Solid-state nanochannels functionalized with polypeptide probes, methods of making and using the same

Abstract

The application provides a polypeptide probe functionalized solid-state nanochannel and a preparation method and application thereof. The polypeptide probe functionalized solid-state nanochannel is constructed by constructing a metal modification layer on the outer surface of a conventional solid-state nanochannel, and then modifying a polypeptide probe on the outer surface of the solid-state nanochannel with the gold layer containing the metal modification layer through a gold-sulfur bond. The polypeptide probe functionalized solid-state nanochannel is used for detecting matrix metalloproteinase 2 (MMP-2), and not only can the MMP-2 in a solution be detected, but also specific detection of MMP-2 secreted by HeLa cancer cells and MMP-2 secreted by HeLa cancer cells in different cell cycles can be realized.

Description

Technical Field

[0001] This invention relates to the field of protein macromolecule detection technology, specifically to a solid-state nanochannel functionalized with a peptide probe, its preparation method, and its application. Background Technology

[0002] Most existing functionalized solid-state nanochannels are based on probe molecules modified on the inner wall of the solid-state nanochannel. This means that the target biomolecule must enter the interior of the solid-state nanochannel to interact with the probe molecules. When dealing with protein biomolecules such as matrix metalloproteinase 2, the limited space within the solid-state nanochannel makes it difficult for relatively large protein biomolecules to reach the probe recognition site, or to simultaneously recognize a large number of probes, resulting in low detection efficiency. Simply increasing the pore size of the solid-state nanochannel will lead to poor reproducibility of the detection results. Summary of the Invention

[0003] Therefore, it is necessary to provide a solid-state nanochannel functionalized with a peptide probe, its preparation method and application, which is suitable for the detection of matrix metalloproteinase 2 (MMP-2) with good specificity.

[0004] The present invention adopts the following technical solution:

[0005] This invention provides a polypeptide probe that can specifically bind to matrix metalloproteinase 2, the amino acid sequence of which includes: CRRRRRPLGLAGFFFF.

[0006] The present invention also provides a solid nanochannel functionalized with a peptide probe, comprising: a solid nanochannel body; an outer membrane metal-modified layer located on one side of the solid nanochannel body; and a peptide probe located on the same side of the outer membrane metal-modified layer and grafted onto the solid nanochannel body, wherein the peptide probe is the aforementioned peptide probe capable of specifically binding to matrix metalloproteinase 2.

[0007] In some embodiments, the solid nanochannel body is anodized aluminum oxide with hydroxyl groups on its surface.

[0008] In some embodiments, the outer metallization layer comprises a gold layer.

[0009] The present invention also provides a method for preparing the above-mentioned peptide probe functionalized solid nanochannel, comprising the following steps: providing a solid nanochannel body; forming an outer metal modification layer on one side surface of the solid nanochannel body; reducing the peptide probe with a reducing agent to obtain a thiol-containing peptide; and then reacting the thiol-containing peptide with the outer metal modification layer of the solid nanochannel to obtain the final product.

[0010] In some embodiments, the outer metallized layer is formed by physical vapor deposition.

[0011] In some embodiments, the reducing agent is selected from tris(2-carboxyethyl)phosphine. Preferably, the molar ratio of tris(2-carboxyethyl)phosphine (TCEP) reducing agent to the peptide is 100:1.

[0012] In some embodiments, the reaction ratio of the 5 nM peptide probe solution to the outer surface of the solid nanochannel is 500 μL:(0.2–0.3) cm⁻¹. 2 .

[0013] The present invention also provides the application of peptide probe-functionalized solid nanochannels or peptide probe-functionalized solid nanochannels prepared by the above preparation method in the detection of matrix metalloproteinase 2.

[0014] In some embodiments, the concentration of matrix metalloproteinase 2 (MMP-2) can be 1 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 150 ng / mL, respectively. The ratio of MMP-2 to the outer membrane surface of the peptide probe-functionalized solid nanochannel is 500 μL:(0.2–0.3) cm. 2 .

[0015] In some embodiments, the matrix metalloproteinase 2 may be derived from matrix metalloproteinase 2 secreted by HeLa cancer cells at different stages.

[0016] Preferably, HeLa cancer cells were induced to four cycles: G0 / G1, G1 / S, S, and G2 / M, respectively, using DMEM medium without serum, medium containing L-mimosine, medium containing thymidine, and medium containing nocodazole.

[0017] In some of these embodiments, the density of the cultured HeLa cancer cells was 4 × 10⁻⁶. 1 cells / mL, 4×10 3 cells / mL, 4×10 4 cells / mL, 4×10 5 cells / mL, 1×10 6 Cells / mL. Culture media at different cell densities were then collected and centrifuged at 400g for 5 minutes to remove floating cells and debris. The ratio of HeLa cancer cell cultures at different densities to peptide probe-functionalized solid nanochannel outer membranes was then collected at 500 μL:(0.2–0.3) cm. 2 .

[0018] The present invention also provides a method for detecting matrix metalloproteinase 2, comprising the following steps: reacting a solution containing matrix metalloproteinase 2 with a solid nanochannel functionalized with a peptide probe, and detecting the change in ion current before and after the reaction of the solid nanochannel functionalized with the peptide probe.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention develops a peptide probe-functionalized solid-state nanochannel that can specifically bind to matrix metalloproteinase 2 (MMP2). Solutions containing MMP2, MMP2-containing culture media of HeLa cancer cells, and MMP2-containing culture media samples of HeLa cancer cells at different cell cycles can all react with the peptide probe-functionalized solid-state nanochannel, thereby altering the transmembrane ion current of the peptide probe-functionalized solid-state nanochannel system and thus enabling the detection of MMP2. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the principle of detecting matrix metalloproteinase 2 (MMP-2) using a solid-state nanochannel with a peptide probe functionalized according to the present invention.

[0022] Figure 2 This is the mass spectrum of the peptide probe tested in Example 2.

[0023] Figure 3 This is a schematic diagram of the surface (top view) and cross-section (cross view) of the solid nanochannel with a metal-modified outer membrane prepared in Example 2.

[0024] Figure 4 This is a schematic diagram of the surface (top view) and cross-section (cross view) of the solid nanochannel functionalized with peptide probes in Example 2.

[0025] Figure 5 This is a graph showing the change in ion current (current increase ratio) of matrix metalloproteinase 2 (MMP-2) at different concentrations in solution detected using a solid nanochannel functionalized with a peptide probe in Example 3.

[0026] Figure 6 This is a graph showing the change in ion current (Current increase ratio) of other proteases (Trypsin, GOD, Thrombin, HAase, Exonuclease, CB) in solution using solid-state nanochannels functionalized with peptide probes, as described in Example 3.

[0027] Figure 7This is a graph from Example 4 showing the change in ion current (current increase ratio) of matrix metalloproteinase 2 (MMP-2) secreted by HeLa cancer cells at different growth densities, detected using solid-state nanochannels functionalized with peptide probes.

[0028] Figure 8 Example 4 uses a peptide probe-functionalized solid-state nanochannel to detect the change in ion current (current increase ratio) of matrix metalloproteinase 2 (MMP-2) not secreted by normal HFL-1 cells at different growth densities.

[0029] Figure 9 Example 5 uses a peptide probe-functionalized solid-state nanochannel to detect the change in ion current (current increase ratio) of matrix metalloproteinase 2 (MMP-2) secreted by HeLa cancer cells at different cycles. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are all conventional means well known to those skilled in the art.

[0031] The technical concept of this invention is to provide a peptide probe that can specifically bind to matrix metalloproteinase 2 (MMP-2), a method for preparing a solid nanochannel functionalized with the peptide probe, and a method for specifically detecting MMP-2 in solution and MMP-2 secreted by HeLa cancer cells and at different cell cycles using a solid nanochannel functionalized with the peptide probe.

[0032] Specifically, the present invention relates to a solid-state nanochannel functionalized with peptide probes. This involves modifying the outer surface of the solid-state nanochannel with a gold layer and then modifying it with peptide probe molecules of a specific structure. MMP-2 can specifically bind to the peptide probe molecules, causing a change in the transmembrane ion current of the solid-state nanochannel. Different concentrations of MMP-2 result in different rates of change in the transmembrane ion current of the solid-state nanochannel. The schematic diagram is shown below. Figure 1 As shown.

[0033] The method for preparing a peptide probe-functionalized solid nanochannel of the present invention preferably includes the following steps: obtaining a solid nanochannel body, forming an outer metal modification layer on one side surface (outer surface) of the solid nanochannel body, and then modifying the peptide treated with tris(2-carboxyethyl)phosphine (TCEP) reducing agent onto the gold layer in the outer metal modification layer of the solid nanochannel, thereby obtaining the product.

[0034] The following example illustrates this:

[0035] Example 1

[0036] This embodiment provides a polypeptide probe molecule capable of specifically binding to matrix metalloproteinase 2 (MMP-2), with the structure shown in Formula I:

[0037]

[0038] The amino acid sequence of the above-mentioned polypeptide probe molecule is CRRRRRPLGLAGFFFF. The polypeptide probe consists of three parts: a hydrophilic unit (CRRRR) containing cysteine ​​at the end, an MMP-2 recognition unit (PLGLAG), and a hydrophobic unit (FFFF).

[0039] This polypeptide probe was synthesized by Jier Biochemical (Shanghai) Co., Ltd., and the mass spectrometry data are as follows: Figure 2 As shown.

[0040] Example 2

[0041] This embodiment provides a method for preparing solid-state nanochannels functionalized with peptide probes, including the following steps:

[0042] S1, Obtaining the nanoporous membrane substrate: Anodized aluminum oxide (AAO) solid nanochannels with hydroxyl groups on the surface, circular in shape with a diameter of 50 mm, and pore diameters of 20–30 nm on the membrane, with an average pore density of 10. 11 -10 12 pcs / cm 2 .

[0043] S2, The outer metal-modified layer of the solid nanochannel is formed by physical vapor deposition:

[0044] 1) A chromium metal layer with a thickness of 10 nm was pre-deposited on the surface of anodized aluminum oxide (AAO) solid nanochannels using physical vapor deposition (PVD). The process conditions were: atmospheric pressure 8 × 10⁻⁶ Torr, temperature 25 °C, and chromium metal layer deposition rate of [missing information].

[0045] 2) Next, deposit an Au layer on the surface of the chromium metal layer. The Au layer thickness is 240 nm. The process conditions are: gas pressure 8 × 10⁻⁶. -6 Torr, temperature 25℃, Au layer deposition rate is

[0046] 3) Finally, the anodic aluminum oxide solid nanochannels (solid nanochannels with an outer metal modification layer) after the metal layer deposition were characterized, such as... Figure 3As shown.

[0047] S3, Peptide probe molecules modify the outer metal layer of solid nanochannels:

[0048] 1) A 5 nM polypeptide probe molecule containing disulfide bonds (prepared in Example 1) was reacted with a 500 μL mixed solution of 500 nM tris(2-carboxyethyl)phosphine (TCEP) for 10 h to obtain the reduced polypeptide.

[0049] The purpose of this step is to reduce the disulfide-bonded polypeptide probe molecule to a thiol-containing polypeptide molecule using tris(2-carboxyethyl)phosphine (TCEP).

[0050] 2) Then, the 5 nM thiol-containing polypeptide molecule solution obtained from the reduction is reacted with 0.2–0.3 cm⁻¹ 2 After depositing a metal layer, the solid-state nanochannel was reacted for 10 h to obtain a peptide probe-functionalized solid-state nanochannel (abbreviated as: Pep@AAO-Au).

[0051] Structural characterization of the prepared peptide probe-functionalized solid-state nanochannels is as follows: Figure 4 As shown, it can be seen that the inner wall pores and outer surface of the solid nanochannel prepared in this embodiment are clearly distinguished. Here, the side where the gold layer is located is defined as the outer surface of the solid nanochannel, and the other parts besides the outer surface are defined as the inner pore wall.

[0052] Example 3

[0053] This embodiment provides a method for detecting matrix metalloproteinase 2 (MMP-2) in solution using solid-state nanochannels functionalized with peptide probes, comprising the following steps:

[0054] 500 μL of MMP-2 activated at different concentrations (1 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL) was reacted with a peptide probe-functionalized solid nanochannel (Pep@AAO-Au prepared in Example 2) at 37 °C for 2 h, and the changes in ion current were detected.

[0055] The detection results of the ion current change are shown in Figure 5 It can be seen that after the activated matrix metalloproteinase 2 (MMP-2) reacts with the peptide probe-functionalized solid nanochannel, the ion current increases; at the same time, the rate of change of ion current increases with the increase of matrix metalloproteinase 2 (MMP-2) concentration.

[0056] This embodiment further explores the effectiveness of using peptide probe-functionalized solid-state nanochannels to detect other proteases in solution, namely: trypsin, glucose oxidase (GOD), thrombin, hyaluronidase (HAase), exonuclease III, and cathepsin B (CB). The specific testing procedures are the same as those for detecting matrix metalloproteinase 2 (MMP-2) in solution.

[0057] The statistical results of the control experiment are shown in the figure. Figure 6 It can be seen that other protein enzymes, including trypsin, glucose oxidase (GOD), thrombin, hyaluronidase (HAase), exonuclease III, and cathepsin B (CB), do not cause changes in ion current.

[0058] This indicates that the solid-state nanochannels functionalized with the peptide probes prepared in Example 2 have excellent specificity for the detection of matrix metalloproteinase 2 (MMP-2).

[0059] Example 4

[0060] This embodiment provides a method for detecting matrix metalloproteinase 2 (MMP-2) secreted by HeLa cancer cells using solid-state nanochannels functionalized with peptide probes, comprising the following steps:

[0061] Collect different culture densities (4×10) 1 cells / mL, 4×10 3 cells / mL, 4×10 4 cells / mL, 4×10 5 cells / mL, 1×10 6 The culture medium of HeLa cancer cells (cells / mL) was centrifuged at 400g for 5 minutes to remove floating cells and debris. Then, the collected HeLa cancer cells at different culture densities were reacted with peptide probe-functionalized solid-state nanochannels at 37℃ for 2 hours, and the changes in ion current were detected.

[0062] The detection results of the ion current change are shown in Figure 7 It can be seen that after the MMP-2 secreted by HeLa cancer cells reacts with the peptide probe-functionalized solid nanochannel, the ion current increases; at the same time, as the density of HeLa cancer cells increases, the rate of change of ion current increases.

[0063] This embodiment further investigates the effectiveness of using peptide probe-functionalized solid-state nanochannels to detect HFL-1 cells that do not secrete matrix metalloproteinase 2 (MMP-2) as a control experiment. The experimental methods and procedures are the same as those for the detection of MMP-2 in HeLa cancer cell culture medium.

[0064] The results of the control experiment are shown in Figure 8 It can be seen that the substances secreted by HFL-1 cells into the culture medium do not cause changes in ion current.

[0065] Example 5

[0066] This embodiment provides a method for detecting matrix metalloproteinase 2 (MMP-2) secreted by HeLa cancer cells at different stages using solid-state nanochannels functionalized with peptide probes, comprising the following steps:

[0067] The procedures for inducing HeLa cells to G0 / G1, G1 / S, S, and G2 / M are as follows:

[0068] G0 / G1 phase: HeLa cells were cultured to a density of 80-90% and then starved in DMEM medium without serum for 24 hours;

[0069] G1 / S phase: HeLa cells were cultured to a density of 60-70% and then cultured for 16 hours with the addition of L-mimosin (100mM).

[0070] S phase: HeLa cells were cultured to a density of 60-70% and then cultured with thymidine (100mM) for 16 hours.

[0071] G2 / M phase: HeLa cells were cultured to a density of 60-70% and then incubated with nocodazole (100 ng / mL) for 16 h.

[0072] Culture medium components from different stages of HeLa cancer cells were collected and centrifuged at 400g for 5 minutes to remove floating cells and debris. The culture medium from HeLa cancer cells at different stages was then reacted with peptide probe-functionalized solid nanochannels (prepared in Example 2) at 37°C for 2 hours, and changes in ion current were detected.

[0073] The detection results of the ion current change are shown in Figure 9 It can be seen that the rate of change of ion current in the second cycle G1 / S is the largest among the four cycles of G0 / G1, G1 / S, S, and G2 / M in HeLa cancer cells. This indicates that the MMP-2 secreted by HeLa cancer cells is related to the cell cycle, and the content of MMP-2 secreted in the second cycle G1 / S phase is the highest.

[0074] In summary, this invention essentially provides a solid-state nanochannel based on an externally modified peptide probe, its preparation method, and the application of this externally surface-modified peptide probe-based solid-state nanochannel for detecting matrix metalloproteinase 2 (MMP-2). The solid-state nanochannel based on an externally surface-modified peptide probe of this invention can not only detect MMP-2 in solution, but also detect MMP-2 secreted by HeLa cancer cells and MMP-2 secreted by HeLa cancer cells at different cell cycles.

[0075] In particular, this invention designs peptide probe molecules to covalently modify the outer surface of a metal-modified solid nanochannel, while ensuring the specific binding of the target substance to the peptide probe molecules. This alters the wettability of the outer surface of the peptide-functionalized solid nanochannel, thereby changing the transmembrane ion current passing through the solid nanochannel. The peptide probe-functionalized solid nanochannel of this invention enables the detection of matrix metalloproteinase 2 (MMP-2) secreted by HeLa cancer cells in solution.

[0076] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide probe capable of specifically responding to matrix metalloproteinase 2, characterized in that, Its amino acid sequence is: CRRRRPLGLAGFFFF.

2. A solid-state nanochannel functionalized with a peptide probe, characterized in that, Include: Solid-state nanochannel bulk; An outer metal modification layer located on one side of the solid nanochannel body; and A polypeptide probe is located on the same side of the outer membrane metal-modified layer and grafted onto the solid nanochannel body. The polypeptide probe is the polypeptide probe of claim 1 that can specifically respond to matrix metalloproteinase 2.

3. The solid-state nanochannel functionalized with the peptide probe according to claim 2, characterized in that, The solid nanochannel body is anodized aluminum nanochannel with hydroxyl groups on its surface.

4. The solid-state nanochannel functionalized with a peptide probe according to claim 2 or 3, characterized in that, The outer metal modification layer includes a gold layer.

5. The method for preparing a peptide probe-functionalized solid-state nanochannel according to any one of claims 2 to 4, characterized in that, Includes the following steps: Provides solid-state nanochannel body; An outer metal modification layer is formed on one side surface of the solid nanochannel body; The polypeptide probe described in claim 1 was reduced using a reducing agent to obtain a thiol-containing polypeptide; The peptide containing thiol groups is then reacted with the metal-modified layer of the outer membrane of the solid nanochannel to obtain the final product.

6. The method for preparing solid-state nanochannels functionalized with peptide probes according to claim 5, characterized in that, The outer metal modification layer is formed by physical vapor deposition.

7. The method for preparing solid-state nanochannels functionalized with peptide probes according to claim 5, characterized in that, The reducing agent is selected from tris(2-carboxyethyl)phosphine.

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