A surface enhanced Raman scattering sensor and its preparation method and application

Abstract

The present invention provides a surface enhanced Raman scattering sensor and a preparation method and application thereof. The sensor is a three-layer structure, including an aluminum oxide nanoporous membrane, a gold layer composed of a gold nanoparticle layer and a nanogold membrane, and a metal organic framework layer. The sensor is not only convenient and fast to prepare, but also has a good enhancement effect. Under the functionalization of the metal organic framework layer, the detection sensitivity and anti-interference are improved. It is beneficial to the direct and rapid detection of serum samples without processing, and further combined with electrochemical reversible redox probe molecules, it is expected to realize the regeneration cycle detection of related substances.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, in particular to a surface enhanced Raman spectroscopy sensor. Background Art

[0002] As an ultra-sensitive molecular vibrational spectroscopy analysis technique, surface enhanced Raman spectroscopy (SERS) inherits the advantages of traditional Raman spectroscopy such as fast response, non-destructiveness, and no need for sample preparation. It has broad application prospects in the fields of biosensing, material characterization, and environmental analysis. However, because the characteristics of SERS are strongly dependent on the enhanced substrate, it has certain limitations, mainly including: the affinity between the enhanced substrate surface and the analyte; the enhancement effect of the substrate is affected by the detection environment; the selectivity of the substrate for the target analyte is limited; the reproducibility of the substrate is insufficient; the homogeneity and reproducibility of the SERS signal on the substrate, etc. Although the existing research can meet the requirements of SERS substrates in terms of sensitivity, reproducibility, and stability, there are still some limitations such as selectivity and reproducibility that need to be overcome in the detection of actual samples in complex systems.

[0003] Metal-organic frameworks (MOFs) are an emerging class of materials that consist of metal centers and specific coordination polymers and have a porous two-dimensional or three-dimensional crystalline network structure. Therefore, they are also called porous coordination clusters or organic linkers. Due to the flexibility of the structure, large surface area and adjustable pore size, MOFs are suitable for the functionalization of SERS sensors, which is beneficial to improve the sensing performance of the sensor.

[0004] Therefore, the development of an electrochemical metal-organic framework-functionalized surface-enhanced Raman spectroscopy sensor will help to give full play to the technical advantages of surface-enhanced Raman spectroscopy, overcome the technical deficiencies of existing detection methods, and quickly, sensitively and selectively detect target analytes in complex systems such as serum. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a surface enhanced Raman spectroscopy sensor, which is not only easy and fast to prepare but also has a good enhancement effect. Under the functionalization of the metal organic framework layer, the detection sensitivity and anti-interference ability are improved, which is beneficial to the direct and rapid detection of serum samples without processing. It is further combined with electrochemical reversible redox probe molecules and is expected to achieve regenerative cyclic detection of related substances.

[0006] The second object of the present invention is to provide a renewable metal organic framework functionalized surface enhanced Raman spectroscopy sensor based on surface enhanced Raman spectroscopy effect combined with electrochemical regulation technology, which can effectively improve detection efficiency and has strong anti-interference ability.

[0007] The third object of the present invention is to provide an application of a surface enhanced Raman spectroscopy sensor.

[0008] In order to achieve the above object, the present invention provides a method for preparing a surface enhanced Raman spectroscopy sensor, wherein the sensor has a three-layer structure, wherein the bottom layer is an aluminum oxide nanoporous membrane, the middle layer is a gold layer composed of a gold nanoparticle layer and a nanogold membrane, and the top layer is a metal organic framework layer. The sensor is prepared by the following method:

[0009] (1) Preparation of nano-gold film: a clean anodic aluminum oxide (AAO) nanoporous film is placed in a magnetron sputtering coating apparatus to sputter a layer of gold film;

[0010] (2) preparing a gold nanoparticle layer: electrochemical deposition is then performed in a chloroauric acid solution containing sodium perchlorate, the deposited substrate is washed with deionized water, and dried with nitrogen gas;

[0011] (3) Preparing a metal organic framework layer: placing the substrate obtained in step (2) in a precursor solution for electrochemical deposition of a metal organic framework (MOFs) layer, soaking the prepared metal organic framework functionalized sensor in deionized water for aging overnight, then washing with a 50% ethanol solution, and drying with nitrogen gas;

[0012] (4) Raman signal molecule modification: A thiol molecule with Raman activity is used as a Raman signal molecule, and the exposed surface of the untreated aluminum oxide film is modified on the sensor to assemble a metal-organic framework functionalized surface-enhanced Raman spectroscopy sensor.

[0013] As a preferred solution, the nano-gold film has a thickness of 20-60 nm, is prepared by a magnetron sputtering coating apparatus at a current of 20 mA, and has good electrical conductivity.

[0014] As a preferred embodiment, the chloroauric acid solution in step (2) contains 0.01-1M sodium perchlorate and 0.1-10mM chloroauric acid.

[0015] As a preferred embodiment, the electrochemical deposition in step (2) is a two-step process, firstly applying a constant potential of -1.0 to 0 V to the electrode for 10 to 100 s, and then using scanning cyclic voltammetry to scan 10 to 300 cycles at a rate of 10 to 100 mv / s in the potential range of -0.03 to 0.4 V.

[0016] As a preferred embodiment, the precursor solution of step (3) is prepared by the following method: 1.5 mmol zinc nitrate and 0.5 mmol sodium nitrate are dissolved in 5 mL of deionized water to prepare solution I, 0.833 mmol trimesic acid and 0.9 mmol hexadecyltrimethylammonium bromide are dissolved in 5 mL of ethanol to prepare solution II, solution II is added to solution I with stirring, nitrogen is introduced into the solution to deoxygenate, and a metal organic framework precursor solution is prepared.

[0017] As a preferred embodiment, the electrochemical deposition in step (3) refers to applying a constant current of 1.0-2.0 mA to the anodized aluminum substrate for 0.5-3 hours.

[0018] In order to achieve the second objective, the present invention provides a surface enhanced Raman spectroscopy sensor prepared by the above preparation method, which can not only detect target analytes in complex systems such as serum quickly, sensitively and selectively, but also can realize the recycling of the sensor under the regulation of electrochemical constant potential.

[0019] In order to achieve the third objective, the present invention provides the use of a surface enhanced Raman spectroscopy sensor in detecting alkaline phosphatase in serum, wherein the Raman signal molecule used is 2-mercaptobenzoquinone which is sensitive to ascorbic acid.

[0020] The invention provides application of a surface enhanced Raman spectroscopy sensor in detecting glucose in serum, wherein the used Raman signal molecule is 2-mercaptohydroquinone which is sensitive to hydrogen peroxide.

[0021] The advantages of the present invention are that the sensor preparation process is simple, no expensive instruments are required, and the cost is low. The prepared sensor has strong anti-interference ability, which is conducive to direct detection of samples such as serum without pretreatment; it can not only quickly, sensitively and selectively detect target analytes in complex systems such as serum, but also realize the recycling of the sensor under the regulation of electrochemical constant potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a structural schematic diagram of the present invention.

[0023] Figure 2 This is a scanning electron microscope image of the alumina substrate after sputtering a gold film in Example 1.

[0024] Figure 3 This is a scanning electron microscope image of the gold nanoparticle layer on the alumina substrate of Example 1.

[0025] Figure 4 This is a scanning electron microscope image of the metal organic framework film on the sensor of Example 1.

[0026] Figure 5This is the surface enhanced Raman spectroscopy fingerprint of the sensor in Example 1 before responding to alkaline phosphatase.

[0027] Figure 6 This is the surface enhanced Raman spectroscopy fingerprint of the sensor in Example 1 after responding to alkaline phosphatase.

[0028] Figure 7 This is a standard curve of the relative intensity of the characteristic peak of the surface enhanced Raman spectroscopy fingerprint of the sensor in Example 1 in response to alkaline phosphatase - the logarithm of the alkaline phosphatase activity.

[0029] Figure 8 This is the surface enhanced Raman spectroscopy fingerprint of the sensor in Example 1 in response to alkaline phosphatase in serum.

[0030] Fig. 9 This is a scanning electron microscope image of the metal organic framework film on the sensor of Example 2.

[0031] Fig.10 Surface enhanced Raman spectroscopy fingerprint of the sensor in Example 2 before detecting glucose

[0032] Fig.11 This is the surface enhanced Raman spectroscopy fingerprint of the sensor in Example 2 after detecting glucose

[0033] Fig.12 This is a logarithmic standard curve of relative intensity of characteristic peaks of the surface enhanced Raman spectroscopy fingerprint of glucose detected by the sensor of Example 2 - glucose concentration. DETAILED DESCRIPTION

[0034] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following specific embodiments are only used to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0035] Example 1

[0036] (1) Preparation of gold nanofilm

[0037] A clean piece of anodized aluminum oxide (AAO) nanoporous membrane was placed in a magnetron sputtering coating apparatus, the current was set to 20 mA, and a 40 nm thick gold film was coated as a conductive layer.

[0038] (2) Preparation of gold nanoparticle layer

[0039] Subsequently, gold nanoparticles were deposited in a two-step method in a three-electrode system consisting of a working electrode (AAO), an Ag / AgCl reference electrode, and a platinum wire counter electrode in a solution containing 0.1 M sodium perchlorate and 1 mM chloroauric acid. The deposition was first performed at an overpotential of -0.8 V for 10 s, followed by cyclic voltammetry in the potential range of -0.03 to 0.4 V at a rate of 50 mV / s for 80 scans, and finally the substrate was washed with deionized water and dried with a nitrogen stream.

[0040] (3) Preparation of metal organic framework layer

[0041] 1.5mmol zinc nitrate and 0.1mmol sodium nitrate were dissolved in 5ml deionized water to prepare a mixture of solution I, and 0.833mmol trimesic acid and 0.9mmol hexadecyltrimethylammonium bromide were dissolved in 5ml ethanol to prepare solution II. Solution II was added to solution I under vigorous stirring, the pH value was adjusted to 2.1, stirred for 2.5 hours, and nitrogen was introduced for 10 minutes to prepare a metal organic framework precursor solution.

[0042] The substrate obtained in step (2) was used as a working electrode, and a three-electrode system consisting of an Ag / AgCl reference electrode and a platinum wire was applied in the precursor solution at a constant current of 1.2 mA for 2 hours to prepare the metal organic framework. The substrate with the metal organic framework film deposited was then placed in deionized water and allowed to stand for 12 hours, then washed with a 50% ethanol solution for 10 minutes and dried with nitrogen.

[0043] (4) Raman signal molecule modification

[0044] Ascorbic acid-sensitive 2-mercaptobenzoquinone was used as a Raman signal molecule, which was prepared into a 100 μM solution, and 60 μL was dropped on the bare surface of the anodized aluminum substrate without any treatment for modification.

[0045] (5) Establishment of standard curve for alkaline phosphatase activity detection

[0046] Alkaline phosphatases of different activities were added to a Tris-HCl buffer containing 2 mM L-ascorbic acid-2-trisodium phosphate, and incubated at 37°C for 30 minutes. Subsequently, 60 μL of the incubated buffer was dripped onto the sensor obtained in step (4) and reacted for 1 minute. After being taken out, the sensor was subjected to surface Raman spectrum detection using a portable Raman spectrometer to obtain its surface enhanced Raman spectrum fingerprint for qualitative analysis, and a standard curve of the relative intensity of the characteristic peak-logarithm of the alkaline phosphatase activity was established for quantitative analysis.

[0047] (6) Obtaining the surface enhanced Raman spectrum of the sensor after responding to alkaline phosphatase in serum

[0048] Different alkaline phosphatases were added to fetal bovine serum containing 2 mM L-ascorbic acid-2-trisodium phosphate and incubated at 37°C for 30 minutes. Subsequently, 60 μL of the incubated buffer was dripped onto the sensor obtained in step (4) and reacted for 1 minute. After that, the sensor was washed with deionized water and the surface Raman spectrum of the sensor was detected using a portable Raman spectrometer.

[0049] (7) Comparison and analysis

[0050] The surface enhanced Raman spectrum obtained in step (6) is compared with the surface enhanced Raman spectrum fingerprint obtained in step (5) and the standard curve of the logarithm of the characteristic peak relative intensity-alkaline phosphatase activity to determine that the content of alkaline phosphatase in the serum is 100 U / L.

[0051] Example 2

[0052] (1) Preparation of gold nanofilm

[0053] Take a clean anodized aluminum oxide (AAO) nanoporous membrane and place it in a magnetron sputtering coating apparatus, set the current to 20 mA, and coat it with a 30 nm thick gold film as a conductive layer.

[0054] (2) Preparation of gold nanoparticle layer

[0055] Then, gold nanoparticles were deposited in a two-step method in a three-electrode system consisting of a working electrode (AAO), an Ag / AgCl reference electrode, and a platinum wire counter electrode in a solution containing 0.1 M sodium perchlorate and 1 mM chloroauric acid. The deposition was first performed at an overpotential of -0.8 V for 50 s, followed by cyclic voltammetry in the potential range of -0.03 to 0.4 V at a rate of 50 mV / s for 90 times, and finally the substrate was washed with deionized water and dried with a nitrogen stream.

[0056] (3) Preparation of metal organic framework layer

[0057] 1.5mmol zinc nitrate and 0.1mmol sodium nitrate were dissolved in 5ml deionized water to prepare a mixture of solution I, and 0.833mmol trimesic acid and 0.9mmol hexadecyltrimethylammonium bromide were dissolved in 5ml ethanol to prepare solution II. Solution II was added to solution I under vigorous stirring, the pH value was adjusted to 2.1, stirred for 2.5 hours, and nitrogen was introduced for 10 minutes to prepare a metal organic framework precursor solution.

[0058] The substrate obtained in step (2) was used as a working electrode, and a three-electrode system consisting of an Ag / AgCl reference electrode and a platinum wire was applied in the precursor solution at a constant current of 1.4 mA for 1.5 hours to prepare the metal organic framework. The substrate with the metal organic framework film deposited was then placed in deionized water and allowed to stand for 12 hours, then washed with a 50% ethanol solution for 10 minutes and dried with nitrogen.

[0059] (4) Raman signal molecule modification

[0060] 2-Mercaptohydroquinone, which is sensitive to hydrogen peroxide, was used as a Raman signal molecule. A 100 μM solution was prepared and 60 μL was dropped onto the bare surface of the anodized aluminum substrate without any treatment for modification.

[0061] (5) Establishment of glucose detection standard curve

[0062] Different concentrations of glucose (0-10 mM) were mixed with 500 U / L glucose oxidase and incubated for 30 minutes. Subsequently, 60 μL of the incubated buffer was dripped onto the sensor obtained in step (4) and reacted for 5 minutes. After being taken out, the sensor was tested for surface Raman spectrum using a portable Raman spectrometer to obtain its surface enhanced Raman spectrum fingerprint for qualitative analysis, and a standard curve of characteristic peak relative intensity-glucose activity logarithm was established for quantitative analysis.

[0063] (6) Obtaining the surface enhanced Raman spectrum after the sensor detects glucose in serum

[0064] Different concentrations of glucose and 500 U / L of glucose oxidase were added to fetal bovine serum and incubated at 37°C for 30 minutes. Subsequently, 60 μL of the incubated buffer was dripped onto the sensor obtained in step (4) and reacted for 1 minute. After that, the sensor was washed with deionized water and the surface Raman spectrum of the sensor was detected using a portable Raman spectrometer.

[0065] (7) Comparison and analysis

[0066] The surface enhanced Raman spectrum obtained in step (6) was compared with the surface enhanced Raman spectrum fingerprint obtained in step (5) and the standard curve of the logarithm of the characteristic peak relative intensity-glucose activity to determine the glucose content in the serum to be 1038.37 and 3061.26 μM.

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a surface enhanced Raman spectroscopy sensor, characterized in that: The sensor has a three-layer structure, the bottom layer is an aluminum oxide nanoporous membrane, the middle layer is a gold layer composed of a gold nanoparticle layer and a nanogold membrane, and the top layer is a metal organic framework layer. The sensor is prepared by the following method: (1) Preparation of nano-gold film: a clean anodized aluminum oxide nanoporous film is placed in a magnetron sputtering coating apparatus to sputter a layer of gold film; (2) preparing a gold nanoparticle layer: electrochemical deposition is then performed in a chloroauric acid solution containing sodium perchlorate, the deposited substrate is washed with deionized water, and dried with nitrogen gas; (3) Preparing a metal organic framework layer: placing the substrate obtained in step (2) in a precursor solution for electrochemical deposition of a metal organic framework layer, soaking the prepared metal organic framework functionalized sensor in deionized water for aging overnight, then washing with a 50% ethanol solution, and drying with nitrogen gas; (4) Raman signal molecule modification: A thiol molecule with Raman activity is used as a Raman signal molecule, and the exposed surface of the untreated aluminum oxide film is modified on the sensor to assemble a metal-organic framework functionalized surface-enhanced Raman spectroscopy sensor.

2. The method for preparing a surface enhanced Raman spectroscopy sensor according to claim 1, characterized in that: The thickness of the nano-gold film is 20-60nm.

3. The method for preparing a surface enhanced Raman spectroscopy sensor according to claim 1, characterized in that: The chloroauric acid solution in step (2) contains 0.01-1M sodium perchlorate and 0.1-10mM chloroauric acid.

4. The method for preparing a surface enhanced Raman spectroscopy sensor according to claim 1, characterized in that: The electrochemical deposition in step (2) is a two-step process, firstly applying a constant potential of -1.0 to 0 V to the electrode for 10 to 100 seconds, and then using scanning cyclic voltammetry to scan 10 to 300 cycles at a rate of 10 to 100 mv / s in the potential range of -0.03 to 0.4 V.

5. The method for preparing a surface enhanced Raman spectroscopy sensor according to claim 1, characterized in that: The precursor solution of step (3) is prepared by the following method: 1.5 mmol zinc nitrate and 0.5 mmol sodium nitrate are dissolved in 5 mL of deionized water to prepare solution I, 0.833 mmol trimesic acid and 0.9 mmol hexadecyltrimethylammonium bromide are dissolved in 5 mL of ethanol to prepare solution II, solution II is added to solution I with stirring, nitrogen is introduced into the solution to deoxygenate, and a metal organic framework precursor solution is prepared.

6. The method for preparing a surface enhanced Raman spectroscopy sensor according to claim 1, characterized in that: The electrochemical deposition described in step (3) refers to applying a constant current of 1.0-2.0 mA on the anodized aluminum substrate for 0.5-3 hours.

7. A surface enhanced Raman spectroscopy sensor prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the surface enhanced Raman spectroscopy sensor according to claim 7 in detecting alkaline phosphatase in serum, characterized in that: The Raman signal molecule used was 2-mercaptobenzoquinone which is sensitive to ascorbic acid.

9. Use of the surface enhanced Raman spectroscopy sensor according to claim 7 in detecting glucose in serum, characterized in that: The Raman signal molecule used was 2-mercaptohydroquinone, which is sensitive to hydrogen peroxide.

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