CN114199958A

CN114199958A - Super-assembly sensing platform for label-free specific detection and preparation and detection methods thereof

Info

Publication number: CN114199958A
Application number: CN202111429128.6A
Authority: CN
Inventors: 孔彪; 曾晖; 周姗; 何彦君; 曾洁
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-11-28
Filing date: 2021-11-28
Publication date: 2022-03-18

Abstract

The invention relates to a super-assembly sensing platform for label-free specific detection and a preparation and detection method thereof, wherein the preparation method comprises the following steps: firstly, an ultrathin and ordered mesoporous silica layer is constructed on the surface of AAO by utilizing a super-assembly method to serve as an ion selective layer, then a three-step modification strategy is adopted, APTES is utilized to carry out amination modification on the mesoporous silica layer, glutaraldehyde is utilized to serve as a connecting agent, amino and aldehyde group covalent reactions are utilized, tyramine is modified on the mesoporous silica layer, and therefore the final Tyr-MS/AAO can be obtained, the surface of the Tyr-MS/AAO contains abundant phenolic hydroxyl groups, and abundant functional groups are provided for follow-up intelligent sensing analysis application. Compared with the prior art, the heterojunction nanochannel prepared by the invention has the detection limit of 2 U.ml for detecting tyrosinase^‑1With other detection partiesCompared with the method, the heterojunction nano-channel has the advantages of short time consumption, simple detection process and the like, and is beneficial to the application of the heterojunction nano-channel in the aspect of sensing analysis.

Description

Super-assembly sensing platform for label-free specific detection and preparation and detection methods thereof

Technical Field

The invention belongs to the technical field of nano ion channels, and relates to a super-assembly sensing platform for label-free specificity detection and a preparation and detection method thereof.

Background

In recent years, artificial nanochannels are widely researched in the aspect of constructing nanofluid sensing devices, and due to the advantages of controllable size and surface function, the bionic solid-state nano ion channels have great potential in the fields of sensing, ultrafiltration, desalination, energy conversion and the like.

However, current research is always faced with some problems, such as how to make nanochannels intelligent by decoration is still a challenge. In the face of the current existing bottleneck, the development of a preparation method and technology of a functionalized nanoparticle channel material is urgently needed.

Tyrosinase is a multifunctional copper-containing enzyme, widely distributed in plants, animals and microorganisms. Tyrosinase can catalyze monophenol hydroxylation reaction to generate catechol, is a key enzyme in the biosynthesis pathway of endogenous melanin, and is also a factor influencing the appearance, taste and nutritional value of vegetables and fruits. Therefore, more accurate methods of monitoring tyrosinase are of great importance in both clinical diagnostics and the food industry. In recent years, there have been various methods for detecting tyrosinase, such as colorimetry, fluorescence, and electrochemical methods. Current methods typically use fluorescently/chromogenically labeled substrates to demonstrate enzyme activity, which is expensive and time consuming. Therefore, there is a need to develop a simple, low cost, label-free, high performance sensor platform for detecting tyrosinase activity.

Disclosure of Invention

The invention aims to provide a simple and high-performance super-assembly sensing platform for label-free specific detection and a preparation and detection method thereof.

The purpose of the invention can be realized by the following technical scheme:

a preparation method of a super-assembly sensing platform for label-free specific detection comprises the following steps:

m1: oxidizing mesoporous silicon oxide/anodeSoaking the aluminum (MS/AAO) heterojunction nano-channel in 3-Aminopropyltriethoxysilane (APTES) solution, taking out and performing heat treatment to obtain an amination-modified MS/AAO heterojunction nano-channel, which is marked as NH₂-MS/AAO；

M2: reacting NH₂Placing the MS/AAO in a glutaraldehyde solution for dark dipping, and taking out to obtain a glutaraldehyde modified MS/AAO heterojunction nano-channel which is marked as Glu-MS/AAO;

m3: and (3) placing Glu-MS/AAO in tyramine solution to be dipped in dark place, and taking out to obtain a tyramine modified MS/AAO heterojunction nano-channel which is marked as Tyr-MS/AAO, namely a super-assembly sensing platform for label-free specific detection.

Further, in the step M1, the concentration of the 3-aminopropyltriethoxysilane solution is 5-10 wt%, the dosage of the 3-aminopropyltriethoxysilane solution is preferably excessive relative to the dosage of MS/AAO, the dipping temperature is 40-50 ℃, and the dipping time is 12-14 h;

in the heat treatment process, the treatment temperature is 100-120 ℃, and the treatment time is 1-2 h.

Further, in step M2, the concentration of the glutaraldehyde solution is 2 to 10 wt% with respect to NH₂The amount of MS/AAO is preferably in excess, the impregnation temperature is room temperature and the impregnation time is from 5 to 7 h.

Further, in the step M3, the tyramine solution has a concentration of 10-50 μ M, and is preferably used in an excess amount relative to the amount of Glu-MS/AAO, and the soaking temperature is room temperature and the soaking time is 5-7 h.

A super-assembly sensing platform for label-free specific detection is prepared by the method.

An application based on the super-assembly sensing platform comprises that the super-assembly sensing platform is provided with a functionalized and modified ion selective layer and a phenolic hydroxyl group, so that the super-assembly sensing platform can be used in the field of sensing analysis.

A tyrosinase specificity detection method based on the super-assembly sensing platform comprises the following steps:

n1: dropwise adding a solution to be detected containing tyrosinase on Tyr-MS/AAO, standing, and washing to obtain Tyr-MS/AAO catalyzed by TYR, wherein the Tyr-MS/AAO + TYR is marked as Tyr-MS/AAO;

n2: soaking Tyr-MS/AAO + TYR in a protective solution, and taking out to obtain Tyr-MS/AAO + TYR with stable bisphenol hydroxyl;

n3: placing Tyr-MS/AAO + TYR with stable bisphenol hydroxyl in a connecting channel between an anode pool and a cathode pool of an electrochemical testing device, enabling the Tyr-MS/AAO layer to face to the anode side, and measuring the current change of the Tyr-MS/AAO before catalysis of tyrosinase;

n4: and D, acquiring tyrosinase concentration information in the solution to be detected according to the current change obtained in the step N3 and the current change/tyrosinase concentration standard curve.

Further, in step N1, the concentration of tyrosinase in the solution to be tested is 2-50U/mL, and the dosage is 100-² Tyr-MS/AAO；

And in the standing process, the standing temperature is room temperature, and the standing time is 20-40 min.

Further, in step N2, the protection solution is 5-15mM ascorbic acid solution, and the dosage of the ascorbic acid solution is preferably excessive relative to Tyr-MS/AAO + TYR;

in the dipping process, the dipping temperature is room temperature, and the dipping time is 10-20 min.

Further, in step N3, the electrochemical testing device includes an anode cell and a cathode cell that are arranged in parallel, a connecting channel for communicating the anode cell and the cathode cell, an anode electrode disposed in the anode cell, a cathode electrode disposed in the cathode cell, and a power supply and a picometer disposed between the anode electrode and the cathode electrode;

the anolyte in the anode pool is 10^-60.1mol/L potassium chloride solution, the catholyte in the cathode cell is 10^-6-0.1mol/L potassium chloride solution; the anode electrode and the cathode electrode are both Ag/AgCl electrodes; the test voltage was-2V to 2V.

Further, in step N4, the method for drawing the current variation/tyrosinase concentration standard curve includes:

preparing a plurality of tyrosinase solutions with different concentrations as solutions to be detected, obtaining corresponding current changes by adopting the method of the same step N1-N3, and then respectively drawing by using the tyrosinase concentration and the current changes as horizontal and vertical coordinates to obtain a current change/tyrosinase concentration standard curve.

Changes in surface charge density and wettability are major factors affecting changes in ionic current. In the invention, under the catalytic action of TYR, Tyr is converted from phenolic hydroxyl to an o-diphenol group, so that the hydrophilicity and the surface charge density of a heterogeneous channel are increased, the ion transmission behavior is adjusted, higher ion current is caused, and the phenomenon of current increase in Tyr-MS/AAO + TYR is generated. Meanwhile, different amounts of phenolic hydroxyl groups can be catalyzed by TYR with different concentrations to be converted into o-diphenol groups, so that different ion current changes are caused, and further the quantitative detection of TRY is realized.

Compared with the prior art, the invention has the following characteristics:

1) the invention constructs an ultrathin and ordered mesoporous silicon oxide layer on the surface of AAO as an ion selective layer by using an interface super-assembly method, adopts a three-step modification strategy, utilizes 3-Aminopropyltriethoxysilane (APTES) to carry out amination modification on the mesoporous silicon oxide layer, utilizes glutaraldehyde as a connecting agent, and modifies tyramine on the mesoporous silicon oxide layer through amino-aldehyde covalent reaction to finally obtain a heterojunction nano-channel Tyr-MS/AAO with ordered ion transfer channels and high channel density, wherein the heterojunction nano-channel Tyr-MS/AAO contains better ion selectivity and phenolic hydroxyl groups with rich surfaces, thereby providing a new material and considerable thinking for an intelligent nano-fluidic nano-channel device in the field of sensing analysis and having application prospects;

2) the invention prepares tyramine modified mesoporous silicon oxide/anodic aluminum oxide heterojunction nano-channel by a super-assembly method, the surface charge density of an ion selective layer of the system, namely a Tyr-MS layer, is increased after being catalyzed by tyrosinase, and the current signal in the nano-channel is increased along with the increase of the current signal, thereby realizing the high sensitivity and selectivity detection of the tyrosinase, and experiments show that the detection limit of the heterojunction nano-channel reaches 2 U.ml when the heterojunction nano-channel is used for the detection of the tyrosinase^-1Compared with other detection methods, the heterojunction nano-channel has the advantages of short time consumption, simple detection process and the like, and has better reference value for the application of the intelligent nano-fluidic nano-channel device in the field of sensing analysis.

Drawings

FIG. 1 is a flow chart of the preparation process of tyramine-modified mesoporous silica/anodized alumina heterojunction nanochannel (Tyr-MS/AAO) in example 1;

FIG. 2 is a Transmission Electron Microscope (TEM) image of Tyr-MS/AAO in example 1;

FIG. 3 is a Scanning Electron Microscope (SEM) image of the section (a) and surface (b) of Tyr-MS/AAO in example 1;

FIG. 4 is a nitrogen adsorption desorption curve (a) and a pore size distribution curve (b) of Tyr-MS/AAO in example 1;

FIG. 5 shows MS/AAO, NH in example 1₂-an X-ray photoelectron spectrum of MS/AAO, Tyr-MS/AAO;

FIG. 6 is a graph showing the contact angle measurements of MS/AAO, Glu-MS/AAO and Tyr-MS/AAO in example 1;

FIG. 7 is a diagram showing the detection mechanism of Tyr-MS/AAO;

FIG. 8 is a schematic structural view of an electrochemical test device;

FIG. 9 is a TYR concentration/current change standard curve of example 2;

FIG. 10 is a Zeta potential diagram of heterojunction nanochannels before and after TYR catalysis;

FIG. 11 is a contact angle test chart of heterojunction nanochannels before and after TYR catalysis;

FIG. 12 is a test chart of the specificity recognition performance of Tyr-MS/AAO for tyrosinase;

the notation in the figure is:

1-anode pool, 2-cathode pool, 3-anode electrode, 4-cathode electrode, 5-power supply and 6-picoammeter.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

m1: soaking the MS/AAO heterojunction nano-channel in excessive 5-10 wt% 3-aminopropyl triethoxy silicon (APTES) alkyl solution at 40-50 deg.C for 12-14h, taking out, washing, and heat treating at 100-120 deg.C for 1-2h to obtain aminated modified MS/AAO heterojunction nanochannel, denoted as NH₂-MS/AAO；

M2: reacting NH₂Placing the MS/AAO in an excessive 2-10 wt% glutaraldehyde solution, soaking for 5-7h at room temperature in a dark place, taking out and washing to obtain a glutaraldehyde modified MS/AAO heterojunction nanochannel, and marking as Glu-MS/AAO;

m3: and (3) placing Glu-MS/AAO in an excessive 10-50 mu M tyramine solution, soaking for 5-7h at room temperature in a dark place, taking out and washing to obtain a tyramine modified MS/AAO heterojunction nano-channel, which is marked as Tyr-MS/AAO, namely a super-assembly sensing platform for label-free specific detection.

A super-assembled sensing platform for label-free specific detection is prepared by the method, and preferably, the heterojunction nanochannel comprises a one-dimensional anodic alumina nanochannel with the thickness of about 60 mu m and the average pore size of about 20nm and a tyramine-modified mesoporous silica layer with the thickness of about 130-150nm and the average pore size of about 5-6 nm.

n1: adopting tyrosinase solution to be detected with the concentration range of 2-50U/mL, dripping 500 mu L of the solution to be detected with the volume of 100-; (ii) a

N2: soaking Tyr-MS/AAO + TYR in 5-15mM ascorbic acid solution at room temperature for 10-20min, and taking out to obtain Tyr-MS/AAO + TYR with stable bisphenol hydroxyl; wherein the amount of the ascorbic acid solution is preferably in excess relative to Tyr-MS/AAO + TYR; (ii) a

N3: placing Tyr-MS/AAO + TYR with stable bisphenol hydroxyl in a connecting channel between an anode pool 1 and a cathode pool 2 of an electrochemical testing device, enabling the Tyr-MS/AAO layer to face to one side of an anode, and measuring the current change of the Tyr-MS/AAO before catalysis of tyrosinase;

the electrochemical testing device is shown in fig. 8 and comprises an anode pool 1 and a cathode pool 2 which are arranged in parallel, a connecting channel for communicating the anode pool 1 with the cathode pool 2, an anode electrode 3 arranged in the anode pool 1, a cathode electrode 4 arranged in the cathode pool 2, a power supply 5 and a picoammeter 6 which are arranged between the anode electrode 3 and the cathode electrode 4;

the anolyte in the anode tank 1 is 10^-60.1mol/L potassium chloride solution, the catholyte in the cathode cell 2 is 10^-6-0.1mol/L potassium chloride solution; the anode electrode 3 and the cathode electrode 4 are both Ag/AgCl electrodes; the picometer 6 is a Keithley 6487 picometer, and the test voltage is-2V to 2V;

The method for drawing the current change/tyrosinase concentration standard curve comprises the following steps: preparing a plurality of tyrosinase solutions with different concentrations as solutions to be detected, obtaining corresponding current changes by adopting the method of the same step N1-N3, and then respectively drawing by using the tyrosinase concentration and the current changes as horizontal and vertical coordinates to obtain a current change/tyrosinase concentration standard curve.

The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.

Example 1:

a tyramine modified mesoporous silica/anodic alumina heterojunction nano-channel (Tyr-MS/AAO) is prepared by the following steps as shown in figure 1:

1) a piece of MS/AAO heterojunction nanochannel (membrane) with a diameter of 15mm was prepared in a similar way as in example 1 of CN202010640001.8, except that in step 2, the time for spin-coating the silicon precursor solution was 60 s;

2) placing MS/AAO in 30mL 5 wt% 3-Aminopropyltriethoxysilane (APTES) ethanol solution, soaking at 45 deg.C for 12h, taking out, washing with ethanol and deionized water for 3 times, removing unreacted APTES, and further washing at 120 deg.CHeating for 2h to stabilize the action of surface silane and MS/AAO nano-channel and obtain amination-modified MS/AAO heterojunction nano-channel (NH)₂-MS/AAO)；

3) Reacting NH₂Placing the MS/AAO in 50mL of 2.5 wt% glutaraldehyde aqueous solution, soaking for 6h in the dark at room temperature, taking out, washing for 3 times by using deionized water, and removing redundant glutaraldehyde on the surface to obtain a glutaraldehyde-modified MS/AAO heterojunction nano-channel (Glu-MS/AAO);

4) and (3) placing Glu-MS/AAO into 100mL of 25 mu M tyramine ethanol solution, soaking for 6h at room temperature in a dark place, taking out, washing for 3 times by using ethanol and deionized water respectively, and removing unreacted tyramine to obtain the Tyr-MS/AAO heterojunction nano-channel.

As shown in FIG. 2, which is a TEM image of Tyr-MS/AAO, it can be seen that the prepared mesoporous silicon has a regular pore structure and a high pore density.

As shown in FIG. 3, the SEM images of the section (a) and the surface (b) of the Tyr-MS/AAO show that the Tyr-MS/AAO has a regular pore structure, and the modified mesoporous silicon is tightly arranged on the upper layer of the AAO, and the thickness is about 150 nm.

As shown in FIG. 4, which shows the nitrogen adsorption and desorption curve (a) and the pore size distribution curve (b) of Tyr-MS/AAO, it can be seen that Tyr-MS/AAO has a high specific surface area and an average pore size of about 6.1 nm.

FIG. 5 shows MS/AAO, NH₂The X-ray photoelectron spectra of MS/AAO, Tyr-MS/AAO, from which it can be seen that MS/AAO shows a typical N1s peak (3.1%) after APTES modification, which was not observed before amino functionalization; and after the Tyr is modified, the content of N1s on the surface of the nano channel is increased to 5.2 percent, and more nitrogen content is introduced due to the modification of the Tyr.

As shown in FIG. 6, which is a contact angle test chart of MS/AAO, Glu-MS/AAO and Tyr-MS/AAO, it can be seen that the wettability of the heterogeneous channel changes after each modification. For MS/AAO, the water contact angle is only 6.1 + -1.8 deg. due to the rich silicon hydroxyl groups of the MS layer. After modification with glutaraldehyde, the contact angle increased to 45.0 ± 2.8 °, with more hydrophobic surface due to the introduction of glutaraldehyde aldehyde groups. After modification of the MS/AAO with Tyr, the contact angle reached 56.9. + -. 2.6. this is due to the phenyl group in the tyramine molecule.

These results indicate that this example successfully modified tyramine to MS/AAO.

Example 2:

a Tyrosinase (TYR) concentration detection method of Tyr-MS/AAO prepared based on example 1 comprises the following steps:

1) and (3) drawing a TYR concentration/current change standard curve:

1-1) dripping 200 mu L of 2-50U/mL tyrosinase solution on a piece of Tyr-MS/AAO heterojunction nano-channel (circular membrane) with the diameter of 15mm, standing and soaking for 30min at room temperature to finish catalytic reaction on tyramine (as shown in figure 7), then washing for 3 times by using deionized water, removing redundant tyrosinase solution on the surface, and obtaining Tyr-MS/AAO (Tyr-MS/AAO + TYR) catalyzed by TYR;

1-2) placing the heterojunction nano channel obtained after washing in the step 1-1) into 50mL of 10mM ascorbic acid solution, soaking for 15min at room temperature to prevent bisphenol hydroxyl on the channel from being oxidized, taking out, and washing with deionized water to obtain Tyr-MS/AAO + TYR with stable bisphenol hydroxyl;

1-3) preparation 10^-5M potassium chloride solution is respectively used as anolyte and catholyte, an electrochemical performance test (recording current value under voltage of-1V) is carried out by adopting an electrochemical testing device (shown in figure 8, comprising a picometer and a pair of Ag/AgCl electrodes, and the details of the rest devices are the same) in CN202010640001.8, wherein Tyr-MS/AAO + TYR with stable bisphenol hydroxyl is arranged at a connecting channel between an anode pool 1 and a cathode pool 2, the Tyr-MS/AAO layer faces to one side of the anode, and the effective ion transport area is about 3 multiplied by 10⁴μm²Approximately 60 μm in thickness, the change in current measured compared to Tyr-MS/AAO before tyrosinase catalysis;

1-4) plotting the measured current as ordinate and the tyrosinase solution concentration as abscissa to obtain a standard curve of TYR concentration/current variation, as shown in FIG. 9, with the standard curve equation of Y ═ 8.69 × 10^-4X +0.0068(2-50U/mL), wherein Y is the change value of the absolute value of the current under-1V, X is the concentration of tyrosinase, R²Is 0.998;

in addition, as shown in FIG. 10, the Zeta potential diagram of the heterojunction nano-channel before and after 50U/mL TYR catalysis, it can be seen that the Zeta potential in the channel after tyrosinase catalysis is increased from 6.95mV to 12.4 mV.

As shown in fig. 11, which is a contact angle test chart of the heterojunction nanochannel before and after the TYR catalysis of 50U/mL, it can be seen that the contact angle of the heterojunction nanochannel after the TYR catalysis is decreased from 56.9 ± 2.6 ° to 53.1 ± 2.0 °. It can be seen that changes in surface charge density and wettability are the main factors affecting changes in ionic current. The phenomenon of current increase in Tyr-MS/AAO + TYR is probably due to the fact that the TYR is converted from phenolic hydroxyl groups to o-diphenol groups, the hydrophilicity and the surface charge density of heterogeneous channels are increased, the ion transmission behavior is adjusted, and higher ion current is caused. Different concentrations of TYR can catalyze different amounts of phenolic hydroxyl groups to be converted into o-diphenol groups, resulting in different ion current changes

FIG. 12 shows a specific recognition performance test chart of Tyr-MS/AAO on tyrosinase, the test process is the same as the steps 1-1) to 1-3), except that: respectively dropping 200 mu L-50U/mL Glucose Oxidase (GOD), 200 mu L0.02 mg/mL cytochrome C (CYT-C), 200 mu L50U/mL hyaluronidase (HAase), 200 mu L50U/mL Catalase (CAT) and 200 mu L0.2 mg/mL histamine (Hm) on a Tyr-MS/AAO heterojunction nano-channel with the diameter of 15mm, and standing and infiltrating for 30min at room temperature.

In conclusion, the Tyr-MS/AAO prepared in the embodiment 1 can realize the concentration detection of tyrosinase with specificity, high sensitivity and strong repeatability.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. A preparation method of a super-assembly sensing platform for label-free specific detection is characterized by comprising the following steps:

m1: soaking the mesoporous silica/anodic alumina heterojunction nano-channel in a 3-aminopropyltriethoxysilane solution, taking out and carrying out heat treatment to obtain an amination-modified MS/AAO heterojunction nano-channel which is marked as NH₂-MS/AAO；

2. The method for preparing the super-assembled sensing platform for label-free specific detection according to claim 1, wherein in the step M1, the concentration of the 3-aminopropyltriethoxysilane solution is 5-10 wt%, the dipping temperature is 40-50 ℃, and the dipping time is 12-14 h;

3. The method for preparing the super-assembled sensing platform for label-free specific detection according to claim 1, wherein in the step M2, the concentration of the glutaraldehyde solution is 2-10 wt%, the dipping temperature is room temperature, and the dipping time is 5-7 h.

4. The method for preparing a super-assembled sensor platform for label-free specific detection according to claim 1, wherein in the step M3, the tyramine solution has a concentration of 10-50 μ M, the dipping temperature is room temperature, and the dipping time is 5-7 h.

5. A super-assembled sensor platform for label-free specific detection, prepared by the method of any one of claims 1 to 4.

6. A tyrosinase specific detection method based on the super-assembled sensing platform of claim 5, which comprises the following steps:

n3: placing the Tyr-MS/AAO + TYR with stable bisphenol hydroxyl in a connecting channel between an anode pool (1) and a cathode pool (2) of an electrochemical testing device, enabling the Tyr-MS/AAO layer to face to the anode side, and measuring the current change of the Tyr-MS/AAO before catalysis of tyrosinase;

7. The tyrosinase specificity detection method according to claim 6, wherein in step N1, the concentration of tyrosinase in the solution to be detected is 2-50U/mL, and the dosage is 100-²Tyr-MS/AAO；

8. The tyrosinase-specific detection method according to claim 6, wherein in step N2, the protection solution is 5-15mM ascorbic acid solution;

9. The tyrosinase-specific detection method according to claim 6, wherein in step N3, the tyrosinase is detectedThe anolyte in the anode pool (1) is 10^-6-0.1mol/L potassium chloride solution, the catholyte in the cathode cell (2) being 10^-6-0.1mol/L potassium chloride solution; the anode electrode (3) and the cathode electrode (4) are Ag/AgCl electrodes; the test voltage was-2V to 2V.

10. The tyrosinase-specific detection method according to claim 6, wherein in step N4, the method for drawing the current variation/tyrosinase concentration standard curve comprises: