Application of lipid droplet-targeted AIE molecule in seaweed staining imaging and screening

Abstract

The present application relates to a kind of lipid droplet targeted AIE molecule in seaweed dyeing imaging and screening etc., the AIE molecule is a kind of aldehyde group substitution triphenylamine skeleton aggregation-induced emission organic fluorescent molecule, it has bright blue-green fluorescence, good biocompatibility, excellent seaweed lipid droplet targeting nature.Compared with commercial probe Nile red, it has better specific marking effect. After using this probe to dye seaweed, the size, content and change of lipid droplet in seaweed can be clearly observed.Combined with flow cytometry screening technology, seaweed with strong fluorescent signal is screened, and seaweed with more and larger lipid droplets is obtained, and the culture result shows that the yield of seaweed biological lipids and the proportion of lipid material in dry weight are significantly improved, about twice of the original culture before screening.Therefore, it has important significance and value in marine microalgae research and biological crude oil production etc.

Description

Technical Field

[0001] This invention relates to the field of bioimaging and screening technology, specifically to the application of a lipid droplet-targeted AIE molecule in seaweed staining, imaging, and screening. Background Technology

[0002] Aggregation-induced emission (AIE) fluorescent materials have wide applications in fields such as bioimaging due to their excellent aggregated luminescence properties, good biocompatibility, and photostability.

[0003] Lipid droplets are organelles that store neutral lipids (triglycerides) and are widely found in animal and plant cells and microorganisms. In seaweed, lipid droplets originate from sugar molecules, a product of photosynthesis. The formation, growth, and dissolution of lipid droplets is a dynamic process. The size and abundance of lipid droplets in seaweed are closely related to the yield and content of its oil. Generally, algae with more and larger lipid droplets have higher oil yields and are therefore very important in the production of biocrude oil.

[0004] In existing technologies, Nile Red, a commercially available probe, is typically used to specifically stain seaweed for the observation and screening of lipid droplets. However, this method suffers from poor stability and sensitivity, making it unsuitable for screening high-performing seaweed. Therefore, a technique is needed to label lipid droplets in seaweed to clearly understand their size, content, and changes, enabling the screening of high-performing seaweed and addressing the problem of low biocrude oil yield from seaweed. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a fluorescent molecule with AIE properties, which can specifically image lipid droplets in seaweed. Combined with flow cytometry screening technology, it can screen for seaweed with superior performance and solve the problem of low yield of seaweed biocrude oil.

[0006] The technical solution adopted by this invention to solve the technical problem is: the application of a lipid droplet-targeted AIE molecule in seaweed staining imaging, wherein the AIE molecule is an aggregation-induced emission organic small molecule material with an aldehyde-substituted triphenylamine backbone, and the structural formula of the AIE molecule is as follows:

[0007]

[0008] R1 and R2 are selected from one or more of H, alkyl chains of 1-10 carbons, glycol chains of 1-10 carbons, phenyl, bromophenyl, iodophenyl, allyl, 2-hydroxyethyl, 2-aminoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl or 2-morpholinoethyl.

[0009] This invention also provides an application of lipid droplet-targeted AIE molecules in seaweed screening, wherein the AIE molecule is an aggregation-induced emission organic small molecule material with an aldehyde-substituted triphenylamine backbone, and the structural formula of the AIE molecule is as follows:

[0010]

[0011] R1 and R2 are selected from one or more of H, alkyl chains of 1-10 carbons, glycol chains of 1-10 carbons, phenyl, bromophenyl, iodophenyl, allyl, 2-hydroxyethyl, 2-aminoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl or 2-morpholinoethyl.

[0012] Preferably, flow cytometry analysis is performed on stained algae to monitor the consumption, generation, and expansion of algal lipid droplets.

[0013] Preferably, different subgroups are obtained by flow cytometry analysis of stained seaweed.

[0014] The application of a lipid droplet-targeting AIE molecule of the present invention in seaweed imaging and screening has the following beneficial effects: The AIE molecule of the present invention is an aggregation-induced emission organic fluorescent molecule with an aldehyde-substituted triphenylamine backbone, exhibiting bright blue-green fluorescence, good biocompatibility, and excellent targeting of seaweed lipid droplets; therefore, it has better specific labeling effect compared with the commercial probe Nile Red; after staining seaweed with this probe, the size, content, and changes of lipid droplets within the seaweed can be clearly observed; combined with flow cytometry screening technology, seaweed with strong fluorescence signals was screened, resulting in seaweed with more and larger lipid droplets. The culture results showed that the yield of seaweed biolipids and the proportion of lipid substances in dry weight were significantly improved, approximately twice that of the original culture before screening; therefore, it has important significance and value in marine microalgae research and biocrude oil production. Attached Figure Description

[0015] Figure 1 The emission distribution and aggregation-induced emission coefficient of compound TPA-A in aqueous tetrahydrofuran solutions with different water contents are shown.

[0016] Figure 2 The absorption and emission distribution of compound TPA-A in a 0.1% DMSO aqueous solution;

[0017] Figure 3 The water and particle size distribution of compound TPA-A in a 0.1% DMSO aqueous solution;

[0018] Figure 4 It is an imaging of Chlorella by the compound TPA-A;

[0019] Figure 5 The image is an image of Chlorella vulgaris co-stained with compound TPA-A and Nile red.

[0020] Figure 6 This is an image of Chlorella vulgaris at different growth stages, showing the compound TPA-A.

[0021] Figure 7 These are different subgroups of Chlorella obtained by flow cytometry sorting after staining with compound TPA-A.

[0022] Figure 8 This is a schematic diagram of Chlorella vulgaris stained with compound TPA-A after two generations of screening.

[0023] Figure 9 This shows the lipid droplet imaging and flow cytometry distribution of Chlorella vulgaris after TPA-A staining;

[0024] Figure 10 The photosynthetic rate, growth status, and lipid content of the primary and screened Chlorella species;

[0025] Figure 11 It is compound TPA-A 1 H NMR spectrum. Detailed Implementation

[0026] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Typical embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

[0027] Unless otherwise defined, 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 pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0028] The application of a lipid droplet-targeted AIE molecule of the present invention in the formation and screening of seaweed is further described below with reference to the accompanying drawings and embodiments:

[0029] Flow cytometry is a single-cell fluorescence sorting technique. Cells are dispersed into individual particles by flowing sheath fluid. As the single-cell suspension passes through a laser channel, its fluorescence signal is collected and displayed. Combined with fluorescent labeling technology, flow cytometry can separate target cells from the whole cell while maintaining high biological activity, making it a powerful tool for biological screening.

[0030] This invention provides a fluorescent material with aggregation-induced emission properties by constructing a molecule with an aldehyde group replacing the triphenylamine backbone. This material is widely available and inexpensive. The fluorescent material developed in this invention has broad applications in seaweed imaging and screening, including seaweed growth research, seaweed lipid droplet formation and tracking, and increasing seaweed biocrude oil production.

[0031] This invention relates to an aggregation-induced emission material TPA-A having an aldehyde-substituted triphenylamine backbone, the structural formula of which is:

[0032]

[0033] R1 and R2 are selected from one or more of H, alkyl chains of 1-10 carbons, glycol chains of 1-10 carbons, phenyl, bromophenyl, iodophenyl, allyl, 2-hydroxyethyl, 2-aminoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl or 2-morpholinoethyl.

[0034] The following detailed explanation uses Chlorella as an example to illustrate the experiment. Those skilled in the art will understand that, in other embodiments, the method provided by this invention is also applicable to single-celled microalgae such as cyanobacteria and green algae, as well as macroalgae such as kelp; this invention is not limited thereto.

[0035] The following detailed description is provided through specific embodiments.

[0036] Purchase 98% pure 4-(Di-p-tolylamino)benzaldehyde from commercial companies such as Aladdin, and recrystallize it in a hexane / dichloromethane mixed solvent to obtain a pale yellow crystalline compound, TPA-A. Figure 11 It shows 1 H NMR (400MHz, CDCl3) δ9.80 (s, 1H), 7.67 (d, J = 8.8Hz, 2H), 7.18 (d, J = 8.3Hz, 4H), 7.10 (d, J = 8.3Hz, 4H), 6.98 (d, J = 8.8Hz, 2H), 2.38 (s, 6H).

[0037] The purified TPA-A was dissolved in tetrahydrofuran to prepare a concentration of 10. -5 Mixtures of M in different proportions of tetrahydrofuran / water were used to determine the fluorescence of M under 365 nm excitation using a PerkinElmer fluorometer. The M exhibited blue-green fluorescence and was a typical aggregation-induced emission molecule. Figure 1 The emission distribution and aggregation-induced emission coefficient of compound TPA-A in aqueous tetrahydrofuran solutions with different water contents are shown.

[0038] Further, TPA-A was dissolved in dimethyl sulfoxide (DMSO) to prepare a concentration of 10. -5 M in a 0.1% DMSO aqueous solution. Figure 2 The absorption and emission distribution of compound TPA-A in a 0.1% DMSO aqueous solution is shown. Figure 2 As can be seen, TPA-A exhibits significant absorption in ultraviolet and short-wavelength blue light, and displays blue-green fluorescence with a maximum emission peak around 500 nm.

[0039] Figure 3 The water and particle size distribution of compound TPA-A in a 0.1% DMSO aqueous solution are described. TPA-A is dissolved in DMSO to prepare a concentration of 10... -5 In a 0.1% DMSO aqueous solution of M, it can be seen that TPA-A forms nanoparticles with a particle size of about 180 nanometers.

[0040] Figure 4 This is an image of Chlorella vulgaris using compound TPA-A. TPA-A was dissolved in a stock solution of DMSO (10...). -2 M) added to Chlorella (10 7 Prepare a 0.1% DMSO aqueous solution (number of cells / mL), stain for 15 minutes, centrifuge, and wash three times with phosphate buffer. Then, perform laser confocal imaging using a Zeiss LSM810 microscope. The green signal excitation wavelength is 405 nm, and the emission wavelength is 420-500 nm; the red signal excitation wavelength is 640 nm, and the emission wavelength is 650-700 nm. The green signal is the fluorescence signal of TPA-A, and the red signal is the autofluorescence of algal chloroplasts. Morphological observation shows that TPA-A is mainly concentrated within the lipid droplets of the algae.

[0041] Figure 5 Imaging of Chlorella vulgaris co-stained with compound TPA-A and Nile Red, using a DMSO stock solution of TPA-A and Nile Red (10... -2 M) added to Chlorella (10 7Prepare a 0.1% DMSO aqueous solution (per droplet / mL), stain for 15 minutes, centrifuge, and wash three times with phosphate buffer. Then, use a Zeiss LSM810 laser confocal imaging system. The green signal excitation wavelength is 405 nm, and the emission wavelength is 420-500 nm; the orange signal excitation wavelength is 488 nm, and the emission wavelength is 550-600 nm; the red signal excitation wavelength is 640 nm, and the emission wavelength is 650-700 nm. The green signal is the fluorescence signal of TPA-A, the red signal is the autofluorescence of algal chloroplasts, and the orange signal is the Nile red signal. The image shows a high degree of overlap between TPA-A and Nile red, and a small number of lipid droplets exhibit only TPA-A fluorescence, indicating that TPA-A can efficiently and specifically stain and fluoresce algal lipid droplets.

[0042] Figure 6 This is an image of Chlorella at different growth stages, showing the compound TPA-A. The Chlorella is arranged in 10... 6 Chlorella cells / mL were inoculated into fresh culture medium and cultured under 12-hour light / 12-hour dark conditions. Chlorella cells at different culture times were stained and photographed under the above conditions, including images at 24, 48, 96, 120, 168, and 192 hours after inoculation. The stained Chlorella cells were then analyzed by flow cytometry using BDFACSAria™ III, with a sample size of 20,000 cells. The analysis signals were APC-H (chloroplast fluorescence, excitation wavelength 633 nm, emission wavelengths 660 / 20 nm) and DAPI-H (TPA-A fluorescence, excitation wavelength 365 nm, emission wavelengths 450 / 50 nm). The images show that the fluorescence imaging of Chlorella, including the size and fluorescence intensity of chloroplasts and lipid droplets, clearly monitors the consumption, formation, and expansion of algal lipid droplets at different stages.

[0043] Figure 7 These are different subgroups of *Chlorella* stained with compound TPA-A, obtained after flow cytometry sorting. *Chlorella* in the plateau phase (approximately 168 hours post-inoculation) were stained and analyzed by flow cytometry under the above conditions. It can be seen that *Chlorella* stained with TPA-A exhibits a certain size and lipid droplet content distribution after reaching the plateau phase. Subsequently, flow cytometry sorting was performed using BDFACSAriaTMIII, selecting region Q2 (chloroplast fluorescence intensity greater than 10). 4 The fluorescence intensity of TPA-A is greater than 2*10. 4 ) and Q4 (chloroplast fluorescence intensity less than 10) 4 The fluorescence intensity of TPA-A is less than 2*10. 4 Algae Q2, which has larger chloroplasts and higher lipid droplet content, and algae Q4, which has smaller chloroplasts and lower lipid droplet content, can be divided into different subgroups.

[0044] Figure 8 This is a schematic diagram of Chlorella vulgaris stained with compound TPA-A and screened for two generations. The primary cells in the plateau phase were screened by flow cytometry as described above. The cells were divided into 50% high and 50% low TPA-A signal intensity. The half with high signal intensity were selected as the first generation. The second generation was obtained by culturing the first generation and performing the same screening on the first generation of algae.

[0045] Figure 9 This describes the lipid droplet imaging and flow cytometry distribution of Chlorella vulgaris stained with compound TPA-A after screening. Primary, first-generation, and second-generation Chlorella vulgaris were sorted according to the above... Figure 6 Staining and flow cytometry analysis under the specified conditions showed that the number of lipid droplets and the intensity of TPA-A fluorescence in Chlorella increased with the number of screening generations.

[0046] Figure 10 The photosynthetic rate, biomass, and lipid content of primary and screened Chlorella were measured in different groups: primary (high-broad-root type), primary-1 (primary generation 1st generation), primary-2 (primary generation 2nd generation), first-generation screened, and second-generation screened. The photosynthetic rate was determined by relative electron transport rate using a PAM fluorescence spectrometer; the number of Chlorella cells was determined by hemocytometer counting; biomass was determined by weighing dried Chlorella cells after the plateau phase; and lipid yield was determined by weighing after methanol extraction. The results show a slight increase in cell number and biomass, while the photosynthetic rate, lipid content, and yield significantly increased. The lipid content of the second-generation screened Chlorella was approximately double that of the primary generation.

[0047] This invention details the application of aggregation-induced emission materials with aldehyde-substituted triphenylamine skeletons in seaweed staining, imaging, and screening through the above embodiments. However, this invention is not limited to the above methods, meaning that it does not necessarily rely on the above reaction conditions to be implemented. Those skilled in the art should understand that equivalent substitutions of seaweed species and changes in application methods in this invention fall within the scope of protection and disclosure of this invention.

[0048] It should be understood that those skilled in the art can make improvements or modifications based on the above description, and all such improvements or modifications should fall within the protection scope of the appended claims.

Claims

1. The application of a lipid droplet-targeted AIE molecule in seaweed staining imaging, wherein the AIE molecule is an aggregation-induced emission organic small molecule material with an aldehyde-substituted triphenylamine backbone, and the structural formula of the AIE molecule is as follows: R1 and R2 are selected from one or more of H, alkyl chains of 1-10 carbons, glycol chains of 1-10 carbons, phenyl, bromophenyl, iodophenyl, allyl, 2-hydroxyethyl, 2-aminoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl or 2-morpholinoethyl.

2. Application of a lipid droplet-targeted AIE molecule in seaweed screening, wherein the AIE molecule is an aggregation-induced emission organic small molecule material with an aldehyde-substituted triphenylamine backbone, and the structural formula of the AIE molecule is as follows: R1 and R2 are selected from one or more of H, alkyl chains of 1-10 carbons, glycol chains of 1-10 carbons, phenyl, bromophenyl, iodophenyl, allyl, 2-hydroxyethyl, 2-aminoethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl or 2-morpholinoethyl.

3. Use according to claim 2, characterized in that, Flow cytometry analysis was performed on stained algae to monitor the consumption, formation, and expansion of algal lipid droplets.

4. The application according to claim 2, characterized in that, Different subgroups were obtained by flow cytometry analysis of stained seaweed.

Images