CN113604039A - Flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy - Google Patents
Flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy Download PDFInfo
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Abstract
The invention discloses a flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy, which is prepared by carrying out in-situ reaction on noble metal salt, cationic hyperbranched polymer and dialdehyde organic matter, has high stability, can be stored for a long time, has enhancement effect on protein Raman spectroscopy, can be used as a flexible substrate with surface enhanced Raman scattering effect for analyzing and detecting protein biomacromolecules, and has the characteristics of higher SERS activity, good 'hot spot' effect and reproducibility.
Description
Technical Field
The invention relates to the technical field of biological macromolecule spectroscopy analysis and detection, in particular to a flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy.
Background
Raman spectroscopy is a scattering spectrum which provides information on molecular vibration and rotation, is an important spectroscopic analysis method for studying molecular structures and quantitative analysis, and has been widely used in the fields of chemistry, physics, biology, medicine, and the like.
The amide I and II bands and secondary structures (alpha-helix, beta-sheet, random coil, etc.) of protein amino acid residues all have characteristic Raman signals, and therefore, Raman spectroscopy is commonly used to study the structure of proteins and the effects of various environments (such as pH, temperature, buffer type and ionic strength, etc.) on protein conformation (Zibao Kun, et al, food science, 2018,39(18), 15-20; Liu \25035, etc., oral medicine, 2021,41(06), 481-485..
Water is an environment in which proteins live, and its raman scattering intensity is extremely weak, so that the raman spectrum of an aqueous protein solution can exclude the influence of an aqueous solvent. On the other hand, the protein in the aqueous solution state is closer to the natural state, so that the Raman spectroscopy analysis of the structure of the protein in the aqueous solution state and the relation between the structure and the function have important scientific research significance and application value. However, raman signals of proteins are weak, so that research of raman spectroscopy in the field of proteins is limited to some extent.
The Surface-enhanced Raman scattering (SERS) technology overcomes the disadvantage of low signal intensity of conventional Raman spectroscopy, and makes Raman spectroscopy rapidly develop in the aspect of analysis and application. An important mechanism of Surface-enhanced raman scattering is Surface Plasmon Resonance (SPR), i.e. incident light forces free electrons on the Surface of a conductor to form collective vibration, which resonates when the collective vibration frequency coincides with the incident light frequency, resulting in a very large enhancement of the electromagnetic field, thereby inducing a strong Surface plasmon enhancement effect, so that the absorption and scattering light of a test sample are enhanced, i.e. a Surface raman enhancement effect is generated (zhuyiying et al, application of raman spectroscopy in chemistry, northeast university press, 1998; liu yu et al, optical scattering bulletin, 2010,22(1), 29-33). For the commonly used SERS noble metal substrate, whether roughened metal surface, metal particle sol or metal nanoparticle array, the structure surface will become some sharp points with extremely large curvature (chenley et al, spectroscopy and spectrum analysis, 2016,36(10), 3087-. In the regions with the extremely large curvature, because the number of surface atoms is large, the surface charges are also large, a strong local electric field (called as a "hot spot") is formed, the more "hot spot" regions are generated, and the stronger the surface enhanced raman scattering effect of the substrate material is.
The research has found that the noble metal nano-structure material with SERS effect mainly comprises nano silver, nano copper, nano platinum, nano palladium, nano rhodium, nano iridium and the like (Bao Y, et al analytical Chemistry 2020, 92(21), 14325-. SERS substrate materials (simply referred to as substrates) are broadly divided into two categories:
(1) conventional solid SERS substrates are typically silicon wafers and glass sheets covered with a noble metal layer. In many practical samples to be tested, the molecules of the tested object are difficult to collect, such as some artworks and cultural relics, and the possibility of damaging the sample exists when the tested object is collected. The rigid SERS substrate is not easy to bend and break and is difficult to fully contact with a sample to be detected with an irregular surface, and the collection and SERS detection of target molecules on the surface of an object to be detected are limited. (Liu Si Jia et al, analytical laboratory, 2021,3(45), 1-11).
(2) A traditional liquid phase synthesis method of a noble metal nanoparticle dispersion system mainly adopts one or more reducing agents to reduce noble metal ions into noble metal simple substances in the presence of a stabilizer (or called a protective agent) to generate noble metal nanoparticles (Wangyuan and the like, Anhui agricultural science, 2013,41(36),13888 and 13889). Furthermore, Lee et al have reported the reduction of silver nitrate to nanosilver particles having a diameter of about 60nm under heating using citrate as a reducing agent and stabilizer (Lee P, et al journal of Physical Chemistry,1982, 86(17), 3391-3395). However, the above-mentioned noble metal nanoparticle dispersion system also has some problems (royal small leaf, etc., noble metal, 2011,32(02),14 to 19): (I) the preparation process is complex and time-consuming, and generally needs a plurality of procedures such as solvent washing, stirring, centrifugal precipitation, purification and the like; (II) the amount of the reducing agent and the stabilizing agent is difficult to control, so that nano particles with uniform appearance and size are difficult to prepare; the dispersion system of the noble metal nanoparticles prepared in the step (III) has poor stability, for example, the noble metal nanoparticles are precipitated and separated out after a nano gold sol (AuNPs) system synthesized by a tannic acid-sodium citrate reduction method is placed for 1 day (Luofusan and the like, Gansu science and technology, 2020,36(05), 43-44).
In order to realize the functional SERS substrate integrating the collection and detection of a protein solution sample, the flexible SERS substrate attracts the attention of scientific researchers. The flexible SERS substrate can be flexible and changeable, is convenient for in-situ detection on an irregular curved surface, and can even be directly used for detection of wiping sampling (Tian L, et al advanced Materials Interfaces,2016,3(15), 1600214). Although the literature reports that flexible SERS substrates of papers (such as filter paper, printing paper and chromatography paper) carrying nano silver (or gold) are used for raman spectroscopic detection for detecting 1, 4-benzenedithiol (Chang H, et al cs Applied Materials & Interfaces,2010,2(12), 3429-: (1) the compact surface of the paper is not favorable for adsorbing a protein sample to be detected, (2) the paper SERS substrate is easy to be dissolved when being soaked in a liquid sample (Absdenour A, et al, analytical Chemistry,2013, 85(8), 3977-. Other flexible polymer SERS substrates (such as Polydimethylsiloxane (PDMS) flexible substrates and the like) (Alexandre RP, et al. materials Letters,2019,255,126557) have the problems of complex preparation method, high cost and the like, so that the application value of the SERS substrates is difficult to realize. Therefore, preparing SERS substrates with high SERS activity, flexibility, good "hot spot" effect and reproducibility still faces a great challenge with simple preparation process and low cost requirements.
Chinese patent CN108760717A (published japanese 2018.11.06) discloses a flexible surface enhanced raman substrate compounded by noble metal nanoparticles and organic polymer and a preparation method thereof, wherein the flexible surface enhanced raman substrate is prepared by mixing noble metal nanoparticles and organic polymer in a solvent and drying, and although the substrate prepared by the method has flexibility, the stability is poor, and the noble metal nanoparticles are easy to precipitate and separate out.
Disclosure of Invention
The invention aims to overcome the defect and the defect that the stability of the conventional flexible surface enhanced Raman substrate is not high enough, and provides a flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy, which is prepared by carrying out in-situ reaction on noble metal salt, cationic hyperbranched polymer and dialdehyde organic matter at 15-39 ℃, has high stability, and has the characteristics of high SERS activity, good 'hot spot' effect and reproducibility as a flexible SERS substrate.
The invention also aims to provide a preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy.
The invention also aims to provide application of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy.
The above purpose of the invention is realized by the following technical scheme:
a flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy is prepared by carrying out in-situ reaction on noble metal salt, a cationic hyperbranched polymer containing amino, dialdehyde organic matter and a reducing agent at 15-39 ℃ for 1-10 h, wherein the molar ratio of the amino in the cationic hyperbranched polymer to the dialdehyde organic matter to the noble metal salt to the reducing agent is 1: 1-2: 0.5-2: 1-2, and the reducing agent is NaBH3CN、(CH3COO)3BHNa or NaBH4One or more of them.
According to the invention, noble metal salt, cationic hyperbranched polymer and dialdehyde organic matter are subjected to in-situ reaction at 15-39 ℃ to prepare the cationic hyperbranched polymer/nano noble metal composite material, wherein the cationic hyperbranched polymer contains a large amount of amino groups, so that noble metal cations can be reduced into noble metal simple substances, the hyperbranched structure of the cationic hyperbranched polymer has the functions of stabilizing and dispersing noble metal nanoparticles, nano noble metals in the material can be uniformly and stably dispersed, the material can be stored for a long time, the stability of the material is improved, the dialdehyde organic matter is added, aldehyde groups contained in the cationic hyperbranched polymer can further react with the cationic hyperbranched polymer through Schiff base (Schiff base) to form carbon-nitrogen (C-N) cross-linked bonds, the noble metal cations can be further reduced into the noble metal simple substances, the material has better flexibility, and convenience in realization, The device can quickly and efficiently wipe and sample, and is suitable for detecting protein samples by protein surface enhanced Raman spectroscopy, but not limited to the test of the protein samples.
Preferably, the noble metal salt is one or more of soluble salts of silver, gold, copper, platinum, palladium, rhodium and iridium.
Preferably, the noble metal salt is AgNO3、HAuCl4、CuSO4One or more of them.
Preferably, the cationic hyperbranched polymer is hyperbranched polyethyleneimine and/or polyamidoamine.
Preferably, the weight average molecular weight of the hyperbranched polyethyleneimine (bPEI) is 1800-75000.
Preferably, the algebraic G of the polyamide-amine (PAMAM) is 1-10.
Preferably, the organic dialdehyde is one or more of glyoxal, succinaldehyde, glutaraldehyde and hexanedial.
The invention protects the preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy, which comprises the following steps:
s1, adding a noble metal salt aqueous solution into a cationic hyperbranched polymer aqueous solution, and uniformly mixing at 15-39 ℃ for 0.5-1 h until the solution is clear; the concentration of the noble metal salt aqueous solution is 0.5-2 mol/L; the mass volume fraction of the cationic hyperbranched polymer aqueous solution is 5-40%;
s2, adding a dialdehyde organic matter aqueous solution with the mass volume fraction of 5-10% into the solution obtained in the step S1, and uniformly mixing at 15-39 ℃ for 0.5-1 h; obtaining a solid jelly;
s3, adding a reducing agent aqueous solution with the concentration of 0.1-0.5 mol/L into the solid jelly obtained in the step S2, and soaking for 5-12 hours at the temperature of 15-39 ℃;
s4, taking out the solid jelly soaked in the step S3, soaking and cleaning for 1-2 hours by using water, and repeating the process for 2-5 times;
s5, freeze drying the solid jelly obtained in the step S4 to obtain the cationic hyperbranched polymer/nano precious metal composite material.
Preferably, the manner of mixing is by vortexing.
Preferably, the noble metal salt aqueous solution is dripped into the cationic hyperbranched polymer aqueous solution in the step S1, and the dripping speed is 5-15 seconds per droplet. The cation hyperbranched polymer contains a large amount of amino, one purpose of the cation hyperbranched polymer is to reduce noble metal cations into noble metal simple substances through the amino, and the other purpose of the cation hyperbranched polymer is to play a role in stabilizing and dispersing noble metal nanoparticles through the hyperbranched structure.
Preferably, the aqueous solution of the dialdehyde organic matter is dripped into the solution obtained in the step S1 at a dripping speed of 30-60 seconds per droplet. Adding dialdehyde organic matter, wherein one purpose of the dialdehyde organic matter is to react aldehyde groups contained with the cationic hyperbranched polymer through Schiff base to form carbon-nitrogen double bond (C ═ N) crosslinking bonds, and the other purpose of the dialdehyde organic matter is to further reduce the noble metal cations into noble metal simple substances.
Preferably, the amount of the noble metal salt aqueous solution used in step S1 is 0.1-1 mL.
Preferably, the dosage of the cationic hyperbranched polymer aqueous solution in the step S1 is 0.5-2 mL.
Preferably, the amount of the aqueous solution of the dialdehyde organic matter in the step S2 is 0.5-2 mL.
Preferably, the amount of the reducing agent aqueous solution used in step S3 is 5 to 10 mL. Adding NaBH3CN、(CH3COO)3BHNa or NaBH4With the aim of reducing unstable carbon-nitrogen double bonds (C ═ N) to stable carbon-nitrogen single bonds (C — N).
Preferably, the temperature of the freeze drying in the step S5 is-40 ℃ to-80 ℃, and the time is 0.5 to 2 days.
Preferably, the solvent of the aqueous solution is distilled water, deionized water, pure water or high-purity water.
And (S4) soaking and cleaning with water, wherein the purpose is to remove unreacted noble metal salt and dialdehyde organic matter.
The invention protects the application of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy in protein surface enhanced Raman spectroscopy analysis.
The flexible cationic hyperbranched polymer/nano noble metal composite material prepared by the invention can be used as a flexible substrate with a surface enhanced Raman scattering effect for analyzing and detecting protein biological macromolecules.
Compared with the prior art, the invention has the beneficial effects that:
the cationic hyperbranched polymer/nano noble metal composite material is prepared by the in-situ reaction of the noble metal salt, the cationic hyperbranched polymer and the dialdehyde organic matter, has high stability, can be stored for a long time, has an enhancement effect on protein Raman spectrum, can be used as a flexible substrate with a surface enhanced Raman scattering effect for analyzing and detecting protein biomacromolecules, and has the characteristics of higher SERS activity, good 'hot spot' effect and reproducibility.
Drawings
FIG. 1 is a process flow diagram of the preparation method of example 1 of the present invention.
FIG. 2 shows XRD patterns of (a) bPEI-GA-AgNPs and (b) bPEI-GA..
FIG. 3 is a solid UV-vis spectrum of bPEI-GA-AgNPs.
FIG. 4 is a photograph of the flexible behavior of bPEI-GA-AgNPs.
FIG. 5 is a microscopic Raman picture of bPEI-GA-AgNPs (a) a three-dimensional image and (b) a sectional image.
FIG. 6 shows Raman spectra (PBS as solvent for BSA, pH 7.4), (a) BSA (5mmol/L), (b) bPEI-GA-AgNPs/BSA (0.1mmol/L), (c) bPEI-GA-AgNPs/BSA (0.05mmol/L), (d) bPEI-GA-AgNPs/BSA (0.001mmol/L), (e) bPEI-GA-AgNPs.
Annotation of abbreviations:
bPEI-GA-AgNPs: hyperbranched polyethyleneimine/nano-silver composite material.
bPEI-GA: without addition of AgNO3Of (3) hyperbranched polyethyleneimine-glutaraldehyde-NaBH3CN gum product.
BSA: bovine serum albumin.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.
Example 1
A flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy is prepared through AgNO3With hyperbranched polyethyleneimine and glutaraldehyde, NaBH3CN is subjected to in-situ reaction at the temperature of 25 ℃ for 1h to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for the protein surface enhanced Raman spectroscopy, wherein amino in the hyperbranched polyethyleneimine is glutaraldehyde and AgNO3:NaBH3The molar ratio of CN is 1:1:1: 2.
The preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy comprises the following steps:
s1, taking 0.4mL of AgNO3Aqueous solution (1mol/L), dropwise (15 sec/d) to 0.5mL of aqueous hyperbranched polyethyleneimine (bPEI, Mw 75000) solution (5%, w/v), vortexed (0.5h) at 25 ℃Clarifying the solution;
s2, dripping (60 seconds per drop) 0.7mL of glutaraldehyde aqueous solution (7.5%, w/v) into the clear solution obtained in the step S1 at 25 ℃ under vortex (0.5h) until the mixed solution becomes a solid jelly;
s3, adding 8mL of NaBH into the solid jelly obtained in the step S23CN (0.5mol/L) and soaking for 12 hours at 25 ℃;
s4, taking out the solid jelly obtained in the step S3, soaking and cleaning the solid jelly for 2 hours by using water, and repeating the process for 2 times;
s5, freeze-drying the solid jelly obtained in the step S4 (at minus 40 ℃ for 2 days) to obtain a hyperbranched polyethyleneimine/nano-silver composite material (named bPEI-GA-AgNPs); the above preparation scheme is shown in FIG. 1.
Example 2
A flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy is prepared by HAuCl4With hyperbranched polyethyleneimine and succinaldehyde, NaBH3CN is subjected to in-situ reaction at 39 ℃ for 2 hours to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy, wherein amino in hyperbranched polyethyleneimine is succinaldehyde and HAuCl4:NaBH3The molar ratio of CN is 1:1:0.5: 1.
The preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy comprises the following steps:
s1, taking 0.1mL of HAuCl4Aqueous solution (2mol/L), dropwise (5 sec/d) to 2mL of aqueous hyperbranched polyethyleneimine (bPEI, Mw 25000) (20%, w/v), and vortexing (1h) at 39 ℃ until the solution is clear;
s2, dropwise adding (30 seconds/drop) 0.5mL of succinaldehyde aqueous solution (10%, w/v) into the clear solution obtained in the step S1 at 39 ℃ under vortex (1h) until the mixed solution becomes a solid jelly;
s3, adding 5mL of NaBH into the solid jelly obtained in the step S24Soaking the water solution (0.1mol/L) at 39 ℃ for 10 h;
s4, taking out the solid jelly obtained in the step S3, soaking and cleaning the solid jelly for 1h by using water, and repeating the process for 5 times;
s5, freeze drying the solid jelly obtained in the step S4 (at the temperature of minus 80 ℃ for 0.5 day) to obtain the hyperbranched polyethyleneimine/nanogold composite material.
Example 3
A flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy is prepared by CuSO4With hyperbranched polyethyleneimine and adipaldehyde, (CH)3COO)3BHNa is subjected to in-situ reaction at 15 ℃ for 4 hours to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy, wherein amino in hyperbranched polyethyleneimine is adipaldehyde and CuSO4:(CH3COO)3BHNa molar ratio 1:1:2: 2.
The preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy comprises the following steps:
s1, taking 1mL of CuSO4Aqueous solution (0.5mol/L), dropwise (10 sec/L) to 1mL of aqueous hyperbranched polyethyleneimine (bPEI, Mw 1800) (40%, w/v), and vortexing (2h) at 15 ℃ until the solution is clear;
s2, dripping (45 seconds/drop) 2mL of hexanedial aqueous solution (5 percent, w/v) into the clear solution obtained in the step S1 at 15 ℃ under vortex (2h) until the mixed solution becomes a solid jelly;
s3, adding 10mL (CH) of solid jelly obtained in step S23COO)3BHNa aqueous solution (0.5mol/L) is soaked for 5 hours at 15 ℃;
s4, taking out the solid jelly obtained in the step S3, soaking and cleaning the solid jelly for 2 hours by using water, and repeating the process for 2 times;
s5, freeze drying the solid jelly obtained in the step S4 (at the temperature of minus 40 ℃ for 2 days) to obtain the hyperbranched polyethyleneimine/nano copper composite material.
Example 4
Flexible Raman spectroscopy method for protein surface enhancementCationic hyperbranched polymer/noble metal nanoparticle composite material prepared by AgNO3With hyperbranched polyamidoamines and glyoxal, NaBH3CN is subjected to in-situ reaction at the temperature of 20 ℃ for 10 hours to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy, wherein the amino group in the hyperbranched polyamide-amine is glyoxal and AgNO3:NaBH3The molar ratio of CN is 1:1:1.5: 2.
The preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy comprises the following steps:
s1, taking 0.5mL of AgNO3Aqueous solution (1.5mol/L), dropwise (10 sec/L) to 1.5mL of aqueous hyperbranched polyamidoamine (PAMAM, G ═ 1) (20%, w/v), and vortexed at 20 ℃ for 5h until the solution is clear;
s2, dripping (45 seconds/drop) 1.5mL of glyoxal aqueous solution (8%, w/v) into the clear solution obtained in the step S1 at 20 ℃ under vortex (5h) until the mixed solution becomes a solid jelly;
s3, adding 10mL of NaBH into the solid jelly obtained in the step S23CN water solution (0.3mol/L) is soaked for 12 hours at the temperature of 20 ℃;
s4, taking out the solid jelly obtained in the step S3, soaking and cleaning the solid jelly for 1h by using water, and repeating the process for 4 times;
s5, freeze drying the solid jelly obtained in the step S4 (-40 ℃,2 days) to obtain the hyperbranched polyamide-amine/nano-silver composite material.
Example 5
A flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy is prepared by HAuCl4With hyperbranched polyamidoamines and glutaraldehyde, (CH)3COO)3BHNa at 25 deg.C according to the amino group of hyperbranched polyamidoamine glutaraldehyde: HAuCl4:(CH3COO)3BHNa molar ratio is 1:2:0.5:2, and the in-situ reaction is carried out for 5h to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopyAnd (5) feeding.
The preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy comprises the following steps:
s1, taking 0.5mL of HAuCl4Aqueous solution (1mol/L), dropwise (5 sec/L) to 1mL of aqueous hyperbranched polyamidoamine (PAMAM, G ═ 10) (20%, w/v), vortexed (2.5h) at 25 ℃ until the solution is clear;
s2, dropwise adding (30 seconds/drop) 1.5mL of glutaraldehyde aqueous solution (5%, w/v) into the clear solution obtained in the step S1 at 25 ℃ under vortex (2.5h) until the mixed solution becomes solid jelly;
s3, adding 8mL (CH) of solid gum obtained in step S23COO)3BHNa aqueous solution (0.5mol/L) is soaked for 10h at 25 ℃;
s4, taking out the solid jelly obtained in the step S3, soaking and cleaning the solid jelly for 2 hours by using water, and repeating the process for 3 times;
s5, freeze drying the solid jelly obtained in the step S4 (-80 ℃, 0.5 day) to obtain the hyperbranched polyamide-amine/nanogold composite material.
Example 6
A flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy is prepared by CuSO4With hyperbranched polyamidoamines and succinaldehyde, NaBH4At 30 ℃, according to the amino group of the hyperbranched polyamide-amine, succinaldehyde and CuSO4:NaBH4And (3) carrying out in-situ reaction for 3h according to the molar ratio of 1:2:1.5:2 to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for the protein surface enhanced Raman spectroscopy.
The preparation method of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy comprises the following steps:
s1, taking 0.5mL of CuSO4Aqueous solution (1.5mol/L), dropwise (15 sec/L) to 1.5mL of aqueous hyperbranched polyamidoamine (PAMAM, G ═ 6) solution (25%, w/v), vortexed (1.5h) at 30 ℃ until the solution is clear;
s2, dripping (60 seconds/drop) 1.5mL of succinaldehyde aqueous solution (10 percent, w/v) into the clear solution obtained in the step S1 at the temperature of 30 ℃ under vortex (1.5h) until the mixed solution becomes solid jelly;
s3, adding 5mL of NaBH into the solid jelly obtained in the step S24Soaking the aqueous solution (0.5mol/L) at 30 ℃ for 10 h;
s4, taking out the solid jelly obtained in the step S3, soaking and cleaning the solid jelly for 2 hours by using water, and repeating the process for 2 times;
s5, freeze drying the solid jelly obtained in the step S4 (-60 ℃,1 day) to obtain the hyperbranched polyamide-amine/nano-copper composite material.
Comparative examples 1 to 3
Comparative example 1 differs from example 1 in that the preparation is carried out without addition of glutaraldehyde.
Comparative example 2 differs from example 1 in that the hyperbranched polyethyleneimine is replaced by a linear polyethyleneimine.
Comparative example 3 differs from example 1 in that NaBH is added3Replacement of CN by LiAlH4。
Performance testing
FIG. 2a is an XRD spectrum of the prepared bPEI-GA-AgNPs composite material, and XRD diffraction peaks of the simple substance silver appear at 38.5 degrees, 44.7 degrees, 64.8 degrees and 77.6 degrees and are respectively assigned to the (111), (200), (220) and (311) crystal faces of the simple substance silver. In contrast, the XRD pattern of bPEI-GA showed only one broad peak in the unoriented state (18.5, FIG. 2 b). These results demonstrate that the bPEI-GA-AgNPs composite contains elemental silver.
In the UV-vis spectrum of the bPEI-GA-AgNPs composite material (fig. 3), a characteristic UV absorption peak of the silver nanoparticles appears around λ 417nm, further indicating that the prepared sample contains silver nanoparticles.
The photograph of FIG. 4 shows that the bPEI-GA-AgNPs composite material can still maintain an intact morphology after being bent, indicating that the composite material has good flexibility. The elastic modulus can be regarded as an index for measuring the difficulty of the material generating elastic deformation, and the calculation method is shown as formula (1).
E＝σ/ε (1)
In the formula: e is the elastic modulus, σ is the stress in the unidirectional stress state, and ε is the strain.
The smaller the value of E, the less stress indicating a certain elastic deformation of the material, i.e. the more flexible the material. The E value of the bPEI-GA-AgNPs composite material tested by an electronic universal tester is 0.95MPa and is far lower than the E value (2MPa) of wearable flexible equipment reported In the literature (Kyung-In Jang et al Nature Communications,2014,5,4779), and the result shows that the bPEI-GA-AgNPs composite material has better flexibility.
The cationic hyperbranched polymer/noble metal nanoparticle composite materials prepared in the embodiments 2-6 have good flexibility, and can have the effect of surface enhanced Raman scattering on BSA solution.
FIG. 5 is a microphotograph of the bPEI-GA-AgNPs composite material taken by a micro-Raman imager, and the surface of the bPEI-GA-AgNPs composite material can be observed to be in an uneven peak structure, so that the contact chance of BSA molecules and nano silver particles is improved, the 'hot spot' effect of SERS is enhanced, and the Raman enhancement effect of the bPEI-GA-AgNPs composite material serving as an SERS substrate material on BSA is further improved.
FIG. 6a is a Raman spectrum of BSA (5mmol/L) showing Raman scattering signal peaks at the amino acid residues and the secondary structure of BSA. The bPEI-GA-AgNPs composite material does not have obvious Raman scattering peaks in the Raman spectrogram area of BSA (FIG. 6e), so that the bPEI-GA-AgNPs composite material is suitable for the Raman spectrum of the BSA. In general, it is difficult to detect the Raman scattering signal when the BSA concentration is less than 5 mmol/L. However, strong Raman scattering signal peaks still appeared after the bPEI-GA-AgNPs composite adsorbed a BSA solution with a lower concentration (FIG. 6b and FIG. 6c, BSA concentrations of 0.1 and 0.05mmol/L, respectively), and no Raman scattering signal peak was observed for BSA until BSA concentrations were as low as 0.001mmol/L (FIG. 6 d). The results prove that the bPEI-GA-AgNPs composite material shows a strong Raman spectrum enhancement effect on BSA, and effectively reduces the lower limit of the detection concentration of the BSA to 0.001-0.05 mmol/L. In the enhanced Raman spectra shown in FIGS. 6b and 6C, the peaks showing stronger Raman signals were assigned to the S-S stretching vibration of BSA, the C-C stretching vibration, the amide I band, the amide III band and the α -helix backbone structure of tryptophan (Tyr) and phenylalanine (Phe), respectively.
The bPEI-GA-AgNPs composite material is placed at 25 ℃ for a week, and a Raman spectrum test is carried out after BSA solution is adsorbed, so that the result shows that the Raman signal of BSA molecules still shows an enhancement effect, and the bPEI-GA-AgNPs composite material has good stability and reproducibility.
In comparative example 1, the product obtained is a nano silver solution due to no chemical crosslinking of glutaraldehyde, and a flexible hyperbranched polyethyleneimine/nano silver composite material cannot be obtained.
In comparative example 2, since linear polyethyleneimine is replaced without a hyperbranched structure, agglomerated elemental silver appears in the obtained product, and a flexible hyperbranched polyethyleneimine/nano silver composite material cannot be obtained.
In comparative example 3, due to LiAlH4The C ═ N double bond cannot be reduced to a stable C — N single bond, the obtained solid jelly is gradually melted into liquid after being left at room temperature for about 5 hours, the stability is poor, and a flexible hyperbranched polyethyleneimine/nano silver composite material cannot be obtained.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for the protein surface enhanced Raman spectroscopy is characterized in that a noble metal salt, a cationic hyperbranched polymer containing amino, a dialdehyde organic matter and a reducing agent are subjected to in-situ reaction at 15-39 ℃ for 1-10 hours to prepare the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for the protein surface enhanced Raman spectroscopyThe cationic hyperbranched polymer belongs to a nanoparticle composite material, wherein the molar ratio of amino, dialdehyde organic matter, noble metal salt and reducing agent in the cationic hyperbranched polymer is 1: 1-2: 0.5-2: 1-2, and the reducing agent is NaBH3CN、(CH3COO)3BHNa or NaBH4One or more of them.
2. The method according to claim 1, wherein the noble metal salt is one or more of soluble salts of silver, gold, copper, platinum, palladium, rhodium and iridium.
3. The cationic hyperbranched polymer according to claim 1 or 2, wherein the molar ratio of the amino group, the dialdehyde organic substance, the noble metal salt and the reducing agent in the cationic hyperbranched polymer is 1:1: 1.5-2: 1-2.
4. The method according to claim 1, wherein the cationic hyperbranched polymer is a hyperbranched polyethyleneimine and/or a polyamidoamine.
5. The method according to claim 4, wherein the weight average molecular weight of the hyperbranched polyethyleneimine is 1800-75000.
6. The method according to claim 1, wherein the organic dialdehyde is one or more of glyoxal, succinaldehyde, glutaraldehyde and adipaldehyde.
7. The method for preparing the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced Raman spectroscopy according to any one of claims 1 to 6, wherein the method comprises the following steps:
s1, adding a noble metal salt aqueous solution into a cationic hyperbranched polymer aqueous solution, and uniformly mixing at 15-39 ℃ for 0.5-1 h until the solution is clear; the concentration of the noble metal salt aqueous solution is 0.5-2 mol/L; the mass volume fraction of the cationic hyperbranched polymer aqueous solution is 5-40%;
s2, adding a dialdehyde organic matter aqueous solution with the mass volume fraction of 5-10% into the solution obtained in the step S1, and uniformly mixing at 15-39 ℃ for 0.5-1 h; obtaining a solid jelly;
s3, adding a reducing agent aqueous solution with the concentration of 0.1-0.5 mol/L into the solid jelly obtained in the step S2, and soaking for 5-12 hours at the temperature of 15-39 ℃;
s4, taking out the solid jelly soaked in the step S3, soaking and cleaning for 1-2 hours by using water, and repeating the process for 2-5 times;
s5, freeze drying the solid jelly obtained in the step S4 to obtain the cationic hyperbranched polymer/nano precious metal composite material.
8. The method according to claim 7, wherein the noble metal salt aqueous solution is added dropwise to the cationic hyperbranched polymer aqueous solution in step S1 at a rate of 5 to 15 seconds per drop.
9. The method according to claim 7, wherein the aqueous solution of the dialdehyde organic substance is added dropwise into the solution obtained in step S1 at a rate of 30-60S/drop.
10. Use of the flexible cationic hyperbranched polymer/noble metal nanoparticle composite material for protein surface enhanced raman spectroscopy of any one of claims 1 to 6 in protein surface enhanced raman spectroscopy analysis.
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