C with different known pH values ​​were prepared, and pH response curves between the fluorescence intensity ratio and pH were obtained by in vitro fluorescence intensity testing. S2. Acquire real-time cell images The fluorescent probe AF-C was co-incubated with E. coli during the expression of heterologous proteins to obtain real-time cell images of E. coli cells. S3, Intracellular pH Test The real-time cell images from step S2 are processed to calculate the fluorescence intensity of the Red channel and the NIR channel for a single cell. The ratio of the fluorescence intensity of the Red channel to the fluorescence intensity of the NIR channel is then substituted into the pH response curve from step S1 to obtain the intracellular pH value of E. coli.

2. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 1, characterized in that, In step S1, the solution containing the fluorescent probe AF-C is formed from fluorescent probe AF-C powder, dimethyl sulfoxide, and buffer solution.

3. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 2, characterized in that, The buffer solution is an aqueous solution containing 10 mM sodium dodecyl sulfate and 10 mM HEPES.

4. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 1, characterized in that, In step S1, the specific process of obtaining the pH response curve by in vitro fluorescence intensity testing is as follows: S1.

1. Measure the fluorescence absorption intensity I of the solution containing the fluorescent probe AF-C at 630 nm under an excitation wavelength of 537 nm. 630nm ; S1.

2. Measure the fluorescence absorption intensity I of the solution containing the fluorescent probe AF-C at 740 nm under an excitation wavelength of 718 nm. 740nm ; S1.3 Calculate the I corresponding to solutions containing the fluorescent probe AF-C at different known pH values. 740nm / I 630nm ratio; S1.4, Plot pH value on the x-axis and I value on the y-axis. 740nm / I 630nm Using the vertical axis as the ordinate, construct a pH response curve.

5. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 1, characterized in that, In step S2, the specific incubation process is as follows: S2.1 Pretreatment of Escherichia coli using the streak plating method to obtain Escherichia coli bacterial culture; S2.

2. Inoculate the Escherichia coli culture into LB liquid medium containing Kan resistance and culture until OD. 600 =0.6~0.8; S2.3 Add a dimethyl sulfoxide solution containing the fluorescent probe AF-C to the cultured system, and then incubate.

6. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 1, characterized in that, In step S2, the incubation is carried out at 37°C and 220 rpm for 45 min to 120 min.

7. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 1, characterized in that, In step S2, a laser confocal microscope is used to acquire real-time cell images of E. coli cells.

8. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 7, characterized in that, The excitation wavelengths of the laser confocal microscope were set to 561 nm and 640 nm.

9. The method for detecting intracellular pH during the expression of heterologous proteins in *E. coli* according to claim 1, characterized in that, In step S3, ImageJ image processing software is used to denoise and remove background from the cell image, and the fluorescence intensity of the Red channel and the NIR channel of a single cell are calculated.

10. Application of a fluorescent probe AF-C in detecting intracellular pH during the expression of heterologous proteins in Escherichia coli.

Images