CN115308186B

CN115308186B - fluorescence-pH biosensor and preparation method and application thereof

Info

Publication number: CN115308186B
Application number: CN202211243531.4A
Authority: CN
Inventors: 吴嵩; 张志乾; 陈西朋; 罗元廷; 刘丽花; 杨敏; 江翱; 王帆; 吴奕瑞
Original assignee: Tichuang Biotechnology Guangzhou Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Current assignee: Tichuang Biotechnology Guangzhou Co ltd; Guangzhou Qianxiang Biotechnology Co Ltd
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2023-01-17
Anticipated expiration: 2042-10-12
Also published as: CN115308186A

Abstract

The invention discloses a fluorescence-pH biosensor, comprising: polymerized sensing protein formed by crosslinking monomer sensing protein; a pH-responsive fluorescent mixture covalently cross-linked to a polymeric sensor protein. Also discloses a preparation method and application thereof. The fluorescence-pH biosensor has the advantages of sensitive pH sensing, wide sensing range, good sensing linearity, low toxicity and the like, is suitable for detecting substances generating pH change in the high-throughput screening process, and is particularly suitable for being applied to microfluidic high-throughput screening.

Description

fluorescence-pH biosensor and preparation method and application thereof

Technical Field

The invention relates to a fluorescence-pH biosensor and a preparation method and application thereof.

Background

pH sensors have been an important subject of research in the scientific research and commercialization field. The traditional pH sensor is a color sensor based on Raman spectrum, such as phenol red, methyl orange, malachite green and the like, which is also the principle of pH test paper for measuring pH. The pH detection method can determine the approximate range of pH through visual color comparison, and is widely applied to pH detection of environments and solutions. However, the throughput of data processing in high-throughput screening by the color sensor based on Raman spectroscopy is low, additional image recognition and processing software is needed, and the processing rate is only about 10 Hz/s. While fluorescence-based biosensors can process at rates of thousands of Hz/s. Therefore, pH biosensors based on raman spectroscopy are difficult to apply on large-throughput bio-screening platforms.

pH changes are a common phenomenon for many catalytic reactions. For example, acid-producing bacteria or alkali-producing bacteria have obvious pH shift in the fermentation process, and the shift degree is in obvious proportion to the yield of the product. For example, formate dehydrogenase produces the end product of formate when fixing carbon dioxide, which eventually causes the pH of the reaction system to shift to acidity. For example, in the gene diagnosis process, the loop-mediated isothermal amplification (LAMP) technique is used, pyrophosphate is continuously generated in the polymerase extension process, and the excessive accumulation of pyrophosphate causes the rapid decrease of pH, and finally the color change occurs in the color sensor based on Raman spectroscopy, thereby achieving the purpose of rapidly detecting genes. Therefore, catalytic reactions based on pH changes have commercially significant application value. High throughput screening of highly active variants of such enzymes is also an urgent problem to be solved in the industry.

In order to solve the problems of low throughput, difficult data processing, etc. in pH screening of Raman spectrum-based color sensors, many companies are also developing fluorescence-based pH sensors, such as pHrodo. TM. PH Sensor Dyes of Sammerlow. The reagent can enter cells, monitors the pH change in different subcellular organelles in real time, and is widely concerned. But still has the problems of low sensitivity, unobvious pH linearity, narrow detection range, low specificity, large cytotoxicity and the like. Therefore, it is difficult to apply the method to high-throughput cell screening.

Disclosure of Invention

The invention aims to provide a fluorescence-pH biosensor which can be used for micro-fluidic or flow sorting.

The purpose of the invention is realized by the following technical scheme:

a fluorescence-pH biosensor, comprising:

the polymeric sensing protein is formed by crosslinking monomeric sensing protein;

a pH-responsive fluorescent mixture covalently cross-linked to a polymeric sensor protein.

The monomeric sensing protein is protein rich in aspartic acid, glutamic acid, lysine and arginine, wherein carboxyl branched chains of asparagine and glutamic acid are used for crosslinking the protein, and amino branched chains of lysine and arginine are used for labeling fluorescent substances.

Preferably, the monomeric sensor protein is bovine serum albumin, human serum albumin, concanavalin, desmin, ovalbumin, bovine globulin or mucin.

Preferably, the crosslinking method of the monomeric sensor protein is a diazotization method, a glutaraldehyde method, a formaldehyde method, a periodate oxidation method, a mixed anhydride method, a carbodiimide method, an active ester method, a diisocyanate method, an azobenzoic acid method, an O-carboxyhydroxylamine method, a para-hydrazinobenzoic acid method, a polybasic acid anhydride method, an azide method, a sodium chloroacetate method, an imidic acid ester method, or a halogenated nitrobenzene method.

Preferably, the pH responsive fluorescent mixture is selected from 7-methoxy-3-carboxycoumarin succinimide ester, 7-methoxy-3-carboxycoumarin, 7-methoxy-4-carboxycoumarin, 7-hydroxy-3-carboxycoumarin succinimide ester, 7-hydroxy-4-methylcoumarin-3-acetic acid, succinimidyl 7-hydroxy-4-methylcoumarin-3-acetate, 4-aminocoumarin, fluorescein isothiocyanate, fluorescein 5 (6) -carboxydiacetate succinimide ester, fluorescein diacetate 5 (6) -carboxytetramethyl rhodamine succinimide ester, rhodamine B, and 2- (2' -hydroxyphenyl) benzothiazole. The pH response fluorescent substance refers to a substance capable of generating fluorescence in response to pH change, but the response range of a fluorescent substance monomer is narrow, so that the invention selects a multi-fluorescent substance combination when preparing the sensor so as to improve the sensitivity and the response range of the pH response.

Preferably, the pH responsive fluorescent mixture is a mixture of 7-methoxy-3-carboxycoumarin succinimide ester, 7-methoxy-3-carboxycoumarin, fluorescein isothiocyanate, 5 (6) -carboxyfluorescein diacetate succinimide ester, and 2- (2' -hydroxyphenyl) benzothiazole.

Preferably, the molecular weight of the fluorescence-pH biosensor is above 100 KDa.

The application of the fluorescence-pH biosensor in microfluidic cell sorting comprises the following steps:

(1) Mixing the cell mixture with a fluorescence-pH biosensor to prepare single cell droplets, and culturing single cells;

(2) The liquid drops pass through a laser, the wavelength range of exciting light is 200nm-450nm, and the wavelength range of emitting light is 510-650 nm;

(3) And applying voltage to the liquid drop according to the threshold value of the emission light intensity, and changing the sorting channel of the liquid drop to obtain the target cell.

The fluorescence-pH biosensor is applied to high-throughput screening of strains or targets, wherein the strains are acid-producing strains or alkali-producing strains and acid-resistant or alkali-resistant strains, and the targets are CO2 reductase variants or Bst polymerase variants.

The invention also discloses a preparation method of the fluorescence-pH biosensor, which comprises the following steps:

(1) Adding a cross-linking agent into the monomeric sensing protein to react so as to cross-link the monomeric sensing protein into polymeric sensing protein;

(2) Adding a crosslinking terminator to terminate the crosslinking reaction;

(3) Mixing the polymeric sensing protein and the pH response fluorescent mixture, adjusting the pH to 7, and reacting by adjusting the proportion of each fluorescent agent until the system is colorless;

(4) Recovery of fluorescence-pH biosensor: recovering the fluorescence-pH biosensor with molecular weight above 100kDa by molecular sieve chromatography or ultrafiltration.

The purpose of adjusting to colorless under the condition of pH7 (both the naked eye and the fluorescence are nearly colorless) is to reduce the interference of background color and to make the fluorescent material show different wavelengths under acidic condition and alkaline condition respectively, so that the fluorescent material can be distinguished according to the wavelength.

Preferably, in the step (1), the concentration of the monomer sensing protein is 1-30 g/L, the used crosslinking agent is glutaraldehyde or carbodiimide, the used concentration of the crosslinking agent is 0.1-5 mM, the crosslinking time is 5-60 min, and the crosslinking temperature is 4-30 ℃.

Preferably, the crosslinking terminator in the step (2) is glycine, sodium bicarbonate or sodium carbonate, the use concentration of the crosslinking terminator is 0.1-5 mM, the treatment time is 5-20 min, and the treatment temperature is 4-30 ℃.

Preferably, the pH responsive fluorescent mixture is added in step (3) at a concentration of 0.05 to 1 mM, a reaction time of 0.5 to 5 h, and a reaction temperature of 20 to 37 ℃.

The crosslinking refers to covalently crosslinking the fluorescent substance to a branched amino group of lysine or arginine through a chemical reaction.

The fluorescence-pH biosensor has the advantages of sensitive pH sensing, wide sensing range, good sensing linearity, low toxicity and the like, is suitable for detecting substances generating pH change in the high-throughput screening process, and is particularly suitable for being applied to microfluidic high-throughput screening.

Drawings

FIG. 1 fluorescence-pH biosensor fluorescence signals at different pH.

Fig. 2 is a structure of a microfluidic sorting chip suitable for use in a fluorescence-pH biosensor.

FIG. 3 shows a chip structure prepared by micro-droplet.

FIG. 4. Procedure for microfluidic sorting of acidogenic or alkaligenic strains.

FIG. 5 acid resistance of the strains after selection and not selected is compared.

Detailed Description

Example 1

The fluorescence-pH biosensor of this example was implemented as follows:

(1) And (3) cross-linking of the sensing protein: 1 mg/mL bovine serum albumin or concanavalin was added to glutaraldehyde to a final concentration of 1 mM, and after incubation at room temperature for 10 min, glycine to a final concentration of 1 mM was added.

(2) Coupling fluorescent agent: adding 7-methoxy-3-carboxycoumarin succinimide ester, 7-methoxy-3-carboxycoumarin, fluorescein isothiocyanate, 5 (6) -carboxydiacetic acid fluorescein succinimide ester and 2- (2 '-hydroxyphenyl) benzothiazole, wherein the final concentration of the 7-methoxy-3-carboxycoumarin, the fluorescein isothiocyanate, the fluorescein 5 (6) -carboxydiacetic acid succinimide ester and the 2- (2' -hydroxyphenyl) benzothiazole are all 0.1 mM, adjusting the pH to 7, and adjusting the proportion of each fluorescent agent until the system is colorless. The reaction was carried out at room temperature for 4 h.

(3) Recovery of fluorescent protein sensor: and (3) performing ultrafiltration by using a 100kDa ultrafiltration tube to remove small molecules and recovering the large cross-linked fluorescence biosensor.

Example 2

This example tests the effect of the invention on color development at different pH's with pHrodo. The implementation mode is as follows:

buffers (1-14) with different pH values are prepared, and a paradigm pH test paper is used for testing to ensure the accuracy of the pH value. 10 ng/ml fluorescence-pH biosensor or pHrodo is added into the buffer solution, mixed uniformly and placed at room temperature for 10 min. After stabilization, the pH fluorescence biosensor of the present invention detects the excitation light with 310 nm and the emission wavelength with 595 nm. The excitation wavelength of pHrodo is 505 nm, and the emission wavelength is 525 nm.

As a result, as shown in FIG. 1, the fluorescence-pH biosensor according to the present invention has better sensitivity and pH sensing range.

Example 3

This example tests the cytotoxicity of the invention with pHrodo. The implementation mode is as follows:

in the logarithmic phase of Escherichia coli, add 10 ng/mL final concentration of fluorescence-pH biosensor or pHrodo, after mixing, room temperature treatment of 1 h. After the strain was diluted in a gradient, 200 ul was spread on a plate and cultured overnight at 37 ℃. Colonies on the plates were counted.

The results are shown in table 1, and the fluorescence-pH sensor prepared by the invention has low toxicity and good biological safety.

TABLE 1

Number of colonies	1:10000	1:100000	1:1000000
				Water (W)	548	76	9
The invention	517	73	9
				pHrodo	443	51	5

Example 4

The embodiment discloses the application of the invention in the microfluidic high-throughput screening process, and the specific implementation mode is as follows:

(1) And (4) designing a microfluidic chip. The structure of the microfluidic chip is shown in fig. 2. The chip comprises two oil phase inlets, a strain liquid drop injection inlet, a fluorescence-pH biosensor injection inlet, a bent uniform mixing channel and a sorting channel. A laser detection system is arranged in front of an inlet of the sorting channel, and voltage is applied to the liquid drops according to the fluorescence intensity in the liquid drops, so that the purpose of sorting the liquid drops with the shifted pH value is achieved.

(2) And (4) preparing a strain library. Mutant libraries, such as acid-producing and alkali-producing bacterial libraries, CO2 reductase mutant bacterial libraries, bst polymerase bacterial libraries, and the like, are prepared by using a mutagenesis technique or error-prone PCR.

(3) Preparation of micro-droplets and culture of monoclonal strains. The production of microdroplets was performed using cross-shaped microchannels, a schematic of which is shown in fig. 3. The oil (oil phase) is produced by introducing liquid drops from both ends, and the strain (aqueous phase) containing the diluted culture medium is introduced in the middle. The water phase produces separate microdroplets upon cutting of the oil phase. The micro-droplets were collected in an incubator and subjected to 37-degree static culture for 24 h.

(4) And (4) sorting the micro-droplets. And under the action of the oil phase, micro liquid drops are fed into the micro channels. The microchannel structure is schematically shown in fig. 2 and 4. At the outlet of the fluorescence-pH biosensor injection channel, an electrode is inserted to conduct 1000V electrification, so that a fused droplet of the monoclonal micro-droplet and the fluorescence-pH biosensor micro-droplet is generated. After the fused droplets are uniformly mixed through the bent uniformly-mixing channel, the fluorescence-pH biosensor inside the micro-droplets generates corresponding fluorescence change according to the pH of the monoclonal droplets. Before the sorting channel, an ultraviolet laser emits ultraviolet light to detect micro liquid drops, fluorescence signals after fluorescence excitation are transmitted to a receiver, the liquid drops are sorted by controlling electrode voltage in front of the sorting channel through the receiver, and the sorting proportion is about 1/1000.

(5) And (5) verifying the activity of the strain. After the sorted strains are collected, amplified fermentation is carried out. After completion of the fermentation, the supernatant was centrifuged at 8000 rpm, and the pH of the fermentation solution was measured with a pH meter. The results show that the pH value of the strain which is not sorted is 5.24 after fermentation, and the pH value of the strain which is sorted by the sensor of the invention is 4.06 after fermentation. The invention can screen out bacterial strains with high acid production in micro-flow control.

(6) And (5) verifying the tolerance of the strain. After the sorted strains are collected, amplification culture is carried out. The same cell amount of the strain was taken and subjected to amplification culture in a pH 3.0 medium. The OD value was measured by taking the strain every 1 h. As a result, as shown in FIG. 5, the screened strains of the present invention in microfluidics can have higher acid resistance.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Claims

1. A fluorescence-pH biosensor, comprising:

a pH-responsive fluorescent mixture covalently cross-linked to a polymeric sensor protein, said fluorescence-pH biosensor having a molecular weight above 100 KDa;

the monomer sensing protein is bovine serum albumin, human serum albumin, concanavalin, complex protein, ovalbumin, bovine globulin or mucin;

the pH responsive fluorescent mixture is selected from 7-methoxy-3-carboxycoumarin succinimide ester, 7-methoxy-3-carboxycoumarin, 7-methoxy-4-carboxycoumarin, 7-hydroxy-3-carboxycoumarin succinimide ester, 7-hydroxy-4-methylcoumarin-3-acetic acid succinimide ester, 4-aminocoumarin, fluorescein isothiocyanate, 5 (6) -carboxydiacetic acid fluorescein succinimide ester, 5 (6) -carboxyfluorescein diacetate, 5 (6) -carboxytetramethylrhodamine succinimide ester, rhodamine B, and 2- (2' -hydroxyphenyl) benzothiazole.

2. The fluorescence-pH biosensor according to claim 1, wherein: the cross-linking method of the monomer sensing protein is a diazotization method, a glutaraldehyde method, a formaldehyde method, a periodate oxidation method, a mixed anhydride method, a carbodiimide method, an active ester method, a diisocyanate method, an azobenzoic acid method, an O-carboxyformic hydroxylamine method, a para-hydrazinbenzoic acid method, a polybasic anhydride method, an azide method, a sodium chloroacetate method, an imidic acid ester method or a halogenated nitrobenzene method.

3. The fluorescence-pH biosensor according to claim 2, wherein: the pH-responsive fluorescent mixture is a mixture of 7-methoxy-3-carboxycoumarin succinimide ester, 7-methoxy-3-carboxycoumarin, fluorescein isothiocyanate, 5 (6) -carboxyfluorescein diacetate succinimide ester, and 2- (2' -hydroxyphenyl) benzothiazole.

4. Use of the fluorescence-pH biosensor according to any one of claims 1-3 in microfluidic cell sorting.

5. Use of the fluorescence-pH biosensor of any one of claims 1-3 in high throughput screening of strains that are acid-producing strains or alkali-producing strains, acid-or alkali-resistant strains, or targets that are CO2 reductase variants or Bst polymerase variants.

6. The method for preparing a fluorescence-pH biosensor according to any one of claims 1 to 3, comprising the steps of:

(2) Adding a crosslinking terminator to terminate the crosslinking reaction;

(4) Recovery of fluorescence-pH biosensor: recovering the fluorescence-pH biosensor with molecular weight above 100kDa by using molecular sieve chromatography or ultrafiltration.

7. The method for preparing a fluorescence-pH biosensor according to claim 6, wherein: the concentration of the monomer sensing protein in the step (1) is 1-30 g/L, the used crosslinking agent is glutaraldehyde or carbodiimide, the used concentration of the crosslinking agent is 0.1-5 mM, the crosslinking time is 5-60 min, and the crosslinking temperature is 4-30 ℃.

8. The method for preparing a fluorescence-pH biosensor according to claim 6, wherein: the crosslinking terminator in the step (2) is glycine, sodium bicarbonate or sodium carbonate, the use concentration of the crosslinking terminator is 0.1-5 mM, the treatment time is 5-20 min, and the treatment temperature is 4-30 ℃.

9. The method for preparing a fluorescence-pH biosensor according to claim 6, wherein: in the step (3), the addition concentration of the pH response fluorescent mixture is 0.05-1 mM, the reaction time is 0.5-5 h, and the reaction temperature is 20-37 ℃.