A biomarker detection device and its production and use method

Abstract

The present invention provides a biomarker detection device and a method for producing and using the same. The present invention constructs a nanochannel response platform for oral squamous cell-related joint markers, and constructs a dual-marker detection system based on the response platform to simultaneously detect the concentration changes of biomarkers in the saliva of patients with oral squamous cell carcinoma, thereby achieving the purpose of early diagnosis, metastasis or recurrence monitoring of oral squamous cell carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of early detection materials for oral squamous cell carcinoma, and in particular to a biomarker detection device and a production and use method thereof. Background Art

[0002] Oral cancer can cause cosmetic and functional defects in patients, seriously affecting their quality of life. 90%-95% of oral cancers are oral squamous cell carcinoma (OSCC). However, approximately 60% of OSCC cases are diagnosed at an advanced stage. Delayed detection of local tumor recurrence and metastasis will significantly reduce the effectiveness of clinical treatment and the survival rate of cancer. Early screening, early diagnosis and treatment of OSCC are the key to improving patient survival. Currently, relevant early diagnosis technologies include tissue biopsy and tissue fluid-related detection and analysis.

[0003] Existing OSCC detection methods include: (1) Tissue biopsy: The surgery is an invasive procedure with complicated steps and long time consumption. It may cause the risk of accelerated tumor growth and metastasis, which is not conducive to high-frequency and large-scale screening diagnosis; (2) Tissue fluid examination: Commonly used body fluid biomarker detection methods mainly include protein spectrum, gene analysis and enzyme-linked immunosorbent assay, but the detection equipment is expensive and the operation steps are complicated. The sensitivity is low and the detection threshold can only reach 10 -9 g / ml, it is impossible to detect early oral cancer tumor markers with saliva levels of ng / ml and pg / ml, and it is not suitable for the popularization of early large-scale screening of OSCC markers. Therefore, exploring a non-invasive and reproducible early assessment method is crucial for the detection and development of oral-related cancers and even the entire cancer field.

[0004] In recent years, relevant studies have shown that biological fluid examinations for the detection of oral cancer biomarkers may be a new technology and method for the early diagnosis of oral cancer. However, the concentration of some markers themselves in saliva is low and difficult to detect through testing. Currently commonly used cancer liquid biopsy methods, including enzyme-linked immunosorbent assay, enzyme-amplified lanthanide luminescence, immunopolymerase chain reaction analysis and selective reaction monitoring mass spectrometry, still cannot detect the concentration increase of biomarkers in saliva specifically and sensitively within the detection range and detection change interval.

[0005] Therefore, there is still a need to develop a salivary dual-marker biopsy technology that is rapid, non-invasive, economical, and simple to meet the needs of clinical applications, so as to improve the predictive prognosis monitoring of oral squamous cell carcinoma and guide clinical treatment strategies. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a biomarker detection device and a method for producing and using the same. Specifically, the present invention includes the following contents.

[0007] According to a first aspect of the present invention, a biomarker detection device is provided, which comprises an electrolytic cell and a first nanochannel array and a second nanochannel array connected in series, wherein: the first nanochannel array and the second nanochannel array each comprise a matrix membrane, a response layer having through holes, and a plurality of nanochannels regularly arranged along the thickness direction of the matrix membrane, the response layer is arranged on one side of the matrix membrane to form a blocking end of a plurality of nanochannels, the surface of the response layer is connected to a marker responsive molecule, and the marker responsive molecule can block ions from passing through the response layer.

[0008] In certain embodiments, according to the biomarker detection device of the present invention, the marker is a disease-related combined marker, which includes a first marker and a second marker.

[0009] In certain embodiments, according to the biomarker detection device of the present invention, the disease includes oral squamous cell carcinoma and / or oral squamous cell carcinoma with lymph node metastasis.

[0010] In certain embodiments, according to the biomarker detection device of the present invention, the response layer surface of the first nanochannel array is covalently linked to the responsive molecules of the first marker, and the response layer surface of the second nanochannel array is covalently linked to the responsive molecules of the second marker.

[0011] In certain embodiments, the biomarker detection device according to the present invention further comprises an anode and a cathode, wherein the first nanochannel array and the second nanochannel array are respectively arranged between the anode and the cathode, and the response layer of the first nanochannel array faces the open ends of the plurality of nanochannels in the second nanochannel array.

[0012] In certain embodiments, the biomarker detection device according to the present invention further comprises an electrolyte.

[0013] In certain embodiments, according to the biomarker detection device of the present invention, the inner diameter of the nanochannel is 40-70 nm, and the inner diameter of the through hole is 0.5-2 nm.

[0014] In certain embodiments, the biomarker detection device according to the present invention further comprises at least one selected from the following:

[0015] a current detection unit configured to detect current intensity and / or changes;

[0016] a determination unit configured to compare the measured current value with a preset value or a standard value, thereby determining the amount of the biomarker in the sample based on the comparison result; and

[0017] The display device is configured to display the detection result or the judgment result of the judgment unit.

[0018] In certain embodiments, according to the biomarker detection device of the present invention, the responsive molecule is a polypeptide or a functional fragment thereof.

[0019] A second aspect of the present invention provides a method for producing a biomarker detection device, comprising:

[0020] (1) Hydroxylating the surface of the response layer of the nanochannel array;

[0021] (2) performing silanization modification on the surface of the hydroxylated response layer;

[0022] (3) Respectively coupling the polypeptides responsive to the first marker and the second marker to the silanized response layer, thereby performing polypeptide modification on the first nanochannel array and the second nanochannel array.

[0023] In certain embodiments, according to the method for producing a biomarker detection device of the present invention, the conditions for modifying the polypeptide include at least one of the following:

[0024] (a) The reaction temperature is 20-45°C;

[0025] (b) the peptide concentration is 0.1-5 μM; and

[0026] (c) The reaction time is 1-5 h.

[0027] According to a third aspect of the present invention, a method for using a biomarker detection device is provided, comprising: applying a processed biological sample to a response layer of the biomarker detection device, applying a voltage to the biomarker detection device, and collecting a scanning current.

[0028] In certain embodiments, according to the method for using the biomarker detection device of the present invention, the sample is a fluid sample.

[0029] The present invention constructs a nanochannel response platform, and constructs a dual-marker detection system based on the response platform to simultaneously detect the concentration changes of biomarkers in the saliva of patients with oral squamous cell carcinoma, thereby achieving the purpose of early diagnosis, metastasis or recurrence monitoring of oral squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The cross section of the porous anodized aluminum nanochannel and the electron microscope morphology at both ends are shown.

[0031] Figure 2 The electrochemical performance characterization of porous anodic aluminum oxide nanochannels is shown.

[0032] Figure 3 A schematic diagram showing the construction of the porous anodized aluminum responsive nanochannel platform.

[0033] Figure 4 The optimization of modification conditions for constructing a porous anodic aluminum oxide responsive nanochannel platform is shown.

[0034] Figure 5 The detection current value detection standard of the matrix metalloproteinase-1 and matrix metalloproteinase-3 responsive nanochannel platform is shown.

[0035] Figure 6 The marker detection performance of the matrix metalloproteinase-1 and matrix metalloproteinase-3 responsive nanochannel platform is shown.

[0036] Figure 7 The current detection standard of the MMP-1 / MMP-3 responsive platform tandem system is shown.

[0037] Figure 8 The marker detection performance of the matrix metalloproteinase-1 / matrix metalloproteinase-3 responsive platform tandem system is shown.

[0038] Fig. 9 The marker detection performance of the matrix metalloproteinase-1 / matrix metalloproteinase-3 responsive platform tandem system for clinical saliva samples is shown. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.

[0041] In addition, the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", "top", "bottom", etc. in the specification and claims indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention. It should be understood that the terms used in this way are interchangeable under appropriate conditions, and the embodiments of the present invention described herein can be operated in other orientations than those described or illustrated herein.

[0042] Detection device

[0043] In one aspect of the present invention, a detection device for oral squamous cell carcinoma combined with biomarkers is provided. The device of the present invention comprises an electrolytic cell and a first nanochannel array and a second nanochannel array connected in series, wherein the first nanochannel array and the second nanochannel array each comprise a matrix membrane, a response layer having through holes, and a plurality of nanochannels regularly arranged along the thickness direction of the matrix membrane, one side of the first nanochannel array is a first opening end, and the other side is a first response layer, and one side of the second nanochannel array is a second opening end, and the other side is a second response layer.

[0044] In the present invention, the first responsive nanochannel array and the second responsive nanochannel array contain a plurality of nanochannels arranged in a honeycomb shape, and the channels are uniformly arranged in a parallel or substantially parallel manner, and the channels are typical hexagonal holes, which can also be channels with other shapes, such as circular or square holes, which are not particularly limited. The channels can be channels with consistent diameters, or channels with non-uniform diameters, such as channels with a diameter on one side greater than the diameter on the other side, thereby forming a channel similar to a truncated cone. The inner diameter of the responsive nanoarray channel is a nanochannel of 40-70 nm, and preferably 45-65 nm, such as 45, 50, 55, 60, 65 nm.

[0045] In the present invention, one end of the nanochannel is an open end for ions to enter, and the other end is a response layer, the response layer having a nanochannel with an inner diameter of 0.5-2 nm, wherein the inner diameter of the response layer needs to be smaller than the inner diameter of the nanoarray channel, so that the ion flow can be blocked after modification on the outside of the response layer. In a preferred embodiment, the inner diameter of the response layer is preferably 0.5-1.5 nm, more preferably 0.5-1.2 nm, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 nm.

[0046] In the present invention, when the series connection is performed, the distance between the first nanochannel array and the second nanochannel array is not particularly limited, and the series connection of the two layers of membrane materials in which they are located can increase the resistance, and the inventors have found that when two joint markers need to be detected at the same time, only setting a single nanochannel array cannot achieve the purpose of the present invention, or when any biomarker exists in the sample, the purpose of the present invention cannot be achieved. In addition, the present invention has conducted a lot of research on the conditions for modification according to the current change rate, and finally determined a modification scheme with a detection limit of 0.1 pg / ml.

[0047] In the present invention, the response layer is connected with a marker responsive molecule, and the marker responsive molecule includes a polypeptide or a functional fragment thereof that can respond to an oral squamous cell carcinoma biomarker. The term "response" means that the molecule located on the surface of the response layer can perform at least one reaction selected from the recognition, binding, and change of the biomarker, such as the marker responsive molecule changes its structure under the interaction with the biomarker, especially cleavage (or cutting), so that the ion flow through the series nanochannel array changes, and this change can be detected by the current change rate. It should be noted that the marker responsive molecule is located on the outside of the response layer, that is, the marker responsive molecule is not located in the nanochannel, but is connected to the outer surface of the response layer. If it is located in the pore, it will cause the detected current change rate to be unclear, which is not conducive to the detection of biomarkers.

[0048] In the present invention, the marker responsive molecule can be a polypeptide or a functional fragment thereof, and the polypeptide or the functional fragment thereof can be connected to the response layer in a suitable manner, preferably connected to the modified response layer, and the modification of the response layer is preferably silanization modification, so that the amino acid residues and the silane can be coupled to each other. In a specific embodiment, the silanization modification is achieved by the following steps:

[0049] (1) using a hydrogen peroxide solution (concentration 10-50%, preferably 20-40%) to heat and boil the porous anodized aluminum membrane for 0.5-5 h (preferably 1-4 h, and more preferably 1-3 h) to hydroxylate the surface of the response layer;

[0050] (2) Treating the response layer with a silanization agent, and then treating the response layer with a cross-linking agent containing thiol and amine reactive groups to complete the silanization treatment. The silanization agent can be any aminosilane capable of introducing an amino group, including but not limited to, for example, APTES (ethanol as solvent, concentration of 0.1-10%, preferably 1-8%, and preferably 3-6%) solution. The cross-linking agent can be any cross-linking agent containing a thiol group capable of reacting with an amino acid residue and a group capable of reacting with an amino group, including but not limited to, for example, N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS, with DMSO as solvent, 0.05-2 nM, preferably 0.1-1 mM, and preferably 0.1-0.5 mM). The silanization agent treatment time is 0.5-5 h, preferably 1-4 h, for example, 1, 2, 3, 4 h. The crosslinking agent treatment time is 5-60 min, preferably 10-60 min, for example 10, 20, 30, 40, 50, 60 min.

[0051] The porous anodized aluminum film in step (1) can be purchased from commercial products, or a response layer having through-holes of 0.5-2 nm can be obtained by anodization. The thickness of the porous anodized aluminum film is 10-100 μm, preferably 20-70 μm.

[0052] In the present invention, after silanization modification, the responsive molecule is further coupled to the treated membrane material to perform polypeptide modification on the material. The present invention conducts in-depth research on the modification process and develops modification conditions that can simultaneously detect combined markers with a lower detection limit and a higher positive detection rate based on the current change rate. The markers of the present invention include a first marker and a second marker, which are used for the detection of oral squamous cell carcinoma and / or oral squamous cell carcinoma with lymph node metastasis. Currently, many studies have shown that the increased levels of two matrix metalloproteinases -1 and matrix metalloproteinase -3 in saliva are relevant markers for predicting the diagnosis and metastasis of oral squamous cell carcinoma. The onset, metastasis and prognosis of oral squamous cell carcinoma can be diagnosed by monitoring the changes in the concentrations of the two in saliva.

[0053] The present invention first designs a specific polypeptide sequence CKPLG↓LKAR for the first marker (matrix metalloproteinase-1) and a specific polypeptide sequence CRPKPVE↓VWRK for the second marker (matrix metalloproteinase-3) (arrows indicate enzyme cleavage sites on the polypeptide sequence). Secondly, the present invention determines the following polypeptide modification conditions, so as to achieve detection with a lower detection limit and a higher positive detection rate when performing joint detection of markers, wherein the reaction temperature of polypeptide modification is 20-45°C, preferably 22-40°C, also preferably 23-35°C, and further preferably 23-30°C, for example 23, 24, 25, 26, 27, 28, 29, 30°C. The concentration of the polypeptide should not be too high or too low, preferably 0.1-5 μM, preferably 0.2-4 μM, further preferably 0.3-1 μM, more preferably 0.35-0.95 μM, such as 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95 μM. The time for the polypeptide to treat the response layer is 1-5 h, preferably 1-4 h, further preferably 1-3.8 h, further preferably 1.2-3.8 h, such as 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8h.

[0054] In a specific embodiment, the first response layer is connected to a polypeptide responsive to matrix metalloproteinase-1, and the second response layer is connected to a polypeptide responsive to matrix metalloproteinase-3. In another embodiment, the first response layer is connected to a polypeptide responsive to matrix metalloproteinase-3, and the second response layer is connected to a polypeptide responsive to matrix metalloproteinase-1.

[0055] In the present invention, the sample to be detected is a fluid sample, in particular a saliva sample.

[0056] In the present invention, the detection device also includes a voltage providing unit and an electrode and an electrolyte solution arranged in the electrolytic cell, wherein the series connection means that the first nanochannel array and the second nanochannel array are respectively arranged between the anode and the cathode, and the electrode is an Ag / AgCl electrode. Preferably, the anode faces the response layer side of the nanochannel array, and the cathode faces the orifice side (open end) of the nanochannel array, that is, the opposite side of the response layer. The electrolyte solution is standard 0.1 M KCl. The voltage providing unit can use any battery component or power supply component that can provide -2 V to +2 V.

[0057] In the present invention, the first nanochannel array and the second nanochannel array in the series structure can be connected in series in any manner. In a preferred embodiment, the first open end of the first nanochannel array is arranged in a manner close to the second response layer of the second nanochannel array, or the second open end of the second nanochannel array is arranged in a manner close to the first response layer of the first nanochannel array.

[0058] The detection device of the present invention may also include at least one selected from the following:

[0059] a current detection unit configured to detect current intensity and / or changes;

[0060] a determination unit configured to compare the measured current value with a preset value or a standard value, thereby determining the amount of the biomarker in the sample based on the comparison result; and

[0061] The display device is configured to display the detection result or the judgment result of the judgment unit.

[0062] In the present invention, the preset value or standard value can be the current change rate detected by the biomarker of healthy people. In addition, the judgment standard of the judgment unit is: when the absolute value of the detected current is <1 μA, the subject is judged as a healthy person, when the absolute value of the detected current is 1-2 μA, the subject is judged as a patient with oral squamous cell carcinoma or a patient at high risk of the disease, and when the absolute value of the detected current is >2 μA, the subject is judged as a patient with oral squamous cell carcinoma with lymph node metastasis or a patient at high risk of the disease.

[0063] Production method

[0064] In one aspect of the present invention, a method for producing or manufacturing a biomarker detection device is provided, comprising:

[0065] (1) Hydroxylating the surface of the response layer of the nanochannel array;

[0066] (2) performing silanization modification on the surface of the hydroxylated response layer;

[0067] (3) Respectively coupling the polypeptides that recognize the first marker and the second marker to the silanized response layer, thereby performing polypeptide modification on the first nanochannel array and the second nanochannel array.

[0068] The biomarker detection device obtained by the above method can be in the form of a kit, that is, it contains at least the following components for completing the detection: an electrolytic cell, electrodes, a first nanochannel array and a second nanochannel array located between the electrodes, or the biomarker detection device obtained by the above method can only contain a first nanochannel array and a second nanochannel array for detecting combined markers.

[0069] How to use

[0070] In one aspect of the present invention, a method for using a biomarker detection device is provided, comprising:

[0071] (1) placing the first nanochannel array and the second nanochannel array between an anode and a cathode to form a series structure in an electrolytic cell, preferably, the anode faces the response layer side of the nanochannel array, and the cathode faces the orifice side (open end) of the nanochannel array, i.e., the opposite side of the response layer;

[0072] (2) adding an electrolyte solution to the electrolytic cell;

[0073] (3) applying the processed biological sample to the response layer of the biomarker detection device, applying a voltage to the biomarker detection device, and collecting a scanning current at the same time.

[0074] The method of use of the present invention further comprises the step of sealing the nanochannel array using a sealant, wherein ions flow only through the nanochannels therein, and the sealant and the sealing operation are known in the art and are not particularly limited thereto.

[0075] In the present invention, the method further includes a step of classifying the sample. When the detection value is lower than 0.316 μA, the sample is determined to be free of the first marker and the second marker. When the detection value is greater than 3 μA, the sample is determined to be present with both the first marker and the second marker.

[0076] Example 1

[0077] 1. Construction of single marker responsive nanochannels for matrix metalloproteinase-1 and matrix metalloproteinase-3

[0078] A porous anodized aluminum nanochannel film is used as the substrate material of the responsive nanochannel. The nanochannel opening end of the selected porous anodized aluminum nanochannel film is a closely arranged honeycomb channel with a diameter of 40-70 nm, and the other end is a response layer (oxide layer). There are a large number of sub-nanopores with a diameter of about 1 nm on the oxide layer. The structural electron microscope image is shown in the figure below. Figure 1 shown.

[0079] A pair of Ag / AgCl electrodes was used to apply a transmembrane potential on the membrane and measure the ion current to detect the ion rectification effect of the porous anodic aluminum oxide nanochannel film. The anode faced the barrier oxide layer side of the membrane, while the cathode faced the pore side. In a concentration gradient KCl electrolyte solution environment, a voltage of -2 V to +2 V was applied. The porous anodic aluminum oxide nanochannel film exhibited an ion rectification performance similar to the diode current-voltage response. And in a standard electrolyte solution of 0.1 M KCl, a voltage of ±1 V was alternately applied for up to 100 minutes. The porous anodic aluminum oxide nanochannel film was able to stably output a transmembrane ion current of about ±0.03 μA, such as Figure 2 shown.

[0080] First, the porous anodized aluminum nanochannel film was ultrasonically cleaned with ethanol and pure water to remove surface impurities. After drying, the ion current value from -2 V to 0 V was measured in the standard electrolyte solution 0.1 M KCl, and the current corresponding to the voltage of -2 V was taken as I0. Then, a 30% hydrogen peroxide solution was heated and boiled for 2 hours to hydroxylate the surface of the oxide layer, and the response layer was treated with a 4% APTES (ethanol as solvent) solution for 2 hours, and the response layer was treated with 0.25 mM GMBS (DMSO as solvent) for 30 minutes to complete the silanization treatment. The matrix metalloproteinase-1 specific peptide sequence CKPLG↓LKAR (matrix metalloproteinase-1 peptide) and the matrix metalloproteinase-3 specific peptide sequence CRPKPVE↓VWRK (matrix metalloproteinase-3 peptide) were designed as recognition motifs, respectively, in which the N-terminal cysteine ​​C was prepared for the silane coupling of the peptide sequence to the response layer surface of the porous anodized aluminum nanochannel film. The modification process is as follows: Figure 3 shown.

[0081] Taking the peptide CKPLG↓LKAR as an example, the optimal reaction conditions for peptide modification were explored. At room temperature 25°C, 0.1 μM, 0.3 μM, 0.5 μM, 1 μM, and 5 μM peptide solutions (pure water as solvent) were used to treat the response layer surface of the porous anodized aluminum nanochannel film for 2 h. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl of peptide solutions with different concentrations, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification, as shown in the figure, when the peptide concentration is 0.5 μM, R1=0.024, which is the optimal modification concentration. The ion current value I2 from -2 V to 0 V was measured after the peptide solution was treated on the response layer surface for 10 min, 30 min, 1 h, 2 h, and 4 h, respectively. The current change rate R2=I2 / I0 corresponding to the -2 V voltage before and after peptide modification, as shown in the figure. Figure 4As shown in the figure, when the reaction time is 2 h, R2=0.024, which is the optimal modification reaction time. Therefore, the preferred peptide concentration is 0.5 μM, and the preferred reaction time is 2 h. Under these conditions, matrix metalloproteinase-1 and matrix metalloproteinase-3 responsive nanochannels were constructed respectively.

[0082] 2. Detection criteria of matrix metalloproteinase-1 and matrix metalloproteinase-3 responsive nanochannels

[0083] According to the above steps, the oxide layer surface was modified with 0.5 μM matrix metalloproteinase-1 peptide for 2 h to construct a matrix metalloproteinase-1 responsive nanochannel. The ion current value of the applied voltage from -2 V to 0 V was measured in the standard electrolyte solution 0.1 M KCl, and the absolute current value I corresponding to -2 V was measured. 基质金属蛋白酶-1peptide =0.718 μA, e.g. Figure 5 Similarly, a matrix metalloproteinase-3 responsive nanochannel was constructed, and the ion current value from the applied voltage of -2 V to 0 V was measured in the standard electrolyte solution 0.1 M KCl, and the absolute value of the current corresponding to -2 V was measured. 基质金属蛋白酶-3peptide =0.616 μA, e.g. Figure 5 shown.

[0084] Then, a concentration gradient of matrix metalloproteinase-1 solution (5*10 -7 , 1*10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 The modified oxide layer of the matrix metalloproteinase-1 responsive nanochannel was immersed in the solution of 0.1 M KCl for 2 h, and the ion current value was measured from the applied voltage of -2 V to 0 V in the standard electrolyte solution. The current I corresponding to -2 V was measured. response They are 9.75, 5.57, 4.94, 4.5, 4.02, 3.42, 3.01, and 2.51 μA, respectively. The regression curve of the current change rate and the concentration gradient of matrix metalloproteinase-1 is drawn. The current change rate refers to: R3=(I esponse -I 基质金属蛋白酶-1peptide ) / I 基质金属蛋白酶-1peptide , and the regression curve of current change rate and matrix metalloproteinase-1 concentration is obtained, such as Figure 6 As shown. Similarly, the concentration gradient matrix metalloproteinase-3 solution (5*10 -7 , 1*10 -7 , 10 -8 , 10 -9 , 10 -10, 10 -11 , 10 -12 , 10 -13 g / ml) response performance, measured I response The current change rate and the concentration gradient of matrix metalloproteinase-3 were plotted as follows: R3=(I response -I 基质金属蛋白酶-3peptide ) / I 基质金属蛋白酶-3peptide , and the regression curve of current change rate and matrix metalloproteinase-3 concentration is obtained, such as Figure 6 As shown, the threshold for detecting the marker reached 0.1 pg / ml.

[0085] 3. Detection criteria for the dual marker response platform for matrix metalloproteinase-1 and matrix metalloproteinase-3

[0086] The matrix metalloproteinase-1 responsive nanochannel was placed on the left side of the electrolyte pool, and the matrix metalloproteinase-3 responsive nanochannel was placed on the right side (the open end was facing the response layer of the matrix metalloproteinase-1 responsive nanochannel, and Ag / AgCl electrodes were placed at both ends to form a series system, and the scanning current was collected when the applied voltage was -2 V to 2 V. In this system, when there was no matrix metalloproteinase-1 and matrix metalloproteinase-3 in the reaction solution at the same time, the detection current was 0.316 μA, and when there was only matrix metalloproteinase-1 or only matrix metalloproteinase-3, the detection current value was still small at 0.331 μA, with almost no change, and the current transmission of the series system was in the closed state. Only when matrix metalloproteinase-1 and matrix metalloproteinase-3 existed at the same time, the ion current value in the series system changed significantly and was in the open state, and the detection current was about 4.77 μA, as shown in Figure 2. Figure 7 As shown, the concentrations of matrix metalloproteinase-1 and matrix metalloproteinase-3 are monitored simultaneously.

[0087] Then, a concentration gradient of MMP-1 / MMP-3 mixed solution (5*10 -7 , 1*10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 The modified oxide layer of the matrix metalloproteinase-1 responsive nanochannel was immersed in the solution of 0.1 M KCl for 2 h, and the ion current value was measured from the applied voltage of -2 V to 0 V in the standard electrolyte solution. The current I corresponding to -2 V was measured. responseThey are 4.55, 2.41, 2.01, 1.71, 1.4, 1.05, 0.716, and 0.496 μA, respectively. The regression curve of the current change rate and the concentration gradient of matrix metalloproteinase-1 is drawn. The current change rate refers to: R3=(I response -I 基质金属蛋白酶-1peptide ) / I 基质金属蛋白酶-1peptide , and obtain the regression curve of current change rate and MMP-1 / MMP-3 mixed concentration, such as Figure 8 As shown, the threshold for detecting the marker reached 0.1 pg / ml.

[0088] 4. Clinical application of the matrix metalloproteinase-1 and matrix metalloproteinase-3 dual marker response platform

[0089] Saliva was collected from OSCC patients (divided into a group without lymph node metastasis and a group with lymph node metastasis based on postoperative pathological results) more than 2 hours after the end of eating. The patients were asked to rinse their mouths with normal saline and then collect 2 ml of saliva. The saliva sample was centrifuged at 1500rcf and 4°C for 5 minutes to obtain the upper supernatant. The upper supernatant was further centrifuged at 15000 rcf and 4°C for 5 minutes to obtain the upper supernatant. The upper supernatant was used to treat the surface of the responsive nanochannel response layer for 2 hours, and the detection current was detected. The detection and judgment criteria are: the absolute value of the detection current (-2 V) of the normal population is 0.56-0.94 μA; the result of patients with oral squamous cell carcinoma is: 1.03-1.73 μA; the result of patients with oral squamous cell carcinoma with lymph node metastasis is: 2.16-2.71 μA, such as Fig. 9 shown.

[0090] Example 2

[0091] Optimization of polypeptide modifications is shown below.

[0092] Optimization scheme 1: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.1 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 4 h. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.6.

[0093] Optimization scheme 2: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.3 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 4 h. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.336.

[0094] Optimization scheme 3: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 1 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 4 h. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.032.

[0095] Optimization scheme 4: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 2 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 4 h. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.045.

[0096] Optimization scheme 5: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.5 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) for 10 minutes at room temperature 25°C. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.47.

[0097] Optimization scheme 6: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.5 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 30 minutes. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.23.

[0098] Optimization scheme 7: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.5 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 1 hour. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.082.

[0099] Optimization scheme 8: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.5 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 2 hours. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.024.

[0100] Optimization scheme 9: After the porous anodic aluminum oxide nanochannel film is silanized, the surface of the response layer of the porous anodic aluminum oxide nanochannel film is treated with a 0.5 μM matrix metalloproteinase-1 peptide solution (pure water as solvent) at room temperature 25°C for 4 hours. By measuring the ion current value I1 from -2 V to 0 V in the standard electrolyte solution 0.1 M KCl with different concentrations of peptide solutions, the current change rate R1=I1 / I0 corresponding to the -2 V voltage before and after peptide modification is as follows: Figure 4 As shown, R1=0.027.

[0101] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A biomarker detection device, characterized in that: The invention comprises an anode, a cathode, an electrolytic cell and a first nanochannel array and a second nanochannel array connected in series, wherein: the first nanochannel array and the second nanochannel array each respectively comprise a matrix membrane, a response layer having through holes and a plurality of nanochannels regularly arranged along the thickness direction of the matrix membrane, the response layer is arranged on one side of the matrix membrane, the outer surface of the response layer is connected with a marker responsive molecule and the marker responsive molecule can block ions from passing through the response layer, one end of the nanochannel is an open end and the other end is a response layer, the first nanochannel array and the second nanochannel array are respectively arranged between the anode and the cathode, and the response layer of the first nanochannel array faces the open ends of the plurality of nanochannels in the second nanochannel array, the inner diameter of the nanochannel is 40-70 nm, and the inner diameter of the through hole is 0.5-2 nm.

2. The biomarker detection device according to claim 1, characterized in that: The marker is a disease-related combined marker, which includes a first marker and a second marker; the disease includes oral squamous cell carcinoma and / or oral squamous cell carcinoma with lymph node metastasis; the response layer surface of the first nanochannel array is covalently linked to the responsive molecule of the first marker, and the response layer surface of the second nanochannel array is covalently linked to the responsive molecule of the second marker.

3. The biomarker detection device according to claim 1, characterized in that: The detection device further includes an electrolyte.

4. The biomarker detection device according to claim 1, characterized in that: Further comprising at least one selected from the following: a current detection unit configured to detect current intensity and / or changes; a determination unit configured to compare the measured current value with a preset value or a standard value, thereby determining the amount of the biomarker in the sample based on the comparison result; and The display device is configured to display the detection result or the judgment result of the judgment unit.

5. The biomarker detection device according to claim 1, characterized in that: The responsive molecule is a polypeptide or a functional fragment thereof.

6. The biomarker detection device according to claim 4, characterized in that: The sample is a fluid sample.

7. The method for producing a biomarker detection device according to any one of claims 1 to 6, characterized in that: include: (1) Hydroxylating the surface of the response layer of the nanochannel array; (2) performing silanization modification on the surface of the response layer after hydroxylation treatment; (3) Respectively coupling the polypeptides responsive to the first marker and the second marker to the silanized response layer, thereby performing polypeptide modification on the first nanochannel array and the second nanochannel array.

8. The method according to claim 7, characterized in that The conditions for polypeptide modification include at least one of the following: (a) The reaction temperature is 20-45°C; (b) the concentration of the polypeptide is 0.1-5 μM; and (c) The reaction time is 1-5 h.

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