CN109060764B

CN109060764B - Preparation method of functionalized SERS platform and application of functionalized SERS platform in ATP detection

Info

Publication number: CN109060764B
Application number: CN201810966524.4A
Authority: CN
Inventors: 杨良保; 李盼; 毛妹; 于建强; 唐祥虎
Original assignee: Anhui Zhongke Saifeier Technology Co ltd
Current assignee: Anhui Zhongke Saifeier Technology Co ltd
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2020-12-29
Anticipated expiration: 2038-08-23
Also published as: CN109060764A

Abstract

The invention discloses a preparation method of a functionalized SERS platform and application of the functionalized SERS platform to ATP detection, and relates to the technical field of biological detection. The preparation method of the SERS platform comprises the following steps: step 1, preparing a gold nanoparticle solution; step 2, modifying the acupuncture needle by gold nanoparticles: immersing the needle body of the acupuncture needle cleaned by ethanol into 10 percent (volume ratio) of 3-mercaptopropyltriethoxysilane ethanol solution for 24 hours at 4 ℃; taking out, cleaning with ethanol, and drying; and immerging the gold nano-particle solution; step 3, functionalizing the acupuncture needle by the Raman probe: soaking the acupuncture needle-shaped SERS substrate prepared in the step 2 in a Raman probe solution at room temperature for treatment; after the treatment, the mixture was dried, washed with pure water and stored at room temperature. According to the invention, gold nanoparticle modification and Raman probe functionalization are sequentially carried out on the acupuncture needle, and the acupuncture needle is applied to ATP detection, so that the detection sensitivity is improved, and a stronger SERS signal is conveniently obtained.

Description

Preparation method of functionalized SERS platform and application of functionalized SERS platform in ATP detection

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a preparation method of a functionalized SERS platform and an application of the functionalized SERS platform in ATP detection.

Background

Acupuncture has been used as a supplement and replacement for drugs in traditional chinese medicine for over 2000 years. Among acupuncture therapies, acupuncture is widely accepted worldwide and is used primarily for the treatment of acute and chronic pain, but the biological basis for its treatment is poorly understood. In a recent study, Goldman et al reported that Adenosine Triphosphate (ATP) was released during acupuncture. After ATP is released, it will act as a transmitter that binds to purine receptors and then rapidly degrades to adenosine, which inhibits pain. In addition to antinociceptive effects, ATP also plays a key role in energy storage and signaling in biological systems. Most importantly, abnormal ATP levels in human body fluids are closely associated with a variety of genetic diseases. Therefore, it is important to develop a rapid and convenient method having sensitivity and selectivity in medical diagnosis and treatment for real-time detection of ATP.

For the selective detection of ATP, the first binding system for ATP is caused by biologically important acyclic polyamines (e.g., putrescine and spermidine), in which the hydrogen bonding of the amine, in addition to electrostatic, can affect the stability and selectivity of the complex interaction with ATP. On the basis of earlier studies, Lehn and Kimura proposed a "macrocyclic effect on anion binding" which is benefited by the fact that macrocyclic analogs of naturally occurring polyamines can strongly bind anions 1-2 orders of magnitude more than linear systems, related to the stability constant between macrocycle and ATP and the number of nitrogen atoms available for electrostatic and hydrogen bonding interactions. Thus, the methods involving classical ultra-high performance liquid chromatography, electrochemical analysis, as well as fluorescence resonance energy transfer and optical surface plasmon resonance have been demonstrated for the sensitive and selective detection of ATP by using different macrocycles. Surface Enhanced Raman Scattering (SERS) was developed as a powerful analytical technique compared to other spectroscopic techniques, and provides ultra-sensitive spectral fingerprint information of features and molecules. In addition, it can reduce photobleaching and peak overlapping, and is suitable for detection of biological samples due to the small Raman scattering cross section of water. Furthermore, it enables non-destructive testing in a variety of matrices without the need for complex sample preparation. These advantages make SERS technology one of the best spectroscopic techniques for bioanalytical applications (e.g., ATP). Acupuncture is a classic chinese medical practice that is inserted into an individual with minimal trauma. The acupuncture needle is an ideal platform for realizing rapid and nondestructive acquisition of samples and monitoring of SERS signals.

Here, we synthesized the compound Macrocyclic Amine (MA) as a Raman probe to achieve sensitive and selective detection with 1-2 orders of magnitude higher detection sensitivity than conventional methods. Wherein, the gold nanoparticles are functionalized by 3-ATPMS, and then surface modification is carried out by a Raman probe MA (Au NPs/MA) to obtain a strong SERS signal. Thus, the needle body surface-bound gold nanoparticles and raman probes can serve as a SERS-active platform, allowing for the enrichment and detection of the target molecule ATP from the mixture. The acupuncture SERS platform can avoid the aggregation of nano particles, and has the sensitivity and reliability of quick reaction and simple operation for detecting the biological molecule ATP. Most importantly, it opens up a rapidly growing market for SERS bioanalytical applications.

Disclosure of Invention

The invention aims to provide a preparation method of a functionalized SERS platform and application of the functionalized SERS platform to ATP detection.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the invention discloses a preparation method of a functionalized SERS platform, which comprises the following steps:

step 1, preparing a gold nanoparticle solution;

synthesizing gold nanoparticles by a method of reducing chloroauric acid by trisodium citrate;

step 2, modifying the acupuncture needle by gold nanoparticles:

(1) cleaning the acupuncture needle with ethanol, and soaking the acupuncture needle body into 10% (volume ratio) 3-mercaptopropyltriethoxysilane ethanol solution for 24 hours at 4 ℃;

(2) washing the acupuncture needle obtained in the step (1) with ethanol for 2-5 times and drying under nitrogen flow;

(3) immersing the needle body of the washed and dried acupuncture needle obtained in the step (2) into the gold nanoparticle solution obtained in the step (1);

step 3, functionalizing the acupuncture needle by the Raman probe:

soaking the acupuncture needle-shaped SERS substrate prepared in the step 2 in the solution with the concentration of 5 multiplied by 10 at room temperature^-52-3 parts of Raman probe solutionHours; after the treatment, the mixture was dried, washed with pure water and stored at room temperature.

Further, the size of gold nanoparticles in the gold nanoparticle solution prepared in the step 1 is 50 nm.

Further, the preparation method of the 50nm gold nanoparticle solution specifically comprises the following steps:

01. respectively adding 99mL of ultrapure water and 1mL of chloroauric acid solution with the mass fraction of 1% into a 250mL three-neck flask, heating by using a heating jacket, quickly adding 10mL of sodium citrate solution with the mass fraction of 1% while stirring when boiling, and continuously keeping boiling for 20 minutes;

02. adding 25mL of prepared gold nanoparticles into a 100mL three-neck flask to serve as a seed solution, and sequentially adding 1mL of 1% PVP solution and 20mL of 0.02% hydroxylamine hydrochloride solution at room temperature under stirring; adding 20mL of chloroauric acid solution with the mass fraction of 0.1% into the mixed solution at the speed of 1mL per minute by a microsyringe; after the addition is finished, stirring is continued for 30 minutes, and the gold nanoparticle solution with the particle size of 50nm is obtained.

Further, the preparation of the raman probe solution in step 3 comprises the following steps:

synthesizing S001, 1, 10-phenanthroline-2, 9-dicarboxaldehyde;

synthesizing S002, 1-alpha-phenyl-n-2, 5-diaza pentane;

s003, preparation of Raman label MA: dissolving 1-alpha-phenyl-n-2, 5-diazapene in a proper amount of ethanol at room temperature under stirring, and then adding 1, 10-phenanthroline-2, 9-diformaldehyde to dissolve under stirring; adding NaBH into ice water bath₄The solvent was rotary evaporated and 5mL water was added;

s004, reacting the aqueous mixture obtained in S003 with CH₂Cl₂Extracting, combining the organic phases and MgSO₄Drying;

s005, MgSO₄The dried mixture was filtered and CH was removed on a rotary evaporator₂Cl₂Obtaining yellow oil;

s006, dissolving the obtained yellow oil in a small amount of ethanol, adding a proper amount of HCl, and collecting light yellow precipitate.

Further, the synthesis of the 1, 10-phenanthroline-2, 9-dicarboxaldehyde comprises the following steps: selenium dioxide is added to a solution of 2, 9-dimethyl-1, 10-phenanthroline in 1, 4-dioxane at room temperature, and then the reaction mixture is heated under reflux at 90 ℃ for 3 hours; the color of the mixed solution changed to green-brown by orange and red; directly filtering the reaction solution after heating and refluxing for 3 hours to remove black precipitates, and slowly cooling the reaction solution to room temperature; forming new precipitate, filtering, washing with ether for 3-5 times, and vacuum drying to obtain yellow solid 1, 10-phenanthroline-2, 9-dicarboxaldehyde.

Further, the synthesis of the 1-alpha-phenyl-n-2, 5-diazapene comprises the following steps: adding a salicylaldehyde methanol solution dissolved with salicylaldehyde into an ethylenediamine methanol solution dissolved with ethylenediamine, stirring to form a mixed solution, and after 1 hour, adding NaBH₄Adding into the mixed solution; after stirring for 3 hours, all volatiles were removed under reduced pressure to give a white solid; the resulting white solid residue was dissolved in saturated NaHCO₃Solution extracted CH₂Cl₂In (1), the aqueous layer is replaced with CH₂Cl₂Extracting for 2-4 times, mixing the organic extracts, washing with salt water, and removing anhydrous MgSO₄Drying, and evaporating CH under reduced pressure₂Cl₂The obtained light yellow solid is 1-alpha-phenyl-n-2, 5-diazapene.

Further, in the preparation process of the S003 Raman label MA, the 1-alpha-phenyl-n-2, 5-diazapene, the 1, 10-phenanthroline-2, 9-dicarboxaldehyde and NaBH₄The dosage ratio of the three is 1-2: 1-2: 1-2.

Application of a functionalized SERS platform in ATP detection.

The invention has the following beneficial effects:

1. according to the invention, gold nanoparticle modification and Raman probe functionalization are sequentially carried out on the acupuncture needle, and the acupuncture needle is applied to ATP detection, so that the detection sensitivity is improved, and a stronger SERS signal is conveniently obtained.

2. The present invention can provide effective SERS enhancement by uniform gold nanoparticles assembled on the needle surface.

3. The functionalized needle obtained by the invention can quickly collect target molecules from a complex sample, can avoid the aggregation of gold nanoparticles with reliable and reproducible signals, and also improves the detection efficiency and has quick response.

4. According to the invention, the gold nanoparticles and the Raman probe are used for modifying the needle body, and the functionalized acupuncture needle can be used as an SERS active sensor for detecting various molecules related in a complex sample, so that the application range is greatly expanded.

Of course, it is not necessary for any product in which the invention is practiced to achieve all of the above-described advantages at the same time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of the synthesis of Raman probe MA;

FIG. 2 is a Scanning Electron Microscope (SEM) picture of gold nanoparticles Au NPs;

FIG. 3 is an SEM picture of the surface of an acupuncture needle functionalized by gold nanoparticle Au NPs;

FIG. 4 is a SERS spectrum of probe MA at a concentration ranging from 100. mu.M to 1. mu.M;

FIG. 5 is a SERS spectrum of a series of concentrations of ATP with 50 μ M MA as probe;

FIG. 6 is a spectrum of the functionalized acupuncture needle for the detection of ATP in serum.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The Au NPs mentioned in examples 1-4 are gold nanoparticles and the MA mentioned is a Raman probe.

Example 1

A preparation method of a functionalized SERS platform comprises the following steps:

step 1, preparing a gold nanoparticle solution;

step 2, modifying the acupuncture needle by gold nanoparticles:

(2) washing the acupuncture needle obtained in the step (1) with ethanol for 3 times and drying under nitrogen flow;

step 3, functionalizing the acupuncture needle by the Raman probe:

soaking the acupuncture needle-shaped SERS substrate prepared in the step 2 in the solution with the concentration of 5 multiplied by 10 at room temperature^-5Treating in a Raman probe solution for 2 hours at mol/L; after the treatment, the mixture was dried, washed with pure water and stored at room temperature.

Preferably, the gold nanoparticles in the gold nanoparticle solution prepared in step 1 have a size of 50 nm.

Preferably, the preparation method of the 50nm gold nanoparticle solution specifically comprises the following steps:

Preferably, the preparation of the raman probe solution in step 3 comprises the steps of:

synthesizing S001, 1, 10-phenanthroline-2, 9-dicarboxaldehyde;

synthesizing S002, 1-alpha-phenyl-n-2, 5-diaza pentane;

s003, preparation of Raman label MA: dissolving 0.08g of 1-alpha-phenyl-n-2, 5-diazapene in 5ml of ethanol at room temperature by stirring, and then adding 0.04g of 1, 10-phenanthroline-2, 9-dimethyl aldehyde by stirring for dissolution; 0.06g of NaBH was added to an ice-water bath₄The solvent was rotary evaporated and 5mL water was added;

s004, the aqueous mixture from S003 was treated with 25ml of CH₂Cl₂Extraction was performed 5 times with each CH₂Cl₂In an amount of 5ml, the organic phases are combined and MgSO₄Drying;

s005, the dried mixture of MgSO4 was filtered and CH was removed on a rotary evaporator₂Cl₂Obtaining yellow oil;

Preferably, the synthesis of 1, 10-phenanthroline-2, 9-dicarboxaldehyde comprises: 0.8007g of selenium dioxide is added to a solution of 0.35g of 2, 9-dimethyl-1, 10-phenanthroline in 25mL of 1, 4-dioxane at room temperature, and the reaction mixture is then heated at 90 ℃ under reflux for 3 hours; the color of the mixed solution changed to green-brown by orange and red; directly filtering the reaction solution after heating and refluxing for 3 hours to remove black precipitates, and slowly cooling the reaction solution to room temperature; a new precipitate was formed, which was then filtered off, washed 3-5 times with diethyl ether and dried in vacuo to give 0.2g of a yellow solid, 1, 10-phenanthroline-2, 9-dicarboxaldehyde.

Preferably, the synthesis of 1-alpha-phenyl-n-2, 5-diaza-pentane comprises: dissolving 0.42mL of salicylaldehyde in 30mL of methanol solution to form a salicylaldehyde methanol solution; dissolving 0.72mL of the aqueous solution in 10mL of methanol to form an ethylenediamine methanol solution, adding the salicylaldehyde methanol solution to the ethylenediamine methanol solution and stirring to form a mixed solution, and after 1 hour, adding 0.303g of NaBH₄Adding into the mixed solution; after stirring for 3 hours, all volatiles were removed under reduced pressure to give a white solid; the resulting white solid residue was dissolved in 130mL of saturated NaHCO₃65mL of CH extracted from solution₂Cl₂In (1), the aqueous layer is replaced with CH₂Cl₂Extraction was performed 3 times with each CH₂Cl₂The combined organic extracts were washed with 100ml brine and anhydrous MgSO 2₄Drying, and evaporating CH under reduced pressure₂Cl₂Obtaining light yellow solid which is 1-alpha-phenyl-n-2, 5-diazapene;

a specific synthetic route is shown in figure 1.

As shown in FIG. 2, the synthesized particles had a uniform particle size and an ultraviolet absorption wavelength of 520 nm.

As shown in fig. 3, it can be seen that the nanoparticles are uniformly decorated on the surface of the needle body.

Example 2

Acupuncture needle detection probe MA modified with Au NPs

Preparing 7 series of MA solutions with concentration ranging from 100umol/L to 1umol/L, respectively soaking 7 acupuncture needles modified by Au NPs in the solution with concentration of 7 in the embodiment 1 for 5min, taking out and drying, and testing under a Raman spectrometer to obtain a spectrum.

The concentration of 7 series of the extract is 1 × 10^-4mol/L、7.5×10^-5mol/L、5×10^-5mol/L、1×10^-5mol/L、7.5×10^-6mol/L、5×10^-6mol/L、1×10^-6mol/L；

As shown in fig. 4, the active probe showed high sensitivity and stable detection for MA detection.

Example 3

ATP detection by Au NPs and MA functionalized acupuncture needle

Preparing 7 series of ATP solutions with the concentration range of 25nM to 1000nM, respectively soaking 7 acupuncture needles modified by Au NPs and MA in 7 solutions with the concentration range of 7 in example 1 for 5min, taking out, drying, and testing under a Raman spectrometer to obtain spectrograms.

The concentration of 7 series of the extract is 2.5 × 10^-8mol/L、5.0×10^-8mol/L、7.5×10^-8mol/L、1.0×10^- ⁷mol/L、2.5×10^-7mol/L、5.0×10^-7mol/L、7.5×10^-7mol/L、1×10^-6mol/L；

As shown in fig. 5, a high SERS signal was exhibited by modifying the probe MA molecule on the SERS-active needle using electrostatic adsorption and Au — N bond; the addition of ATP can effectively inhibit the SERS signal of MA; the affinity between MA and ATP is stronger than the affinity between MA and the substrate, so addition of ATP keeps MA away from the substrate with complex formation, thereby reducing MA signal; the needle decorated with the raman probe not only enables sensitive and selective detection of ATP, but also provides a reliable SERS signal. Most importantly, it strongly simplifies indirectly the detection of a sample, especially for target molecules involved in complex samples.

Example 4

Detection of ATP in serum

Randomly collecting human serum samples from healthy volunteers, putting the acupuncture needle modified by Au NPs and MA in example 1 into a pure serum sample, soaking for 5min, taking out, drying and collecting an SERS spectrogram. Next, ATP was added to the serum in a series of concentrations, and the SERS signal of ATP was collected in the same manner.

As shown in fig. 6, when ATP (100nM) was incorporated into serum, the SERS signature of the MA probe showed a significant ATP signal response with decreasing raman intensity and band shift of the vibrational band. Due to the high chemical interaction between the target molecule ATP and the probe MA, the SERS reaction of ATP can effectively suppress the possibly interfering raman features of the serum sample.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A preparation method of a functionalized SERS platform is characterized by comprising the following steps:

step 1, preparing a gold nanoparticle solution;

step 2, modifying the acupuncture needle by gold nanoparticles:

(1) cleaning the acupuncture needle with ethanol, and immersing the needle body of the acupuncture needle into a 3-mercaptopropyltriethoxysilane ethanol solution with the volume ratio of 10% for 24 hours at 4 ℃;

step 3, functionalizing the acupuncture needle by the Raman probe:

soaking the acupuncture needle-shaped SERS substrate prepared in the step 2 in a Raman probe solution with the concentration of 5 multiplied by 10 < -5 > mol/L for 2-3 hours at room temperature; taking out and drying after treatment, washing with pure water and storing at room temperature;

wherein, the preparation of the Raman probe solution in the step 3 comprises the following steps:

synthesizing S001, 1, 10-phenanthroline-2, 9-dicarboxaldehyde;

synthesizing S002, 1-alpha-phenyl-n-2, 5-diaza pentane;

2. The method of claim 1, wherein the gold nanoparticles in the gold nanoparticle solution prepared in step 1 have a size of 50 nm.

3. The method for preparing a functionalized SERS platform according to claim 2, wherein the method for preparing the 50nm gold nanoparticle solution specifically comprises:

4. The method for preparing the functionalized SERS platform according to claim 3, wherein the synthesis of the 1, 10-phenanthroline-2, 9-dicarboxaldehyde comprises: selenium dioxide is added to a solution of 2, 9-dimethyl-1, 10-phenanthroline in 1, 4-dioxane at room temperature, and then the reaction mixture is heated under reflux at 90 ℃ for 3 hours; the color of the mixed solution changed to green-brown by orange and red; directly filtering the reaction solution after heating and refluxing for 3 hours to remove black precipitates, and slowly cooling the reaction solution to room temperature; forming new precipitate, filtering, washing with ether for 3-5 times, and vacuum drying to obtain yellow solid 1, 10-phenanthroline-2, 9-dicarboxaldehyde.

5. The method of claim 4, wherein the synthesis of 1- α -phenyl-n-2, 5-diazapene comprises: adding a salicylaldehyde methanol solution dissolved with salicylaldehyde into an ethylenediamine methanol solution dissolved with ethylenediamine, stirring to form a mixed solution, and adding NaBH4 into the mixed solution after 1 hour; after stirring for 3 hours, all volatiles were removed under reduced pressure to give a white solid; the resulting white solid residue was dissolved in saturated NaHCO₃Solution extracted CH₂Cl₂In (1), the aqueous layer is replaced with CH₂Cl₂Extracting for 2-4 times, combining the organic extracts, washing with brine and drying over anhydrous MgSO4, and evaporating CH under reduced pressure₂Cl₂The obtained light yellow solid is 1-alpha-phenyl-n-2, 5-diazapene.

6. The method of claim 4, wherein the 1- α -phenyl-n-2, 5-diazepane, 1, 10-phenanthroline-2, 9-dicarboxaldehyde and NaBH are used in the preparation of the S003 Raman tag MA₄Three ofThe dosage ratio is 1-2: 1-2: 1-2.

7. Use of a functionalized SERS platform prepared according to any of the methods of claims 1-6 for ATP detection.