A method for rapid and non-destructive detection of ergothioneine in cells

Abstract

The present invention discloses a method for rapidly and non-destructively detecting ergothioneine in cells. Based on Raman spectroscopy, it rapidly detects ergothioneine in single living cells. The specific operation is as follows: obtain the liquid of the living cells to be tested, wash it, adjust the concentration of the bacterial suspension, add the obtained bacterial suspension to the sample detection cell, use a microscope to find the cells in the solution, irradiate the cells with laser and obtain the Raman spectrum of the cells, perform background removal, smoothing, baseline calibration and normalization on the obtained Raman spectrum data, detect the Raman characteristic peaks. If there are Raman marker peaks at 1210 cm-1 and 1507 cm-1 in the Raman spectrum of the cells, it is determined that the living cells to be tested contain ergothioneine; otherwise, it is determined that the living cells to be tested do not contain ergothioneine. The method of the present invention has simple sample preparation, requires a small amount of samples, does not require cell damage, has a short detection time, and can realize the detection of ergothioneine in single living cells.

Description

Technical Field

[0001] The present invention belongs to the fields of biochemistry and analytical chemistry, and particularly relates to a method for rapidly and non-destructively detecting ergothioneine in cells. Background Art

[0002] The molecular formula of ergothioneine (EGT) is C9H 15 N3O2S, with a molecular weight of 229.3. It is easily soluble in water and not easily decomposed, and is a rare natural chiral amino acid. Ergothioneine is a safe and non-toxic natural antioxidant, with various physiological functions such as relieving inflammation, scavenging free radicals, detoxifying, maintaining DNA biosynthesis, normal cell growth, and cellular immunity. It has wide applications and market prospects in the fields of organ transplantation, cell preservation, food and beverage, cosmetics, animal feed, etc. It is known that natural ergothioneine is mainly produced by limited microbial groups such as actinomycetes, cyanobacteria, and mushrooms (Han et al., 2021). Although there are microbial flora in the intestines of animals and humans, there is currently no conclusive evidence that intestinal microorganisms can synthesize ergothioneine. The ergothioneine in tissues such as red blood cells, bone marrow, liver, and kidney of animals or humans is obtained through exogenous uptake and accumulation (Cheah and Halliwell 2021; Ey et al., 2007). It has been reported that in some pathogenic bacteria, such as Burkholderia (Gamage et al., 2018), Mycobacterium tuberculosis (Cumming et al., 2018), and Aspergillus fumigatus (Sheridan et al., 2016), can also synthesize ergothioneine to resist host defense or enhance antibiotic resistance (Cheah and Halliwell 2021). Ergothioneine can be obtained by two methods: chemical synthesis and biosynthesis. Among them, biosynthesis has the advantages of short fermentation cycle, high yield, and low cost (Chen Jiamin et al., 2022). Currently, ergothioneine can be fermented and produced by genetically recombinant microorganisms such as Escherichia coli, Saccharomyces cerevisiae, and Aspergillus.

[0003] The detection of ergothioneine in cells is crucial for research such as the exploration of germplasm resources of ergothioneine-producing microorganisms, breeding, and the screening of high-yield cells in recombinant strains, as well as for industrial applications. Currently, the main methods for detecting ergothioneine are as follows: (1) Spectrophotometry, that is, in an alkaline medium, copper ions mediate the oxidation of thiols, and then rapidly react with 2,2'-Dipyridyldisulfide at about pH 1 (Carlsson et al., 1974); (2) Thin-layer chromatography, that is, the extracted sample is placed on a glass plate filled with an adsorbent system, and the target compound is distinguished by the difference in the adsorption degree of the compound and the adsorbent (Kaneko et al., 1980); (3) High-performance capillary electrophoresis, the sample to be tested is added to the capillary, and under the action of an electric field, different components migrate at a certain speed due to differences in the size, charge nature, and amount of different chemical molecules. During the migration of different chemical components, different components are detected by laser-induced fluorescence (Sotgia et al., 2013); (4) High-performance liquid chromatography (High performance liquid chromatography, HPLC) (Sotgia et al., 2013; van der Hoek et al., 2022). Currently, the HPLC method is the mainstream method for detecting ergothioneine. First, cells need to be disrupted or the fermentation broth needs to be extracted. The crude ergothioneine in the cells or fermentation broth is extracted by methods such as reflux extraction method / enzyme digestion extraction method / ultrasonic microwave combined extraction method, and finally concentrated to obtain the sample to be tested. Methanol or acetonitrile-water, etc. are used as the mobile phase, and the sample passes through a hydrophilic column and a UV detector (wavelength 254 - 257 nm) to detect whether ergothioneine is present in the sample and its content.However, this detection method requires a complex preparation process for the sample to be tested, damages cells, has high requirements for the purity of the reagents needed in the detection process, and is not suitable for detecting the changes of ergothioneine in single cells; (5) Inductively coupled plasma tandem mass spectrometry combined with HPLC (HPLC-ICP-QQQ-MS). First, collect the cell lysate, remove cell debris and proteins, etc. After the lysate is frozen and thawed with liquid nitrogen, add TCEP and IAM solutions in a certain proportion. After incubating at room temperature for 2 hours, remove the proteins in the lysate through a molecular sieve, etc. Add the filtrate to HPLC / ICP-QQQ-MS and use the 8800-ICP-QQQ-MS system for analysis. For example, when detecting ergothioneine in red blood cells, set the gas temperature at 500 degrees, the flow rate at 65, the spray voltage at 3500V, the capillary temperature at 300 degrees, and the mass-to-charge ratio scanning range at 170-600 (Kroepfletal., 2019); (6) Also combined with HPLC is LC-MS / MS triple quadrupole tandem mass spectrometry. Atomize the sample with nitrogen, introduce it into the detection chamber at 520 degrees, and analyze the optimal collision energy of ergothioneine through positive electrospray ionization. The precursor and product ions are monitored using MRM when passing through the mass spectrometer, and finally analyze the data with analysis software (Wangetal., 2013). All of the above detection techniques require complicated sample preparation work in the early stage, damage cells, and require a large number of samples, and cannot be applied to the detection of ergothioneine in single cells. Summary of the Invention

[0004] The object of the present invention is to provide a method for rapidly and non-destructively detecting ergothioneine in cells. The method of the present invention has simple sample preparation, requires a small amount of sample, does not require cell damage, has a short detection time, and can realize the detection of ergothioneine in single living cells.

[0005] The method for rapidly and non-destructively detecting ergothioneine in cells provided by the present invention is based on Raman spectroscopy for rapid detection of ergothioneine in cells.

[0006] The cells are single or multiple cells within the range of laser irradiation.

[0007] In one embodiment of the present invention, the cells are Cryptococcus neoformans cells of wild type WT, EGT1 gene mutant strain egt1Δ, and EGT1 gene complemented strain PEGT1-EGT1 in the Q phase (resting phase).

[0008] In the study of the virulence and infection mechanism of opportunistic pathogenic fungi, we found that Cryptococcus neoformans can form a dormant state, and the dormant state cells can effectively resist the clearance of the host immune system. Metabolomic studies first discovered that such dormant cells can highly express ergothioneine. The detection of intracellular ergothioneine usually uses HPLC method, while the rapid and non-destructive detection of single-cell ergothioneine is extremely challenging, and there is no report on the rapid and non-destructive single-cell intracellular ergothioneine detection technology. In order to detect intracellular ergothioneine more efficiently, rapidly and non-destructively, we adopted the vibrational spectroscopy (Raman spectroscopy) detection technology that can be used for single-cell level detection.

[0009] The method for rapidly and non-destructively detecting ergothioneine in cells of the present invention comprises the following steps:

[0010] Obtain the live cell fluid to be tested, wash it, adjust the concentration of the bacterial suspension, add the obtained bacterial suspension to the sample detection cell, find the cells in the solution by using a microscope, irradiate the cells with laser, and obtain the Raman spectrum of the cells. If the Raman spectrum of the cells has identification peaks at 1210 cm -1 and 1507 cm -1 , it is determined that the live cells to be tested contain ergothioneine; otherwise, it is determined that the live cells to be tested do not contain ergothioneine.

[0011] Furthermore, if the Raman characteristic peak intensities at 1210 cm -1 and 1507 cm -1 in the Raman spectrum of the cells are high, it is determined that the content of ergothioneine in the live cells is high.

[0012] The Raman identification peaks at 1210 cm -1 and 1507 cm -1 in the Raman spectrum of the cells are limited by the resolution of the instrument equipment, and the identification peaks can have an offset of 1-2 cm -1 .

[0013] The number of cells irradiated by the laser is a single cell or multiple cells within the laser irradiation range.

[0014] In the above method, the washing is to wash the cells in the live cell fluid to be tested with a sodium chloride solution or a cell isotonic solution, and the washing is carried out multiple times, specifically 2-3 times;

[0015] Among them, the sodium chloride solution is 0.8-0.9% NaCl or a NaCl solution suitable for the physiological concentration of the cells;

[0016] Adjust the concentration of the bacterial suspension to 10 4 -10 7 CFU / mL;

[0017] The sample detection cell may specifically be the liquid sample cell for microscopic Raman spectroscopy detection of aerobic or facultative anaerobic pathogenic microorganisms disclosed in Chinese Patent ZL2020101989793; or a sample detection cell with a quartz or calcium fluoride substrate; or a substrate sample detection cell without Raman scattering background interference;

[0018] The Raman spectroscopy system used for cell Raman spectroscopy detection is an upright or inverted microscopic Raman spectroscopy system. Among them, the laser wavelength of the Raman spectroscopy system is any one or combination of wavelengths such as 532nm, 633nm, or 785nm.

[0019] Before cell Raman spectroscopy detection, the Raman spectroscopy system is first calibrated with the characteristic peaks of materials dedicated to Raman spectrometer calibration;

[0020] The materials used for Raman spectrometer calibration are polystyrene microspheres or silicon wafers, etc.;

[0021] During cell Raman spectroscopy detection, the total integration time is more than 3 seconds, and the power used by lasers of different wavelengths is based on not damaging the cells;

[0022] In one embodiment of the present invention, a 785nm continuous laser is used as the light source for exciting cell Raman scattering, and the total integration time is more than 20 seconds.

[0023] The method further includes background removal, smoothing, baseline calibration, and normalization processing of the obtained Raman spectroscopy data,

[0024] Among them, the smoothing method can adopt convolution smoothing method, moving average method, Gaussian filtering, bilateral filtering, or mean filtering, etc.;

[0025] The baseline calibration method can adopt polynomial fitting method, BEADS algorithm, wavelet algorithm, empirical mode decomposition (EMD), etc.;

[0026] The normalization method can adopt minimum-maximum method, area normalization, vector normalization, etc.

[0027] The method of the present invention has simple sample preparation, requires a small amount of sample, does not require cell damage, has a short detection time, and can realize the detection of ergothioneine in a single living cell. Description of the Drawings

[0028] Figure 1 It is the Raman spectroscopy peak diagram of ergothioneine with different concentration gradients, and the concentration unit of ergothioneine is mg / mL.

[0029] Figure 2Results of HPLC detection of ergothioneine in different strains of Cryptococcus neoformans. WT: wild type, egt1Δ: ergothioneine synthesis gene deletion strain, Q: quiescent phase, P: proliferative phase, Standard: ergothioneine standard spectrum.

[0030] Figure 3 Raman spectroscopy was used to detect ergothioneine in different strains of Cryptococcus neoformans in the Q phase. A. Full Raman spectra of different strains. B. Magnified view of the characteristic peak of intracellular ergothioneine. C-D. Statistical chart of the Raman peak intensity of ergothioneine, t-test two-tailed test, ** P < 0.01, **** P < 0.0001, ns indicates no significant difference. Detailed implementation manners

[0031] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided examples are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0033] Example

[0034] Genomic analysis of Cryptococcus neoformans and knockout experiments of key ergothioneine synthesis genes confirmed that the key gene for synthesizing ergothioneine is the EGT1 gene. The mutant strain egt1Δ of this gene causes Cryptococcus neoformans cells to be unable to form ergothioneine. We detected wild type and egt1Δ of Cryptococcus neoformans cultured in vitro for 6 hours (proliferative phase, labeled as P) and 72 hours (quiescent phase, labeled as Q) by HPLC. It was found that only wild type cells cultured to the quiescent phase produced ergothioneine, and the EGT1 gene is the key gene for synthesizing intracellular ergothioneine. To further achieve rapid and non-destructive detection of intracellular ergothioneine, we used Raman spectroscopy to detect the single-cell Raman spectra of Cryptococcus neoformans. The test strains were wild type (WT), the mutant strain egt1Δ of the gene synthesizing ergothioneine, and the mutant strain P EGT1 -EGT1 Raman spectra. At the same time, we used Raman spectroscopy to detect the Raman spectra of ergothioneine compounds in aqueous solution.

[0035] The specific steps for HPLC detection of ergothioneine in Cryptococcus neoformans are as follows:

[0036] Using the CRISPR / Cas9 gene editing system, the gene EGT1 related to ergothioneine synthesis in Cryptococcus neoformans was knocked out. Take the cells in the logarithmic growth phase cultured for 6 hours and the stationary phase cells cultured for 72 hours with a cell concentration of 2.5×10 8 CFU / mL. After centrifuging to remove the supernatant of the culture medium, add ceramic beads to break the bacterial cells, and keep it at low temperature throughout the process. Then add 50% methanol to make up the volume, shake well, cool on ice for 20 min, and centrifuge at 1000 rpm for 2 min. Transfer the supernatant to a 3 kDa ultrafiltration tube and perform ultrafiltration under low temperature conditions (5000 rpm, 60 min). Take the filtrate as the test solution and store it in the refrigerator. Inject the sample into a high performance liquid chromatography (HPLC) system. Mobile phase: 0.1% trifluoroacetic acid - acetonitrile (99:1, v / v); Flow rate: 0.7 mL / min; Detection wavelength: 260 nm.

[0037] The WT, egt1Δ and P EGT1 -EGT1 strains of Cryptococcus neoformans were simultaneously transferred to YPD culture medium and cultured at 37°C for 6 hours and 72 hours. Collect the cell bodies of the above three strains of bacteria. Collect the cell bodies by centrifuging at 8000 rpm for 5 min to obtain the test live cell solution. Wash the cells in the obtained test live cell solution 2 - 3 times with 0.85% saline or cell isotonic solution. Adjust the concentration of the bacterial suspension to 10 4 -10 7 CFU / mL. Pipette 20 μL of the bacterial suspension with a 20 μL pipette and add it to the sample detection cell (Patent No.: ZL2020101989793). The Raman spectroscopy system is a microscopic Raman spectroscopy system, and the excitation wavelength of the Raman spectrometer is 785 nm. Before detecting the Raman spectrum of the cells, first calibrate the Raman spectroscopy system with the characteristic peak of polystyrene microspheres. Find the cells in the solution under the microscope, irradiate a single cell with laser, and the total integration time is more than 20 seconds to obtain the Raman spectrum of a single cell. The number of Raman spectra measured for each strain of bacteria is not less than 50. The Raman spectra of all the obtained cells are subjected to background removal, Savitzky_Golay smoothing, 6th order polynomial fitting baseline calibration, and minimum - maximum normalization. Raman spectral analysis of ergothioneine solutions with different concentrations found that the Raman characteristic peaks of ergothioneine are most prominent at 1210 cm -1 and 1507 cm -1 ( Figure 1Raman spectral peak diagrams of ergothioneine at different concentration gradients, with the concentration unit of ergothioneine being mg / mL. The operation is as follows: Prepare ergothioneine solutions with concentrations of 1.0, 1.4, 1.6, 1.8, and 2.0 mg / mL. Use an inverted microscopic Raman spectrometer to detect the Raman spectra of ergothioneine at different concentrations. The laser wavelength of the Raman spectrometer is 785 nm, and the integration time for laser irradiation of ergothioneine at different concentrations is more than 3 seconds. The average number of spectral acquisitions for each gradient solution is not less than 3. The obtained Raman spectra are subjected to background removal, Savitzky_Golay smoothing, 6th-order polynomial fitting baseline calibration, and minimum-maximum normalization, and can be used as candidate peaks for identifying peaks of intracellular ergothioneine.

[0038] Detection of different strains of Cryptococcus neoformans by HPLC found that only the wild-type WT and cells containing the egt1 gene in the Q phase were able to synthesize ergothioneine ( Figure 2 ). Raman spectral analysis of the corresponding strain cells found that two characteristic peaks of ergothioneine could be observed in the Raman spectra of Cryptococcus neoformans ( Figure 3 A-B), so 1210 cm -1 and 1507 cm -1 could be determined as the identification peaks for detecting intracellular ergothioneine. The wild-type WT had obvious Raman peak intensities at 1210 cm -1 and 1507 cm -1 , while the characteristic peaks at this position in the egt1Δ deletion strain (cells unable to synthesize ergothioneine) were significantly reduced or disappeared, and the identification peak intensity of ergothioneine in the strain with the P EGT1 -EGT1 complemented with the EGT1 gene (able to synthesize ergothioneine) was significantly higher than that of the egt1Δ deletion strain and was similar to that of the wild-type ( Figure 3 C-D). The analysis results of HPLC and cell Raman spectra proved that Raman spectroscopy could be applied to the detection of ergothioneine in single cells, and it was only necessary to determine whether intracellular ergothioneine was present through the identification peaks at 1210 cm -1 and 1507 cm -1 . Raman spectroscopy, as a technique for detecting ergothioneine in living cells, has the advantages of requiring a small amount of sample, no need for damage, rapidity, and single-cell detection.

[0039] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A method for rapid and non-destructive detection of ergothioneine in living cells, which is based on Raman spectroscopy for rapid detection of ergothioneine in living cells; the method for rapid and non-destructive detection of ergothioneine in living cells includes the following steps: Obtain the live cell fluid to be tested, wash it, adjust the concentration of the bacterial suspension, add the obtained bacterial suspension to the sample detection cell, use a microscope to find the cells in the solution, irradiate the cells with laser, and obtain the Raman spectrum of the cells. If there are identification peaks at 1210 cm -1 and 1507 cm -1 , it is determined that the live cells to be tested contain ergothioneine; otherwise, it is determined that the live cells to be tested do not contain ergothioneine; The cells are Cryptococcus neoformans cells in the resting phase.

2. The method according to claim 1, wherein: If the Raman signature peaks at 1210 cm -1 and 1507 cm -1 in the Raman spectrum of the cell have high intensities, it is determined that the content of ergothioneine in the live cell to be measured is high.

3. The method according to claim 1, characterized in that: The Raman signature peaks at 1210 cm in the Raman spectrum of the cells -1 and 1507 cm -1 are limited by the resolution of the instrument, and their signature peaks have an offset of 1 - 2 cm -1 .

4. The method according to claim 1, wherein: The number of cells irradiated by the laser is a single cell or multiple cells within the laser irradiation range.

5. The method according to claim 1, characterized in that: The washing is to wash the cells in the obtained living cell fluid to be tested with a sodium chloride solution or a cell isotonic solution, and the washing is carried out multiple times, specifically 2-3 times; Adjust the concentration of the bacterial suspension to 10 4 -10 7 CFU / mL.

6. The method according to claim 1, characterized in that: The Raman spectroscopy system used for cell Raman spectroscopy detection is an upright or inverted microscope Raman spectroscopy system, wherein the excitation wavelength of the Raman spectroscopy system is any one or a combination of 532 nm, 633 nm and 785 nm wavelengths; When performing cell Raman spectroscopy detection, the total integration time is more than 3 seconds.

7. The method according to claim 6, characterized in that: A 785 nm continuous laser is used as the light source for exciting cell Raman scattering, and the total integration time is more than 20 seconds.

8. The method according to claim 1, wherein: The method also includes background removal, smoothing, baseline calibration and normalization processing of the obtained Raman spectroscopy data.

9. The method according to claim 8, wherein: The smoothing method uses convolution smoothing method, moving average method, Gaussian filtering, bilateral filtering or mean filtering; The baseline calibration method uses polynomial fitting method, BEADS algorithm, wavelet algorithm, empirical mode decomposition; The normalization method uses minimum-maximum method, area normalization, vector normalization.

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