Biomolecular sensor based on cucurbit [7] uril functionalized nanopores as well as preparation method and application of biomolecular sensor

Abstract

The invention discloses a biomolecular sensor based on a cucurbit [7] uril functionalized nanopore. The sensor comprises a conical nanopore formed in a quartz glass tube, and an assembly of mercaptopropyltrimethoxysilane, gold nanoparticles and cucurbit [7] uril, wherein the mercaptopropyltrimethoxysilane, the gold nanoparticles and the cucurbit [7] uril are sequentially modified in the nanopore. The biomolecular sensor has a good application effect in recognition of amino acids, biogenic amines, polypeptides and proteins. The sensor has the advantages of high efficiency, high sensitivity, simplicity and convenience in operation and the like, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of supramolecular materials and detection thereof, and in particular relates to a biomolecular sensor based on cucurbit[7]uril functionalized nanopores and a preparation method and application thereof. Background Art

[0002] As an important component of the human body, biological molecules are widely involved in various physiological activities of the human body. Their content in the human body is related to a variety of diseases. Currently, there are more than 400 diseases related to amino acid imbalance, such as phenylketonuria, diabetes, etc. At present, some researchers use chromatography and spectroscopy to detect various types of biological small molecules, such as the detection of some specific amino acids with unique functional groups. There are also literature reports on the use of fluorescence to detect tryptophan and histidine. However, these reported methods also have limitations such as expensive instruments, complex sample pretreatment, long time consumption, high cost and low sensitivity. Solid-state nanopores are widely used in the field of molecular detection due to their high sensitivity, easy manufacturing and simple operation. They are ideal platforms for instant detection of biological small molecules. Summary of the invention

[0003] The main purpose of the present invention is to provide a sensitive, rapid and accurate biomolecular sensor based on cucurbit[7]uril functionalized nanopores and its preparation method and application in view of the problems and shortcomings of the prior art.

[0004] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0005] A biomolecule sensor based on cucurbit[7]uril functionalized nanopores, characterized in that the sensor comprises a conical nanopore formed on a quartz glass tube and an assembly (GNP@CB[7]) of mercaptopropyltrimethoxysilane (MPTS) and gold nanoparticles (GNP) and cucurbit[7]uril (CB[7]) sequentially modified in the nanopore.

[0006] Preferably, the molecule of the mercaptopropyltrimethoxysilane is C6H 16 O3SSi, its molecular weight is 196.34.

[0007] Preferably, in the cucurbitac[7]uril and gold nanoparticle assembly (GNP@CB[7]), CB[7] is coated on the surface of GNP by a self-assembly process, and the particle size of GNP is 4 to 20 nm, preferably 8 to 12 nm, and more preferably 10 nm.

[0008] Preferably, the biomolecules are various amino acids, biogenic amines, polypeptides and proteins. Further preferably, the amino acids are one or more of tyrosine, histidine, phenylalanine, tryptophan, lysine, proline and glycine; the biogenic amines are one or more of 1,6-hexanediamine, cadaverine, putrescine, n-hexylamine, spermine, spermidine, dihexylamine, tyramine, histamine and creatinine; the polypeptides are one or more of dipeptides Phe-Gly, Gly-Phe, Tyr-Gly, Gly-Tyr and pentapeptides PGGGG, LGGGG, GGGGG; the proteins are one or more of bovine serum albumin, human serum albumin and insulin.

[0009] The present invention provides a method for preparing a biomolecule sensor based on cucurbit[7]uril functionalized nanopores, firstly, a quartz glass tube after hydroxyl activation is formed into a nanopore body by laser firing process, and then thiol modification, GNP@CB[7] modification and other steps are sequentially performed in the nanopore to prepare the biomolecule sensor, specifically comprising the following steps:

[0010] S1: The hydroxyl activation is achieved by immersing the quartz glass tube in a piranha solution, heating, and washing;

[0011] S2: The laser burning process is completed by a laser drawing instrument under specific parameters;

[0012] S3: The thiol modification of the inner wall of the nanopore is achieved by injecting an MPTS solution into the tip of the nanopore for rinsing, soaking, incubating, washing, and drying in sequence;

[0013] S4: The GNP@CB[7] modification process is achieved by mixing the GNP solution with the CB[7] solution to self-assemble into GNP@CB[7], and then injecting it into the thiol-modified nanopore tip for immersion incubation, washing, and drying.

[0014] Preferably, the inner diameter of the quartz tube in step S1 is 0.5-0.8 mm, more preferably 0.7 mm.

[0015] Preferably, the method for activating the hydroxyl group in step S1 is to activate with a piranha solution (H2SO4:H2O2 (30%) = 3:1), the heating temperature is 90°C, the heating time is 0.5 to 5 hours, and more preferably 2 hours; the washing conditions after activation are ultrapure water rinsing for 0.5 to 2 minutes, and more preferably 1 minute; ultrasonic 2 to 5 times, and more preferably 3 times, each time for 5 minutes.

[0016] Preferably, the parameters of the laser drawing instrument in step S2 are as follows: power is 700 W, scanning program is 4, drawing speed is 60 mm / s; drawing time is 170 ms, and drawing force is 180.

[0017] Preferably, the MPTS solution used for thiol modification in step S3 is an ethanol solution of MPTS, and the concentration is 3-30wt%, more preferably 10wt%; the number of rinsing is 2-5 times, more preferably 3 times, and 0.5mL of MPTS solution is used each time; the MPTS solution immersion incubation time is 1-6h, more preferably 3h; the washing condition is to first rinse the inner wall of the nanopore with ethanol solution 2-5 times, more preferably 3 times; the drying condition is vacuum drying at room temperature for 1-3h, more preferably 2h.

[0018] Preferably, the maximum ultraviolet absorption wavelength of the GNP solution in step S4 is 510 nm, and the corresponding absorbance is 0.1 to 0.6, more preferably 0.36; the concentration of the CB[7] solution is 5 to 15×10 -6 M, more preferably 10×10 -6 M; the incubation time of the mixed GNP solution and the CB[7] solution is 15 to 60 minutes, more preferably 30 minutes; the maximum ultraviolet absorption peak of the GNP@CB[7] is 550nm, and the corresponding absorbance is 0.05 to 0.3, more preferably 0.2; the incubation time of the GNP@CB[7] modification is 6 to 24 hours, more preferably 12 hours; the washing condition is rinsing with ultrapure water for 2 to 5 times, more preferably 3 times; the drying condition is vacuum drying at room temperature for 1 to 3 hours, more preferably 2 hours.

[0019] The present invention provides an application of a biomolecule sensor based on a cucurbit[7]uril functionalized nanopore, which detects biomolecules by detecting an I-Vq curve; the process used in the IV detection includes the following conditions: a two-electrode system is used, an Ag / AgCl electrode is inserted into the inside of the cucurbit[7]uril functionalized nanopore as a working electrode, and the Ag / AgCl electrode at the other end is used as a reference electrode, the working electrode and the reference electrode are both inserted into an electrolyte containing an object to be detected, and the detection of biomolecules is completed under a specific voltage range and scanning rate.

[0020] Preferably, the electrolyte is a KCl solution with a concentration of 0.005-0.02M, more preferably 0.01M.

[0021] Preferably, the voltage range is -2.0V to +2.0V, and more preferably -1.0V to +1.0V.

[0022] Preferably, the scanning speed is 0.01-0.1 V / s, more preferably 0.05 V / s.

[0023] Preferably, the biomolecules (various amino acids, biogenic amines and polypeptides) are added to the electrolyte at a concentration range of 10 -15-10 -6 M.

[0024] The principle of the present invention is as follows: the present invention uses MPTS to covalently modify the inner wall of the quartz glass nanopore, and then uses the gold-sulfur bond of MPTS and GNP@CB[7] to modify cucurbitacin[7]uril on the inner wall of the nanopore, thereby forming a biomolecule sensor based on cucurbitacin[7]uril functionalized nanopore. The recognition effect of cucurbitacin[7]uril on biomolecules is used to achieve rapid detection. The response performance of the sensor to amino acids, biogenic amines and peptides is investigated by the change in the ion rectification current.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) The present invention prepares a biomolecule sensor based on cucurbit[7]uril functionalized nanopore, couples the high sensitivity of the nanopore with the specific recognition characteristics of cucurbit[7]uril, and realizes the recognition and detection of the object to be detected by utilizing the host-guest interaction, thus solving the shortcoming that the nanopore sensor cannot perform specific detection.

[0027] 2) The invention proposes to modify cucurbit[7]uril on the inner wall of the nanopore by using gold nanoparticles as a medium, thereby solving the problem that cucurbit[7]uril is difficult to directly modify on the detection device.

[0028] 3) The method proposed in the present invention, which is based on the combination of cucurbit[7]uril functionalized nanopore biomolecular sensor and electrochemical detection technology, can realize the detection of various amino acids, biogenic amines and polypeptides, and has the advantages of high sensitivity, good selectivity, wide adaptability, low cost, etc., and the operation involved is simple, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the preparation process and electrochemical characterization diagram of the cucurbit [7] uril functionalized nanopores described in Example 1 of the present invention;

[0030] Figure 2 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of tyrosine in Example 2 of the present invention (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of tyrosine concentration (right);

[0031] Figure 3 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of histidine in Example 3 of the present invention (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the histidine concentration (right);

[0032] Figure 4The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of tyramine in Example 4 of the present invention (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of tyramine concentration (right);

[0033] Figure 5 The IV curve of the biomolecular sensor based on cucurbit [7] uril functionalized nanopores for different concentrations of histamine (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the histamine concentration (right) in Example 5 of the present invention;

[0034] Figure 6 The IV curves of the biomolecule sensor based on cucurbit [7] uril functionalized nanopores for different concentrations of dipeptides (tyrosine-glycine) in Example 6 of the present invention (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of dipeptide concentration (right);

[0035] Figure 7 The IV curve diagram of the biomolecule sensor based on cucurbit [7] uril functionalized nanopores for different concentrations of pentapeptides (phenylalanine-glycine-glycine-glycine-glycine) in Example 7 of the present invention (left) and the linear relationship diagram of the ion current change rate (△I / I0) and the logarithm of the pentapeptide concentration (right);

[0036] Figure 8 The IV curve of the biomolecule sensor based on cucurbit [7] uril functionalized nanopores for different concentrations of bovine serum albumin (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the bovine serum albumin concentration (right) in Example 8 of the present invention are shown;

[0037] Fig. 9 The IV curve diagram of the biomolecular sensor based on cucurbit[7]uril functionalized nanopores for different insulin concentrations (left) and the linear relationship diagram of the ion current change rate (△I / I0) and the logarithm of insulin concentration (right) in Example 9 of the present invention.

[0038] Fig.10 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of tyrosine in Example 10 of the present invention (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of tyrosine concentration (right);

[0039] Fig.11 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of tyrosine in Example 11 of the present invention (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of tyrosine concentration (right);

[0040] Fig.12 The IV curve of the biomolecular sensor based on cucurbit [7] uril functionalized nanopores for different concentrations of phenylalanine (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of phenylalanine concentration (right) in Example 12 of the present invention are shown;

[0041] Fig.13 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of tryptophan (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of the tryptophan concentration (right) in Example 13 of the present invention are shown;

[0042] Fig.14 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores for different concentrations of lysine (left) and the linear relationship between the rate of change of ion current (△I / I0) and the logarithm of lysine concentration (right) in Example 14 of the present invention are shown;

[0043] Fig.15 The IV curve of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore for different concentrations of proline (left) and the linear relationship diagram of the ion current change rate (△I / I0) and the logarithm of the proline concentration (right) in Example 15 of the present invention are shown;

[0044] Fig.16 IV curves of the biomolecule sensor based on cucurbit[7]uril functionalized nanopores prepared in comparative example 1 of the invention at a ratio of MPTS to anhydrous ethanol of 1:9 (v:v) for different concentrations of dipeptides (tyrosine-glycine) (left) and a linear relationship diagram of the ion current change rate (△I / I0) and the logarithm of the dipeptide concentration (right);

[0045] Fig.17 This is an IV curve diagram of the nanopore at different modification stages under the conditions of MPTS: anhydrous ethanol being 1:19 (v:v) and MPTS incubation time being 12 h in comparative example 2 of the invention.

[0046] Fig.18 This is an IV curve diagram of the nanopore before and after MPTS modification in comparative example 3 of the invention under the conditions of MPTS: anhydrous ethanol being 1:9 (v:v) and the incubation time being 12 h. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0048] The present invention uses MPTS to covalently modify the inner wall of the quartz glass nanopore, and then uses the gold-sulfur bond of MPTS and GNP@CB[7] to modify cucurbitacin[7]uril on the inner wall of the nanopore to form a biomolecule sensor of cucurbitacin[7]uril functionalized nanopore. The recognition effect of cucurbitacin[7]uril on biomolecules is used to achieve rapid detection. The response performance of the sensor to amino acids, biogenic amines and peptides is investigated by the change in the ion rectification current.

[0049] Example 1

[0050] This example determines the process for preparing a biomolecular sensor based on cucurbit[7]uril functionalized nanopores (e.g. Figure 1 ), the specific experimental method is as follows:

[0051] 1) Preparation of cucurbit[7]uril functionalized nanopores

[0052] a. Mix 10 μM CB[7] solution and GNP solution (the particle size of gold nanospheres is 10 nm, the maximum UV absorption peak is 510 nm, and the corresponding absorbance is 0.36) in equal proportions to obtain GNP@CB[7] solution for use (the maximum UV absorption peak is 550 nm, and the corresponding absorbance is 0.2).

[0053] b. The quartz nanotubes were placed in a piranha solution (98% sulfuric acid: 30% hydrogen peroxide = 3:1) and heated at 90°C for 1 hour, and the inside of the nanotubes were repeatedly rinsed with the piranha solution to activate the hydroxyl groups of the nanotubes, and then ultrasonically washed with excess ultrapure water for 3 times, each time for 5 minutes, and finally vacuum dried at room temperature for 2 hours; after drying, a laser drawing instrument (parameters are as follows: power 700W, scanning program 4, drawing speed 60mm / s; drawing time 170ms, drawing force 180) was used to laser fuse the nanotubes into conical nanopores.

[0054] c. Use a micro syringe to inject an ethanol solution of mercaptopropyltrimethoxysilane (MPTS) (MPTS: anhydrous ethanol = 1:19 (v / v)) into the tip of the conical nanopore obtained in step b) and fill the entire tip; then fix it and immerse it in a beaker containing the same solution, place it in a desiccator and incubate it for 3 hours at room temperature for thiolization; then take out the nanopore, rinse it with anhydrous ethanol three times to remove the unreacted silane reagent, and then put it in a vacuum drying oven to dry for 2 hours.

[0055] d. The nanopore obtained in step c) was injected with the GNP@CB[7] solution into the tip of the nanopore using a microsyringe and filled with the entire tip. The nanopore was then fixed and immersed in a beaker containing the same GNP@CB[7] solution and placed in a desiccator at room temperature for 12 h. The nanopore was then taken out and washed with ultrapure water three times and then dried in a vacuum drying oven for 2 h for subsequent electrochemical testing.

[0056] 2) Electrochemical characterization of cucurbit[7]uril functionalized nanopores

[0057] Two Ag / AgCl electrodes were placed in the prepared cucurbit[7]uril functionalized nanopore and in the electrolyte as the working electrode and reference electrode, respectively. During the test, the solution filled in the nanopore and the electrolyte solution were both 0.01M KCl solution. Using a CHI 760E electrochemical workstation, the voltage was swept from -1V to +1V at a sweep rate of 0.05V / s for IV testing; the test temperature was room temperature and the solution pH was 7; the hydroxylated nanopore, thiol nanopore and cucurbit[7]uril functionalized nanopore were used as the nanopores in the working electrode for IV testing.

[0058] Figure 1 The schematic diagram (above) and the IV curve characterization diagram (below) of the preparation process of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore in this embodiment. When the unfunctionalized nanopore is used as the working electrode, the IV curve shows a large rectification effect, and the rectification ratio (r) is 18.16 at this time; after MPTS functionalization, the inner surface of the nanopore is hydrophobic due to the introduction of hydrophobic mercaptosilane, and the rectification is deflected, r is 0.92; after modification of GNP@CB[7], the rectification is deflected, r is 7.71; the change in the rectification ratio of the IV curve proves the successful preparation of the cucurbit[7]uril functionalized nanopore.

[0059] Example 2

[0060] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect tyrosine (Tyr), which specifically includes the following steps:

[0061] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1.

[0062] 2) Detection of Tyr molecules

[0063] 0.0018 g of Tyr was weighed and dissolved in 10 mL of ultrapure water to prepare a 1 mM Tyr aqueous solution, and then Tyr was diluted with 0.01 M KCl solution to make the final concentrations of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, and 0.001 nM, respectively. The above KCl solutions containing different concentrations of Tyr were used as electrolytes. Two Ag / AgCl electrodes were placed in the prepared cucurbit[7]uril functionalized nanopores and in the electrolyte as working electrodes and reference electrodes, respectively. During the test, the solution filled in the nanopores and the electrolyte solution were both 0.01 M KCl solutions. At room temperature, a CHI 760E electrochemical workstation was used to sweep the voltage from -1 V to +1 V at a scan rate of 0.05 V / s for IV testing.

[0064] Figure 2 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Tyr concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of Tyr concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Tyr ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Tyr solution concentration. In the concentration range of 1pM to 50nM, the linear fitting equation for detecting Tyr is △I / I0=0.196lg[C Tyr / 10 -12 M]+0.033, R 2 =0.996; limit of detection (LOD) is 0.998 pM.

[0065] Example 3

[0066] This example determines the feasibility of detecting histidine (His) using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores, which specifically includes the following steps:

[0067] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0068] 2) Detection of His molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of His. The electrolyte is specifically prepared as follows: 0.0016 g of His is dissolved in 10 mL of ultrapure water to prepare a 1 mM His aqueous solution, and then His is diluted with a 0.01 M KCl solution to a final concentration of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, and 0.001 nM, respectively.

[0069] Figure 3 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different His concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of His concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected His ion current change rate (△I / I0) has a good linear relationship with the logarithm of the His solution concentration. In the concentration range of 500pM to 1000nM, the linear fitting equation for detecting His is △I / I0=0.696lg[C His / 10 -12 M]-1.654, R 2 =0.995; LOD is 234.4pM.

[0070] Example 4

[0071] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect tyramine (TA), which specifically includes the following steps:

[0072] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0073] 2) Detection of TA molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of TA. The electrolyte is specifically prepared as follows: 0.0014 g of TA is dissolved in 10 mL of ultrapure water to prepare a 1 mM TA aqueous solution, and then TA is diluted with 0.01 M KCl solution to make the final concentrations of TA 1000 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.5 pM, 0.1 pM, 0.01 pM, 0.001 pM, respectively.

[0074] Figure 4 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different TA concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of TA concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected TA ion current change rate (△I / I0) has a good linear relationship with the logarithm of TA solution concentration. In the concentration range of 1fM to 1nM, the linear fitting equation for detecting TA is △I / I0=0.472lg[C TA / 10 -15 M]+0.495, R 2 =0.995; LOD is 0.125fM.

[0075] Example 5

[0076] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect histamine (HI), which specifically includes the following steps:

[0077] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0078] 2) Detection of HI molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of HI. The electrolyte is specifically prepared as follows: 0.0011 g of HI is dissolved in 10 mL of ultrapure water to prepare a 1 mM HI aqueous solution, and then HI is diluted with a 0.01 M KCl solution to a final concentration of 1000 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.5 pM, 0.1 pM, 0.01 pM, and 0.001 pM, respectively.

[0079] Figure 5 The IV curve of the current response of the biomolecule sensor based on cucurbit [7] uril functionalized nanopore to different HI concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of HI concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected HI ion current change rate (△I / I0) has a good linear relationship with the logarithm of the HI solution concentration. In the concentration range of 500fM to 1nM, the linear fitting equation for detecting HI is △I / I0=0.483lg[C HI / 10 -15 M]-0.626, R 2 =0.989; LOD is 120.4fM.

[0080] Example 6

[0081] This example determines the feasibility of using a biomolecular sensor based on cucurbitacin [7] uril functionalized nanopores to detect dipeptide-glycine (Tyr-Gly), which specifically includes the following steps:

[0082] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0083] 2) Detection of Tyr-Gly molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of Tyr-Gly. The electrolyte is specifically prepared as follows: 0.0024 g of Tyr-Gly is dissolved in 10 mL of ultrapure water to prepare a 1 mM Tyr-Gly aqueous solution, and then Tyr-Gly is diluted with 0.01 M KCl solution to a final concentration of 1000 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.5 pM, 0.1 pM, 0.01 pM, 0.001 pM, respectively.

[0084] Figure 6 The IV curve of the current response of the biomolecular sensor based on cucurbit [7] uril functionalized nanopore to different dipeptide Tyr-Gly concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the dipeptide Tyr-Gly concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected dipeptide Tyr-Gly ion current change rate (△I / I0) has a good linear relationship with the logarithm of the dipeptide Tyr-Gly solution concentration. In the concentration range of 10fM to 1nM, the linear fitting equation for detecting dipeptide Tyr-Gly is △I / I0=0.374lg[C Tyr-Gly / 10 -15 M]-0.174, R 2 =0.996; LOD is 1.172fM.

[0085] Example 7

[0086] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect the pentapeptide phenylalanine-glycine-glycine-glycine-glycine (PGGGG), which specifically includes the following steps:

[0087] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0088] 2) Detection of PGGGG molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of PGGGG. The electrolyte is specifically prepared as follows: 0.0039 g of PGGGG is dissolved in 10 mL of ultrapure water to prepare a 1 mM PGGGG aqueous solution, and then PGGGG is diluted with 0.01 M KCl solution to a final concentration of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, 0.001 nM, respectively.

[0089] Figure 7The IV curve of the current response of the biomolecule sensor based on cucurbit [7] uril functionalized nanopore to different pentapeptide PGGGG concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the pentapeptide PGGGG concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected pentapeptide PGGGG ion current change rate (△I / I0) and the logarithm of the pentapeptide PGGGG solution concentration have a good linear relationship. In the concentration range of 10pM to 500nM, the linear fitting equation for detecting pentapeptide PGGGG is △I / I0=0.522lg[C PGGGG / 10 -12 M]+0.434, R 2 =0.984; LOD is 1.834pM.

[0090] Example 8

[0091] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect bovine serum albumin (BSA), which specifically includes the following steps:

[0092] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0093] 2) Detection of BSA molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of BSA. The electrolyte is specifically prepared as follows: 0.64 g of BSA is dissolved in 10 mL of ultrapure water to prepare a 1 mM BSA aqueous solution, and then BSA is diluted with 0.01 M KCl solution to a final concentration of 1000 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.5 pM, 0.1 pM, 0.01 pM, and 0.001 pM, respectively.

[0094] Figure 8 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different BSA concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the BSA concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected BSA ion current change rate (△I / I0) has a good linear relationship with the logarithm of the BSA solution concentration. In the concentration range of 100fM to 1nM, the linear fitting equation for detecting BSA is △I / I0=0.441lg[C BSA / 10 -15 M]-0.677, R 2 =0.98; LOD is 48.41pM.

[0095] Example 9

[0096] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect insulin, and specifically includes the following steps:

[0097] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1).

[0098] 2) Detection of insulin. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of insulin. The electrolyte is specifically prepared as follows: 0.057 g of insulin is dissolved in 10 mL of ultrapure water to prepare a 1 mM insulin aqueous solution, and then the insulin is diluted with a 0.01 M KCl solution to a final concentration of 1000 pM, 100 pM, 50 pM, 10 pM, 1 pM, 0.5 pM, 0.1 pM, 0.01 pM, and 0.001 pM, respectively.

[0099] Fig. 9 The IV curve of the current response of the biomolecular sensor based on cucurbit[7]uril functionalized nanopore to different insulin concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the insulin concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected insulin ion current change rate (△I / I0) has a good linear relationship with the logarithm of the insulin solution concentration. In the concentration range of 100fM to 1nM, the linear fitting equation for detecting insulin is △I / I0=0.956lg[C 胰岛素 / 10 -15 M]+0.046, R 2 =0.989; LOD is 6.62fM.

[0100] Example 10

[0101] This example determines the feasibility of preparing a biomolecular sensor for detecting amino acids (taking tyrosine as an example) using a cucurbit[7]uril-functionalized nanopore under the condition of MPTS: anhydrous ethanol = 1:14 (v / v). Specifically, the following steps are included:

[0102] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1); the difference is that the ratio of MPTS:anhydrous ethanol is 1:14 (v / v).

[0103] 2) The detection of tyrosine is the same as in Example 2), except that the sensor is a biomolecular sensor with cucurbit[7]uril functionalized nanopore prepared in this example.

[0104] Fig.10 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Tyr concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of Tyr concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Tyr ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Tyr solution concentration. In the concentration range of 0.05nM to 100nM, the linear fitting equation for detecting Tyr is △I / I0=0.072lg[C Tyr / 10 -12 M]-0.033, R 2 =0.990; limit of detection (LOD) is 20.31 pM.

[0105] Embodiment 11

[0106] This example determines the feasibility of preparing a biomolecular sensor for detecting amino acids (taking tyrosine as an example) using a cucurbit[7]uril functionalized nanopore under the condition of MPTS: anhydrous ethanol = 1:24 (v / v). Specifically, the following steps are included:

[0107] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1); the difference is that the ratio of MPTS:anhydrous ethanol is 1:24 (v / v).

[0108] 2) The detection of tyrosine is the same as in Example 2), except that the sensor is a biomolecular sensor with cucurbit[7]uril functionalized nanopore prepared in this example.

[0109] Fig.11 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Tyr concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of Tyr concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Tyr ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Tyr solution concentration. In the concentration range of 0.05nM to 100nM, the linear fitting equation for detecting Tyr is △I / I0=0.040lg[C Tyr / 10 -12 M]+0.042, R 2 =0.995; limit of detection (LOD) is 31.18 pM.

[0110] Example 12

[0111] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect phenylalanine (Phe), which specifically includes the following steps:

[0112] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1.

[0113] 2) Detection of Phe molecules. This step is the same as the Tyr detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of Phe. The electrolyte is specifically prepared as follows: 0.0017 g of Phe is dissolved in 10 mL of ultrapure water to prepare a 1 mM Phe aqueous solution, and then Phe is diluted with a 0.01 M KCl solution to a final concentration of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, 0.001 nM.

[0114] Fig.12 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Phe concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the Phe concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Phe ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Phe solution concentration. In the concentration range of 0.001nM to 50nM, the linear fitting equation for detecting Phe is △I / I0=0.123lg[C His / 10 -12 M]+0.111, R 2 =0.997; LOD is 0.12pM.

[0115] Embodiment 13

[0116] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect tryptophan (Trp), which specifically includes the following steps:

[0117] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1.

[0118] 2) Detection of Trp molecules. This step is the same as the Trp detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of Trp. The electrolyte is specifically prepared as follows: 0.0020 g of Trp is dissolved in 10 mL of ultrapure water to prepare a 1 mM Trp aqueous solution, and then Trp is diluted with a 0.01 M KCl solution to a final concentration of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, and 0.001 nM, respectively.

[0119] Fig.13 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Trp concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the Trp concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Trp ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Trp solution concentration. In the concentration range of 0.01nM to 10nM, the linear fitting equation for detecting Trp is △I / I0=1.891g[C His / 10 -12 M]-1.833, R 2 =0.982; LOD is 9.12pM.

[0120] Embodiment 14

[0121] This example determines the feasibility of using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores to detect lysine (Lys), which specifically includes the following steps:

[0122] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1.

[0123] 2) Detection of Lys molecules. This step is the same as the Lys detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of Lys. The electrolyte is specifically prepared as follows: 0.0015 g of Lys is dissolved in 10 mL of ultrapure water to prepare a 1 mM Lys aqueous solution, and then Lys is diluted with a 0.01 M KCl solution to a final concentration of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, and 0.001 nM, respectively.

[0124] Fig.14The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Lys concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of the Lys concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Lys ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Lys solution concentration. In the concentration range of 0.001nM to 1nM, the linear fitting equation for detecting Lys is △I / I0=0.181g[C His / 10 -12 M]+0.017, R 2 =0.993; LOD is 0.81pM.

[0125] Embodiment 15

[0126] This example determines the feasibility of detecting proline (Pro) using a biomolecular sensor based on cucurbit[7]uril functionalized nanopores, which specifically includes the following steps:

[0127] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1.

[0128] 2) Detection of Pro molecules. This step is the same as the Pro detection step in Example 2, except that the electrolyte is a KCl solution containing different concentrations of Pro. The electrolyte is specifically prepared as follows: 0.0012 g of Pro is dissolved in 10 mL of ultrapure water to prepare a 1 mM Pro aqueous solution, and then Pro is diluted with a 0.01 M KCl solution to a final concentration of 1000 nM, 100 nM, 50 nM, 10 nM, 1 nM, 0.5 nM, 0.1 nM, 0.01 nM, 0.001 nM, respectively.

[0129] Fig.15 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Pro concentrations (left) and the linear relationship between the ion current change rate (△I / I0) and the logarithm of Pro concentration (right) in this embodiment, where the current value taken in the ion current change rate is the current value when the voltage is -1V. The results show that the detected Pro ion current change rate (△I / I0) has a good linear relationship with the logarithm of the Pro solution concentration. In the concentration range of 0.001nM to 10nM, the linear fitting equation for detecting Lys is △I / I0=1.401g[C His / 10 -12 M]+0.056, R 2 =0.985; LOD is 0.91pM.

[0130] Comparative Example 1

[0131] This comparative example is to illustrate the effect of the amount of MPTS on the detection results. The concentration of MPTS is increased to MPTS: anhydrous ethanol = 1:9 (v / v), and a biomolecular sensor based on cucurbitacin [7] uril functionalized nanopore is constructed. The detection of dipeptide amino acid-glycine (Tyr-Gly) is taken as an example to illustrate the detection effect. Specifically, the following steps are included:

[0132] 1) The steps for preparing the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1), except that the concentration of the MPTS ethanol solution in step c) is MPTS: anhydrous ethanol = 1:9 (v / v).

[0133] 2) Detection of Tyr-Gly: This step is the same as the Tyr-Gly detection step in Example 6.

[0134] Fig.16 The IV curve of the current response of the biomolecule sensor based on cucurbit[7]uril functionalized nanopore to different Tyr-Gly concentrations in this comparative example shows that the cucurbit[7]uril functionalized nanopore prepared by this method does not have a linear response to Tyr-Gly molecules at 1nM and 1000nM concentrations, and cannot quantitatively detect Tyr-Gly. This indicates that the nanopore sensor constructed under this condition cannot achieve the purpose of quantitative detection.

[0135] Comparative Example 2

[0136] This comparative example is to illustrate the effect of the modification time of MPTS on the detection results. The incubation time of MPTS is increased to 12 h, and a biomolecular sensor based on cucurbit[7]uril functionalized nanopore is constructed. Specifically, the following steps are included:

[0137] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1, except that: in step c), the incubation time of the MPTS ethanol solution in the nanopores is 12 h.

[0138] 2) Electrochemical characterization of cucurbit[7]uril functionalized nanopores. This step is the same as step 2 in Example 1.

[0139] Fig.17The IV curves of the nanopores at different modification stages for the biomolecule sensor based on cucurbit[7]uril functionalized nanopores in this comparative example under the condition of 12 hours of MPTS incubation time are shown. The blank nanopore exhibits good rectification properties. After incubation with MPTS for 12 hours, the IV curve of the nanopore approaches a straight line and the rectification properties disappear. This is due to excessive MPTS blocking the nanopores due to the long modification time. After modification with GNP@CB[7], there is still no rectification property. This indicates that under this condition (MPTS incubation for 12 hours), an ideal nanopore sensor cannot be obtained and the analyte cannot be detected.

[0140] Comparative Example 3

[0141] This comparative example is to illustrate the effect of the modification time and concentration of MPTS on the detection results. The concentration of MPTS is set to MPTS: anhydrous ethanol = 1:9, the incubation time is 12h, and a biomolecule sensor based on cucurbit [7] uril functionalized nanopore is constructed. Specifically, the following steps are included:

[0142] 1) The preparation steps of the cucurbit[7]uril functionalized nanopores used in this example are the same as those in Example 1, except that: in step c), the concentration of MPTS is MPTS: anhydrous ethanol = 1:9; and the incubation time of the MPTS ethanol solution in the nanopores is 12 h.

[0143] 2) Electrochemical characterization of cucurbit[7]uril functionalized nanopores. This step is the same as step 2 in Example 1.

[0144] Fig.18 For the biomolecule sensor based on cucurbit[7]uril functionalized nanopore in this comparative example, the IV curve of the nanopore before and after modification with MPTSQ under the conditions of MPTS concentration of MPTS: anhydrous ethanol = 1:9 and incubation time of 12 hours. The blank nanopore exhibits good rectification characteristics. After modification with MPTS, the IV curve of the nanopore approaches a straight line and the rectification characteristics disappear. This indicates that an ideal nanopore sensor cannot be obtained under this condition and the analyte cannot be detected.

[0145] The above results indicate that the cucurbit[7]uril-functionalized nanopore biomolecular sensor designed in the present invention can achieve highly sensitive detection of a variety of amino acids, biogenic amines, peptides and proteins through host-guest recognition and electrochemical detection; it can effectively solve the limitations and shortcomings of existing biomolecular detection technologies and has a very important reference significance for the detection and application of disease-related biomarker molecules.

[0146] It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these modifications and improvements all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A biomolecular sensor based on cucurbit[7]uril functionalized nanopores, characterized in that: The sensor comprises a conical nanopore formed on a quartz glass tube and an assembly of mercaptopropyltrimethoxysilane, gold nanoparticles and cucurbit[7]uril which are sequentially modified in the nanopore.

2. The method for preparing a biomolecule sensor according to claim 1, characterized in that: The steps include: Nanopores are formed in a quartz glass tube by a laser firing process; Injecting mercaptopropyltrimethoxysilane solution into the tip of the nanopore, followed by rinsing, soaking, washing, and drying in sequence to achieve mercapto modification of the inner wall of the nanopore; The gold nanoparticle solution and the cucurbitac[7]uril solution are mixed to form an assembly of gold nanoparticles and cucurbitac[7]uril, which is then injected into the thiol-modified nanopore tip for incubation, washing, and drying.

3. The method for preparing a biomolecule sensor according to claim 2, characterized in that: The mercaptopropyl trimethoxysilane solution is prepared by dissolving mercaptopropyl trimethoxysilane in anhydrous ethanol at a volume ratio of 1:14 to 1:24, and the soaking time is 1 to 6 hours.

4. The method for preparing a biomolecule sensor according to claim 2, wherein: The particle size of the gold nanoparticles is 4-20 nm.

5. The method for preparing a biomolecule sensor according to claim 2, wherein: The incubation time is 8-15h.

6. Use of the biomolecular sensor according to claim 1 in identifying amino acids, biogenic amines, peptides and proteins.

7. The use according to claim 5, characterized in that The amino acid is one or more of tyrosine, histidine, phenylalanine, tryptophan, lysine, proline and glycine.

8. The use according to claim 5, characterized in that The biogenic amine is one or more of 1,6-hexanediamine, cadaverine, putrescine, n-hexylamine, spermine, spermidine, dihexylamine, tyramine, histamine and creatinine.

9. The use according to claim 5, characterized in that The polypeptide is one or more of the dipeptides Phe-Gly, Gly-Phe, Tyr-Gly, Gly-Tyr and the pentapeptides PGGGG, LGGGG, GGGGG.

10. The use according to claim 5, characterized in that The protein is one or more of bovine serum albumin, human serum albumin and insulin.

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