A microfluidic-based polypeptide-based helical fiber with circularly polarized luminescence and a preparation method thereof

Abstract

This invention discloses a microfluidic-based polypeptide-based helical fiber with circularly polarized luminescence and its preparation method, belonging to the field of nanotechnology. Utilizing multiple hydrogen bonding interactions between polypeptides and based on the solute conformational confinement and ordered solvent diffusion mechanism of a microfluidic laminar flow chip, this invention develops a polypeptide-based circularly polarized luminescent material with excellent luminescence performance. Circular dichroism spectroscopy and circular polarization spectroscopy tests show that the helical fiber material obtained by this invention exhibits mirror-symmetric signals. Compared with materials obtained by traditional solution stirring, the polypeptide-based helical fibers prepared in the microfluidic chip by this invention not only have a more uniform morphology but also a larger... g lum value( L -Peptide / TBI and D The luminescence asymmetry factor of -Peptide / TBI is ±1.9×10⁻⁶. ‑3 Furthermore, this method has a certain degree of universality and is applicable to the assembly of other macromolecules, including polymers, nucleic acids, and proteins.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology, and in particular to a microfluidic-based polypeptide-based helical fiber with circularly polarized luminescence and its preparation method. Background Technology

[0002] Helical structures are ubiquitous in nature, from nanoscale DNA double helices and secondary protein structures to macroscopic patterns on nautilus shells and tendrils of climbing plants. The helical morphology is considered the most fundamental topological structure in nature. Artificial helical nanostructures with right-handed (P) or left-handed (M) helicity are of great significance for studying the origin of chirality and its potential applications in materials science, nonlinear optics, biological systems, chemical sensing, chiral selective catalysis, and chiral devices.

[0003] For supramolecular self-assembly systems, the reported methods for preparing helical structures mainly involve non-covalent interactions such as hydrogen bonding, π-π stacking, and coordination bonds. While single-component self-assembly is considered an effective method for obtaining helical structures at the nanometer to micrometer scale, it has significant limitations in the flexible modulation of supramolecular chirality. Environmental factors such as temperature, solvent, or concentration are the primary factors controlling the properties of assemblies formed by single-component self-assembly. In contrast, multi-component assembly has attracted increasing attention in recent years because it allows for the control of supramolecular chirality at different levels by introducing different components, enabling synergistic effects between different components to compensate for the shortcomings of single materials and enhance stability, and producing diverse chiral structures and soft materials in a precise and modular manner.

[0004] Amino acids are the basic building blocks of polypeptides and proteins, containing multiple easily modifiable amino and carboxylic acid functional groups. Due to their ease of modification, abundant nonvalent binding sites, and inherent point chirality, they are widely used in the construction of multi-component helical nanosystems, such as helical tubular aggregates, helical polymers, and small molecule helical assemblies.

[0005] Circularly polarized light-emitting (CPL) materials have potential applications in optical sensors, 3D displays, optoelectronic devices, spintronic devices, and information storage.

[0006] One current method for obtaining CPL materials is the self-assembly of chiral organic or inorganic luminescent materials, including small organic molecules, polymers, and lanthanide complexes with chiral centers. However, achieving this often requires lengthy synthetic routes, and the emission characteristics of CPL materials are difficult to predict and control. On the other hand, multi-component self-assembly has been shown to be a key method for constructing CPL materials and improving the luminescence asymmetry factor (g). lumAn effective method for obtaining CPL materials is to address the chiral nature of chiral fragments after multi-component self-assembly. This allows chiral transfer to the luminescent group under photoexcited conditions, potentially leading to high-performance CPL materials. Currently, obtaining CPL materials through multi-component assembly is primarily carried out in open-vial systems.

[0007] However, the spiral fiber structure produced in the existing open vial system is difficult to control precisely and cannot meet the relevant usage requirements. Summary of the Invention

[0008] One of the objectives of this invention is to provide a method for preparing polypeptide-based helical fibers with circularly polarized luminescence, in order to solve the above-mentioned problems.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing polypeptide-based helical fibers with circularly polarized luminescence based on microfluidics, wherein the method is as follows:

[0010] (1) First, prepare DMSO (dimethyl sulfoxide) stock solution of L-peptide or D-peptide, DMSO stock solution of TBI, and NaOH aqueous solution respectively;

[0011] (2) Then, the DMSO mother liquor of the L-peptide or D-peptide and the DMSO mother liquor of TBI flow into the chip from the inner channel of the microfluidic chip, and the NaOH aqueous solution flows into the chip from the outer channel of the microfluidic chip. Then, the assembly is carried out in the microfluidic chip to obtain the assembly.

[0012] (3) The assembly is aged to obtain polypeptide-based helical fibers with circularly polarized light emission.

[0013] This invention employs microfluidic chips for multi-component self-assembly to obtain fiber materials exhibiting AIE (Aggregation-Induced Emission) effects. Specifically, this invention utilizes the co-assembly of chiral peptides and non-chiral aggregation-induced emission molecules to construct circularly polarized luminescent materials. This invention, employing microfluidic devices for multi-component self-assembly, represents a novel strategy for preparing helical fibers with circularly polarized emission.

[0014] As a preferred technical solution, in step (1), the amino acid sequences of the L-peptide and D-peptide are GFFVLK or KLVFF.

[0015] In the above sequence, as is well known to those skilled in the art, G is glycine (Gly), F is phenylalanine (Phe), V is valine (Val), L is leucine (Leu), and K is lysine (Lys).

[0016] The lysine residue at the GFFVLK terminus can be used to adjust its isoelectric point using an acid-base mixture (hydrochloric acid-sodium hydroxide solution), which allows for the construction of multi-component assemblies based on hydrophilic and electrostatic interactions. The L-peptide (L-Peptide) or (D-Peptide) can be synthesized using existing known methods or commissioned to a commercial company to synthesize it according to the sequence.

[0017] As a preferred technical solution, in step (1), the preparation method of TBI (i.e., 1,1,2,2-tetrakis(4-(1H-benzo[d]imidazol-2-yl)phenyl)ethene) is as follows: 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene, o-phenylenediamine and polyphosphoric acid (PPA, CAS No.: 8017-16-1) are added to a container, and then stirred at 160 ℃~200 ℃ for 8h~12h to carry out the reaction; after the reaction is completed, post-processing is performed to obtain TBI; wherein, the molar ratio of 1,1,2,2-tetrakis(4-carboxyphenyl)ethylene to o-phenylenediamine is 1:1~1:10.

[0018] As a further preferred technical solution, the post-processing method is as follows: after cooling to 60 ℃~100 ℃, it is placed in ice water, stirred thoroughly, and then filtered. The filter cake is added to a saturated sodium bicarbonate solution and stirred overnight. After stirring, it is filtered again, and the filter cake is added to pure water and stirred for 8 h~12 h. After stirring, the filter cake is obtained by filtration. The filter cake is recrystallized twice in methanol to obtain a bright yellow solid, which is TBI.

[0019] As a preferred technical solution, in step (1), the concentration of the NaOH aqueous solution is 1.25 mM. This ensures that the pH of the assembly is 8. At pH 8, the peptide is at its isoelectric point, and the TBI remains electrically neutral, allowing intermolecular hydrogen bonds to form between them.

[0020] As a preferred technical solution, in step (2), the microfluidic chip is a laminar flow chip.

[0021] As a preferred technical solution, in step (2), the width and height of the assembly channel of the microfluidic chip are 300 μm and 100 μm, respectively. If the chip channel is too small, it is easy to clog the chip, while if the channel is too large, it is similar to the existing small bottle open system and cannot produce the corresponding technical effect.

[0022] As a preferred technical solution, in step (2), the flow rate of the inner channel of the chip is 1-3 μL / min, and the flow rate of the outer channel of the chip is 4-12 μL / min. The selection of such flow rates is the result of comprehensive consideration of the liquid holding capacity of the chip and whether a stable laminar flow can be maintained. If the flow rate is too low, a stable laminar flow may not be formed, while if the flow rate is too high, the laminar flow will turn into turbulent flow. In addition, if the flow rate is too high, the advantage of laminar flow, namely the uniform and rapid diffusion and collision between molecules that make up the assembly, will also disappear.

[0023] As a further preferred technical solution, in step (2), a four-channel chip is used, with two channels on the inner side and two on the outer side. The flow rate of the inner channel is 2 μL / min, and the flow rate of the outer channel is 8 μL / min, for a total flow rate of 20 μL / min for the microfluidic chip. At this flow rate, the laminar flow is more stable, and the resulting spiral structure is more uniform.

[0024] The second objective of this invention is to provide a polypeptide-based helical fiber with circularly polarized luminescence prepared by the above-described method.

[0025] For multi-component assemblies, helical structures hold promise for efficient chiral transfer from chiral to achiral substances, which is crucial for constructing high-efficiency CPL materials. Unlike the self-assembly behavior of molecules in existing open-system vials, this invention utilizes a microfluidic device to provide a micrometer-scale continuous flow phase, allowing for the regulation of intermolecular interactions at different locations during flow. Because the diffusion mass transfer properties of molecules are enhanced in such a microspace, solvent exchange between solute molecules and intermolecular interactions can proceed very rapidly and uniformly. Furthermore, micrometer-scale laminar flow allows the fiber self-assembly structure to align with the laminar flow, thereby generating micrometer-scale uniform helical fibers.

[0026] Compared with existing technologies, the advantages of this invention are as follows: This invention utilizes multiple hydrogen bonding interactions between peptides, based on the solute conformational confinement and ordered solvent diffusion mechanism of a microfluidic laminar flow chip, to develop peptide-based circularly polarized luminescent materials with excellent luminescence performance. Circular dichroism spectroscopy and circular polarization spectroscopy tests show that the helical fiber material obtained by this invention exhibits mirror-symmetric signals. Compared with materials obtained by traditional solution stirring, the peptide-based helical fibers prepared in a microfluidic chip by this invention not only have a more uniform morphology but also a larger g-value. lum The luminescence asymmetry factors for L-Peptide / TBI and D-Peptide / TBI were ±1.9 × 10⁻⁶. -3 This method has a certain degree of universality and is applicable to the assembly of other macromolecules, including polymers, nucleic acids, and proteins. Attached Figure Description

[0027] Figure 1This is a structural diagram of the microfluidic chip prepared in Example 1;

[0028] Figure 2 This is a schematic diagram of each channel of a microfluidic chip;

[0029] Figure 3 The structural diagram of the L- / D-Peptide prepared in Example 1;

[0030] Figure 4 The 1H NMR spectrum of TBI prepared in Example 1;

[0031] Figure 5 The carbon NMR spectrum of TBI prepared in Example 1;

[0032] Figure 6 The mass spectrum of TBI prepared in Example 1;

[0033] Figure 7 High performance liquid chromatography of L-Peptide in Example 1;

[0034] Figure 8 The mass spectrum of L-Peptide in Example 1;

[0035] Figure 9 High performance liquid chromatography of D-Peptide in Example 1;

[0036] Figure 10 The mass spectrum of D-Peptide in Example 1;

[0037] Figure 11 The image shows the morphology of the Peptide / TBI assembly in the microfluidic chip of Example 1, where a) is a TEM image of L-Peptide / TBI, b) is a TEM image of D-Peptide / TBI, c) is a SEM image of L-Peptide / TBI, and d) is a SEM image of D-Peptide / TBI.

[0038] Figure 12 TEM image of L-Peptide / TBI in a vial of Comparative Example 1;

[0039] Figure 13 a) Circularly polarized emission and b) emission asymmetry factor spectra of the assembly prepared in the microfluidic chip of Example 1;

[0040] Figure 14 a) 1H NMR spectrum and b) X-ray diffraction spectrum of the assembly of Example 1. Detailed Implementation

[0041] The invention will now be further described with reference to the accompanying drawings.

[0042] Example 1:

[0043] A polypeptide-based helical fiber with circularly polarized luminescence based on microfluidics is prepared by the following steps:

[0044] (1) Preparation of TBI: 1,1,2,2-tetra(4-carboxyphenyl)ethylene, o-phenylenediamine and PPA were added to a 100 mL round-bottom flask and stirred at 180 °C for 8 h. After the reaction was completed, the mixture was cooled to 80 °C and placed in ice water. After thorough stirring, the mixture was filtered, and the filter cake was added to a saturated sodium bicarbonate solution and stirred overnight. After stirring, the mixture was filtered again, and the filter cake was added to pure water and stirred for 8 h. After stirring, the filter cake was obtained by filtration. The filter cake was recrystallized twice in methanol to obtain a bright yellow solid, namely TBI. Its 1H NMR spectrum, 1C NMR spectrum and mass spectrum are shown below. Figures 4-6 As shown; wherein, the molar ratio of 1,1,2,2-tetra(4-carboxyphenyl)ethylene to o-phenylenediamine is 1:4, and the reaction formula is as follows:

[0045] ;

[0046] (2) L-Peptide or D-Peptide with the amino acid sequence GFFVLK (e.g. Figure 3 The solution was dissolved in DMSO to a concentration of 8 mM, and the TBI prepared in step (1) was also dissolved in DMSO to a concentration of 2 mM; and a 1.25 mM NaOH aqueous solution was prepared; wherein, the high performance liquid chromatography and mass spectra of L-Peptide and D-Peptide are shown in the figure. Figures 7-10 As shown;

[0047] (3) Preparation of helical fiber assemblies in microfluidic chips: The L-Peptide or D-Peptide DMSO solution and TBI DMSO solution prepared in step (2) flow into the microfluidic laminar flow chip through the inner channel (chip structure as shown in Figure 2). Figure 1 The image shows a four-channel chip; schematic diagrams for each channel are shown below. Figure 2 As shown in the figure, the NaOH aqueous solution prepared in step (2) flows into the microfluidic laminar flow chip through the outer channel and is then assembled in the chip to obtain the L / D Peptide / TBI assembly; wherein, the flow rate of the inner channel of the chip is 2 μL / min; the flow rate of the outer channel of the chip is 8 μL / min; and the total flow rate of the chip is 20 μL / min.

[0048] Comparative Example 1

[0049] Using a pipette, take 100 μL of DMSO solution of L-Peptide or D-Peptide with sequence GFFVLK obtained in step (2) of Example 1 and 100 μL of DMSO solution of TBI into a 5 mL vial. After gently shaking, add 800 μL of NaOH aqueous solution (concentration 1.25 mM) to assemble the assembly and then age it according to the same method as in Example 1.

[0050] Experimental example:

[0051] (1) Morphological characterization of the Peptide / TBI assembly in microfluidics in Example 1

[0052] The assembly that flowed out of the chip in Example 1 was collected in a vial and aged for 8 hours. The morphology of the assembly was then characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

[0053] The TEM sample was prepared as follows: First, a copper grid was placed on filter paper, and 20 μL of the aged L / D Peptide / TBI assembly was dropped onto the copper grid and allowed to dry naturally.

[0054] like Figure 11 As shown in a), L-Peptide / TBI exhibits uniform left-handed helical fibers, demonstrating efficient co-assembly of L-Peptide and TBI within the chip thanks to diffusion and collisions of laminar molecules. Furthermore, the inverse co-assembled D-Peptide / TBI possesses helical fibers in the opposite (right-handed) direction. Figure 11 b)). These results demonstrate that the chirality of the peptide was successfully transferred to the Peptide / TBI co-assembly. For reference peptide structures with pyrene rings (such as... Figure 3 The “L-Referenece” in the text, when co-assembled with TBI, cannot form a helical structure, demonstrating the selective assembly properties of different polypeptide structures.

[0055] To further characterize the morphology of the assembly, this experimental example also performed SEM characterization on the assembly, and the results are as follows: Figure 11 As shown in c) and 11d), the SEM results further demonstrate that the present invention successfully utilized microfluidic laminar flow assembly to obtain uniform helical fibers.

[0056] (2) Morphological characterization of the assembly obtained in Comparative Example 1

[0057] Because the assembly environment in the vial of Comparative Example 1 differs from that in the microfluidic system of Example 1, different assembly morphologies may result. Similarly, the inventors also prepared TEM samples using the same method after aging the assemblies in the vial of Comparative Example 1 for 8 hours, and the results are as follows. Figure 12 As shown, unlike the uniform helical fibers obtained in the microfluidic chip of Example 1, the helical fibers prepared in the vial in Comparative Example 1 are neither obvious nor uniform. This result demonstrates the significant application potential of microfluidic laminar flow systems in controlling the assembly morphology of assemblies.

[0058] (3) Experiment on the circularly polarized light emission properties of the assembly prepared by the microfluidic chip in Example 1

[0059] Since this invention involves the co-assembly of chiral peptides and non-chiral aggregation-induced luminescent molecules, it holds promise for the construction of circularly polarized luminescent materials.

[0060] To verify this, the circularly polarized light-emitting sample was prepared as follows: the assembly obtained from the microfluidic chip in Example 1 was aged for 8 h, centrifuged, and then uniformly coated onto a quartz plate.

[0061] Tests revealed that L-Peptide / TBI and D-Peptide / TBI exhibit positive and negative circularly polarized emission signals, respectively. Figure 13 As shown in a); in addition, the luminescence asymmetry factor (g) lum This is often used to evaluate the magnitude of circularly polarized luminescence. The luminescence asymmetry factors of the L-Peptide / TBI and D-Peptide / TBI prepared in Example 1 are 1.9 × 10⁻⁶. -3 ,like Figure 13 As shown in b), this demonstrates that the present invention has successfully constructed a circularly polarized luminescent material with a large luminescence asymmetry factor.

[0062] (4) Research and experimentation on assembly mechanism

[0063] To study the assembly mechanism of peptides and TBI, the inventors performed 1H NMR spectroscopy (NMR spectroscopy). 1 Characterization by H NMR and X-ray diffraction (XRD):

[0064] The sample preparation for the 1H NMR spectrum is as follows: Weigh a certain amount of L-Peptide from step (2) of Example 1 and TBI from step (1) of Example 1 and dissolve them in DMSO-d6, respectively. Dissolve sodium hydroxide in H2O-d2 with concentrations of 8 mM and 2 mM, respectively. The volume ratio of DMSO-d6 to H2O-d2 is 39 / 1.

[0065] The results are as follows Figure 14As shown in figure a), H1, H2, and H4 of TBI show a significant shift towards lower fields after co-assembly with L-Peptide, while H3 shows a slight shift towards higher fields. The changes in the 1H NMR spectrum suggest that the driving force for assembly may be the intermolecular hydrogen bonds between L-Peptide and TBI.

[0066] In addition, the results of X-ray diffraction are as follows Figure 14 As shown in b), the figure shows that the peak shape changed significantly before and after assembly, which further confirms the successful assembly of L-Peptide and TBI.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing polypeptide-based helical fibers with circularly polarized luminescence based on microfluidics, characterized in that, The method is as follows: (1) First, prepare separately L -peptide or D The preparation methods for TBI include: DMSO mother liquor of the peptide, DMSO mother liquor of TBI, and NaOH aqueous solution; wherein, the preparation method of TBI is as follows: 1,1,2,2-tetra(4-carboxyphenyl)ethylene, o-phenylenediamine and polyphosphoric acid are added to a container, and then stirred at 160 ℃~200 ℃ for 8 h~12 h to carry out the reaction; after the reaction is completed, post-processing is performed to obtain TBI; wherein, the molar ratio of 1,1,2,2-tetra(4-carboxyphenyl)ethylene to o-phenylenediamine is 1:1~1:10; (2) Then make the above L -peptide or D The DMSO stock solution of the peptide and the DMSO stock solution of TBI flow into the microfluidic chip through the inner channel at a flow rate of 1–3 μL / min. The NaOH aqueous solution flows into the microfluidic chip through the outer channel at a flow rate of 4–12 μL / min. The assembly is then carried out in the microfluidic chip. The width and height of the assembly channel are 300 μm and 100 μm, respectively, to obtain the assembly. The pH of the assembly is 8. (3) The assembly is aged to obtain polypeptide-based helical fibers with circularly polarized light emission.

2. The method according to claim 1, characterized in that, In step (1), the L -peptide or D -The amino acid sequence of the peptide is GFFVLK or KLVFF.

3. The method according to claim 1, characterized in that, The post-processing method is as follows: after cooling to 60-100℃, it is placed in ice water, stirred thoroughly, and then filtered. The filter cake is added to a saturated sodium bicarbonate solution and stirred overnight. After stirring, it is filtered again. The filter cake is added to pure water and stirred for 8-12 hours. After stirring, the filter cake is obtained by filtration. The filter cake is recrystallized twice in methanol to obtain a bright yellow solid, which is TBI.

4. The method according to claim 1, characterized in that, In step (1), the concentration of the NaOH aqueous solution is 1.25 mM.

5. The method according to claim 1, characterized in that, In step (2), the microfluidic chip is a laminar flow chip.

6. The method according to claim 1, characterized in that, In step (2), the total flow rate of the microfluidic chip is 20 μL / min.

7. The polypeptide-based helical fiber with circularly polarized luminescence prepared by the method according to any one of claims 1 to 6.

Images