Kidney disease marker sensor, sensor preparation method and kidney disease marker detection method

Abstract

The present invention provides a sensor preparation method and a kidney disease marker detection method, wherein a kidney disease marker sensor for a specific kidney disease marker is formed by continuously introducing hydroxyl, amino, and aldehyde groups into a microchannel on the surface of a silicon nanowire field effect transistor biosensor to covalently connect probe molecules, and after the kidney disease marker is captured by the probe molecule, the kidney disease marker sensor applies a set voltage, and after the current is stable, the detection current is recorded, and according to the monitoring current of a kidney disease marker solution with a known set concentration and a kidney disease marker solution to be tested, it is determined whether the kidney disease marker is contained in the kidney disease marker solution to be tested and the kidney disease marker concentration is calculated. The detection steps are simplified and the detection accuracy is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kidney disease marker sensor, a sensor preparation method, and a kidney disease marker detection method. Background Art

[0002] Kidney diseases are divided into chronic and acute, both of which can damage the kidneys. As an important organ of human metabolism, the kidneys cannot perform normal metabolic functions after damage, and as the disease progresses, it will cause heart damage and cerebral vascular damage. When the disease develops to the late stage, uremia, myocardial infarction, cerebral infarction and other diseases will occur, seriously threatening the patient's life. If early clinical renal function damage can be discovered and treated in time, the early damage to the glomerular blood vessels can be reversed. However, if it is not diagnosed and effectively treated in time, when pathological changes such as extracellular matrix hyperplasia and thickening of the glomerular basement membrane occur, the condition cannot be reversed. Therefore, the detection of early markers for the early diagnosis and evaluation of kidney disease has always been a hot topic in the field of laboratory medicine.

[0003] Existing detection methods require complex sample preparation and handling of the analyte during the test, resulting in high testing costs, cumbersome experimental procedures, and the need for specialized personnel. Most importantly, these methods are time-consuming and lack rapid detection. Therefore, developing a rapid, low-cost, highly specific, and sensitive method for detecting kidney disease markers is of great significance. Summary of the Invention

[0004] In view of this, the embodiments of the present invention provide a kidney disease marker sensor, a sensor preparation method, and a kidney disease marker detection method to eliminate or improve one or more defects in the existing technology and solve the problems of complex preliminary processing and high detection cost for human serum cystatin C detection.

[0005] The technical solutions of the present invention are as follows:

[0006] In one aspect, the present invention provides a renal disease marker sensor, comprising:

[0007] A silicon nanowire field-effect transistor biosensor is disclosed. The silicon nanowires in the microchannels on the surface of the silicon nanowire field-effect transistor biosensor are provided with continuously introduced hydroxyl groups, amino groups and aldehyde groups. The aldehyde groups are covalently linked to probe molecules, which are antibodies for capturing kidney disease markers.

[0008] In some embodiments, the probe molecule is a human serum cystatin C antibody, and the kidney disease marker is a human serum cystatin C antigen.

[0009] In some embodiments, the silicon nanowire field-effect transistor biosensor uses a single crystal silicon layer as a substrate, a silicon dioxide layer is provided on the substrate as an insulating layer, a silicon nanowire is provided on the insulating layer, a metal film is deposited at both ends of the silicon nanowire as a source and a drain, and polydimethylsiloxane is filled between the source, the drain and the silicon nanowire as a microchannel.

[0010] In another aspect, the present invention further provides a method for preparing a renal disease marker sensor, comprising:

[0011] A silicon nanowire field-effect transistor biosensor is obtained, wherein the silicon nanowire field-effect transistor biosensor uses a single crystal silicon layer as a substrate, a silicon dioxide layer as an insulating layer on the substrate, a silicon nanowire on the insulating layer, a metal film deposited at both ends of the silicon nanowire as a source and a drain, and polydimethylsiloxane is filled between the source, the drain, and the silicon nanowire as a microchannel;

[0012] Plasma cleaning the microchannel of the silicon nanowire field-effect transistor biosensor by oxygen plasma treatment to bond hydroxyl groups to the silicon nanowires; introducing an amino group introduction solution into the microchannel, allowing the solution to stand to introduce amino groups to the hydroxyl groups, and performing a thermal annealing treatment;

[0013] introducing an aldehyde group introduction solution into the microfluidic channel and allowing the solution to stand to introduce an aldehyde group onto the amino group;

[0014] A probe molecule solution is introduced into the microchannel and allowed to stand to introduce probe molecules onto the aldehyde groups. The probe molecules are antibodies for capturing kidney disease markers, so as to obtain the kidney disease marker sensor.

[0015] In some embodiments, the amino group introduction solution is 3-aminopropyltriethoxysilane anhydrous ethanol solution; the aldehyde group introduction solution is glutaraldehyde solution, and the probe molecule solution is human serum cystatin C antibody buffer.

[0016] In some embodiments, an amino group-introducing solution is introduced into the microchannel, allowed to stand to introduce amino groups onto the hydroxyl groups, and then subjected to thermal annealing treatment, comprising:

[0017] The 3-aminopropyltriethoxysilane anhydrous ethanol solution was passed into the microchannel, and after being stored at room temperature for 1 hour, it was washed with ethanol and deionized water and placed in a rapid annealing furnace for annealing at 150° C. for 30 minutes;

[0018] An aldehyde group introduction solution is introduced into the microfluidic channel and allowed to stand to introduce an aldehyde group on the amino group, comprising:

[0019] The glutaraldehyde solution was passed into the microchannel and sealed in the dark at 37°C for 45 minutes;

[0020] A probe molecule solution is introduced into the microfluidic channel and allowed to stand to introduce the probe molecule onto the aldehyde group, comprising:

[0021] The probe molecule solution was introduced into the microfluidic channel and cultured in a water bath at 37° C. for 45 minutes.

[0022] In some embodiments, plasma cleaning the microchannel of the silicon nanowire field effect transistor biosensor by oxygen plasma treatment, and bonding hydroxyl groups to the silicon nanowires, comprises:

[0023] The silicon nanowire field effect transistor biosensor was treated with oxygen plasma for 2 minutes using an oxygen plasma cleaning machine at a power of 15 W and a flow rate of 100 sccm;

[0024] The surface of the microchannel was treated with an oxygen plasma gun for 20 seconds to align and bond the microchannel with the channel of the silicon nanowire field effect transistor biosensor.

[0025] In some embodiments, after the probe molecule solution is introduced into the microfluidic channel and allowed to stand to introduce the probe molecules onto the aldehyde groups, the method further comprises:

[0026] introducing a phosphate buffer solution containing bovine serum albumin into the microchannel of the silicon nanowire field-effect transistor biosensor and allowing the solution to stand for treatment;

[0027] The microchannel is cleaned with a phosphate buffer solution.

[0028] On the other hand, the present invention also provides a method for detecting kidney disease markers, comprising:

[0029] A renal disease marker solution of a set concentration is introduced into the microchannel of the renal disease marker sensor, a set voltage is applied to the renal disease marker sensor, and a detection current is recorded as first electrical characteristic data after the current stabilizes;

[0030] Cleaning the renal disease marker sensor with a phosphate buffer solution;

[0031] A renal disease marker solution to be tested is introduced into the microchannel of the renal disease marker sensor, a set voltage is applied to the renal disease marker sensor, and the detection current is recorded as the second electrical characteristic data after the current stabilizes;

[0032] The distribution relationship between the first electrical characteristic data and the second electrical characteristic data is compared to determine whether the renal disease marker solution to be tested contains the renal disease marker and calculate the renal disease marker concentration.

[0033] In some embodiments, a renal disease marker solution of a set concentration is introduced into the microchannel of the renal disease marker sensor, a set voltage is applied to the renal disease marker sensor, and after the current stabilizes, the detected current is recorded as the first electrical characteristic data, including:

[0034] A phosphate buffer solution is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor. After the current stabilizes, the detected current is recorded as initial current characteristic data;

[0035] Cleaning the renal disease marker sensor with a phosphate buffer solution;

[0036] A renal disease marker solution with a first set concentration is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor. After the current stabilizes, the detected current is recorded as the current characteristic data of the first set concentration;

[0037] Cleaning the renal disease marker sensor with a phosphate buffer solution;

[0038] A renal disease marker solution with a second set concentration is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor. After the current stabilizes, the detected current is recorded as the current characteristic data of the second set concentration;

[0039] The distribution characteristics of the detection current corresponding to the kidney disease marker solutions with different concentrations are obtained according to the initial current characteristic data, the first set concentration current characteristic data and the second set concentration current characteristic data.

[0040] The beneficial effects of the present invention are at least:

[0041] In the described kidney disease marker sensor, sensor preparation method, and kidney disease marker detection method, hydroxyl, amino, and aldehyde groups are sequentially introduced onto silicon nanowires in the microfluidic channels on the surface of a silicon nanowire field-effect transistor biosensor. The aldehyde groups are then covalently linked to probe molecules to form a kidney disease marker sensor targeting a specific kidney disease marker. After the probe molecules capture the kidney disease marker, the kidney disease marker sensor applies a set voltage and records the detected current after the current stabilizes. Based on the monitored currents of a kidney disease marker solution with a known set concentration and the kidney disease marker solution to be tested, it is determined whether the kidney disease marker is present in the test solution and the kidney disease marker concentration is calculated. This simplifies the detection process and improves detection accuracy.

[0042] Additional advantages, objects, and features of the present invention will be set forth in part in the following description and will in part become apparent to those skilled in the art upon examination of the following or may be learned by practice of the present invention. The objects and other advantages of the present invention may be realized and attained by the structure particularly pointed out in the written description and claims thereof as well as in the accompanying drawings.

[0043] Those skilled in the art will understand that the purposes and advantages that can be achieved by the present invention are not limited to the above specific descriptions, and the above and other purposes that can be achieved by the present invention will be more clearly understood based on the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings described herein are intended to provide a further understanding of the present invention, constitute a part of this application, and do not constitute a limitation of the present invention. The components in the drawings are not drawn to scale, but are merely for the purpose of illustrating the principles of the present invention. To facilitate the illustration and description of some parts of the present invention, corresponding parts in the drawings may be exaggerated, that is, they may be larger than other components in an exemplary device actually manufactured according to the present invention. In the drawings:

[0045] Figure 1 Schematic diagram of the structure of a kidney disease marker sensor according to an embodiment of the present invention.

[0046] Figure 2 This is a logical diagram of a method for detecting kidney disease markers according to an embodiment of the present invention.

[0047] Figure 3 for Figure 2 Schematic diagram of the logic for generating the first electrical characteristic data.

[0048] Figure 4 This is a schematic diagram of the structure of a silicon nanowire field-effect transistor biosensor used in the kidney disease marker sensor according to one embodiment of the present invention.

[0049] Figure 5 This is a schematic diagram of the preparation and working principle of the kidney disease marker sensor according to one embodiment of the present invention.

[0050] FIG6 (a) is a schematic diagram of the preparation process of a renal disease marker sensor according to an embodiment of the present invention.

[0051] Figure 6(b) is a comparison diagram of component identification in Figure 6(a).

[0052] FIG6( c ) is a schematic diagram of the state of capturing human serum cystatin C by the kidney disease marker sensor according to one embodiment of the present invention.

[0053] FIG7( a ) is a schematic diagram showing the detection current of the renal disease marker sensor according to an embodiment of the invention for different concentrations of human serum cystatin.

[0054] FIG7( b ) is a linear relationship diagram of concentration and current during the detection of various concentrations of human serum cystatin by the renal disease marker sensor in FIG7( a ).

[0055] Figure 8 (a) is a comparison of the detection currents of the unmodified silicon nanowire field-effect transistor biosensor for retinol binding protein solution, phosphate buffer solution, and human serum cystatin solution.

[0056] Figure 8 (b) is a comparison of the detection currents of the modified silicon nanowire field-effect transistor biosensor for retinol binding protein solution, phosphate buffer solution, and human serum cystatin solution. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and the accompanying drawings. Here, the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0058] It should also be noted that, in order to avoid obscuring the present invention due to unnecessary details, the accompanying drawings only show structures and / or processing steps closely related to the solutions according to the present invention, while other details that are not closely related to the present invention are omitted.

[0059] It should be emphasized that the term "include / comprises" when used herein refers to the existence of features, elements, steps or components, but does not exclude the existence or addition of one or more other features, elements, steps or components.

[0060] It should also be noted that, unless otherwise specified, the term "connection" herein may refer not only to a direct connection but also to an indirect connection involving an intermediate.

[0061] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the accompanying drawings, the same reference numerals represent the same or similar components, or the same or similar steps.

[0062] Human serum cystatin C (Cys-C), a 13kDa non-hydrolyzed protein, is considered an early marker associated with glomerular filtration rate (GFR) and exhibits high sensitivity in kidney disease. Studies have shown that Cys-C concentration accurately reflects glomerular filtration function, and the degree of change in Cys-C levels is closely correlated with the severity of kidney disease.

[0063] To date, a variety of analytical strategies have been used to detect serum Cys-C, such as enzyme-linked immunosorbent assay (ELISA), turbidimetry, and turbidimetric immunoassay. However, the detection methods for human serum cystatin C require complex pre-processing of samples, and the analyte needs to be processed during the detection process, the detection cost is high, the experimental operation process is cumbersome, and specialized personnel are required to operate. The most important thing is that these methods are all relatively time-consuming and cannot achieve rapid detection. In view of this, the present invention provides a method for detecting a kidney disease marker that can overcome or make up for the defects of existing detection methods that require labeling of the analyte and long detection time. Therefore, the present invention provides a kidney disease marker sensor, a sensor preparation method, and a kidney disease marker detection method.

[0064] Specifically, the present invention provides a kidney disease marker sensor, such as Figure 1 As shown, including:

[0065] Silicon nanowire field-effect transistor biosensor: Serially introduced hydroxyl groups, amino groups and aldehyde groups are arranged on the silicon nanowires in the microfluidic channels on the surface of the silicon nanowire field-effect transistor biosensor; the aldehyde groups are covalently linked to probe molecules, which are antibodies for capturing kidney disease markers.

[0066] The kidney disease marker sensor in this embodiment primarily uses a label-free detection method to indicate biomolecule charge. A field-effect transistor (FET) is an electronic component whose channel conductivity can be controlled by an external electric field. Because its channel conductivity is also affected by the charge of biomolecules, it can be used in biosensors. The kidney disease marker sensor in this embodiment, based on a silicon nanowire field-effect transistor biosensor, sequentially introduces hydroxyl, amino, and aldehyde groups onto the silicon nanowires, and covalently links the amino groups to probe molecules, enabling the capture and detection of specific kidney disease markers.

[0067] In some embodiments, the probe molecule is a human serum cystatin C antibody, and the kidney disease marker is a human serum cystatin C antigen.

[0068] In this embodiment, the kidney disease marker selected is human serum cystatin C antigen, and is captured using human serum cystatin C antibody as a probe molecule.

[0069] In some embodiments, a silicon nanowire field-effect transistor biosensor utilizes a single-crystal silicon layer as a substrate, a silicon dioxide insulating layer on top of the substrate, and a silicon nanowire on top of the insulating layer. Metal films are deposited at both ends of the silicon nanowires to serve as source and drain electrodes, and polydimethylsiloxane is filled between the source and drain electrodes and the silicon nanowires to form microfluidic channels. In this embodiment, the silicon nanowire field-effect transistor biosensor can utilize a one-dimensional silicon material with a nanometer diameter, a silicon core surrounded by a silicon dioxide outer layer. A silicon nanowire (SiNW) array is arranged on a Si substrate with a SiO2 insulating layer, with metal films deposited at both ends of the silicon nanowires to serve as source and drain electrodes, respectively. A buffer solution is dripped onto the silicon nanowires to form a liquid-gate device. The conductivity of the silicon nanowires can be modulated by applying a gate voltage to the substrate electrode, and this property change is monitored using a circuit between the source and drain electrodes. When the silicon nanowires are modified with target molecule receptors, they can capture specific target molecules in the test solution, thereby sensing changes in the silicon nanowires' conductivity caused by the target molecule's charge. This change is linearly correlated with the logarithm of the target molecule concentration within a certain range, enabling quantitative detection of the target substance.

[0070] On the other hand, the present invention also provides a method for preparing a renal disease marker sensor, referring to Figure 5 6(a) and 6(b), including steps S101 to S104:

[0071] Step S101: Obtain a silicon nanowire field-effect transistor biosensor. The silicon nanowire field-effect transistor biosensor uses a single crystal silicon layer as a substrate, a silicon dioxide layer as an insulating layer on the substrate, a silicon nanowire on the insulating layer, a metal film deposited at both ends of the silicon nanowire as a source and a drain, and polydimethylsiloxane (PDMS) is filled between the source, the drain and the silicon nanowire as a microchannel.

[0072] Step S102: plasma cleaning the microchannel of the silicon nanowire field effect transistor biosensor by oxygen plasma treatment to bond hydroxyl groups to the silicon nanowires.

[0073] Step S103: introducing an amino group introduction solution into the microchannel, allowing the solution to stand to introduce amino groups onto the hydroxyl groups and performing a thermal annealing treatment.

[0074] Step S104: introducing an aldehyde group introduction solution into the microchannel and allowing the solution to stand to introduce aldehyde groups onto the amino groups.

[0075] Step S105: introducing a probe molecule solution into the microchannel and allowing it to stand to introduce probe molecules onto the aldehyde groups. The probe molecules are antibodies for capturing kidney disease markers, so as to obtain the kidney disease marker sensor.

[0076] In some embodiments, in steps S101 to S105 , the amino group introduction solution is 3-aminopropyltriethoxysilane in anhydrous ethanol; the aldehyde group introduction solution is glutaraldehyde solution; and the probe molecule solution is human serum cystatin C antibody buffer.

[0077] In steps S101 to S105, hydrophilic hydroxyl groups are introduced to the surface of the PDMS after oxygen plasma treatment. During the oxygen plasma treatment of the silicon wafer, the Si-O bonds are broken, thereby forming a large number of Si dangling bonds on the surface, which form Si-OH bonds by absorbing hydroxyl groups in the air. The treated PDMS is bonded to the silicon surface, and the following reaction occurs between the Si-OH on the two surfaces: 2Si-OH=Si-O-Si+2H2O. A strong Si-O bond is formed between the silicon substrate and the PDMS, thereby completing the irreversible bond between the two. Furthermore, by continuously introducing the amino group introduction solution, the aldehyde group introduction solution and the probe molecule solution to react, the amino group, the aldehyde group and the probe molecule are further connected to the Si-O bond.

[0078] In some embodiments, in step S101, the microchannel of the silicon nanowire field effect transistor biosensor is plasma cleaned by oxygen plasma treatment to bond hydroxyl groups to the silicon nanowires, including steps S1011 to S1012:

[0079] Step S1011: using an oxygen plasma cleaning machine, the silicon nanowire field effect transistor biosensor was subjected to oxygen plasma treatment for 2 minutes at a power of 15 W and a flow rate of 100 sccm.

[0080] Step S1012: using an oxygen plasma gun to treat the surface of the microchannel for 20 seconds to align and bond the microchannel to the channel of the silicon nanowire field effect transistor biosensor.

[0081] In some embodiments, in step S103, an amino group introduction solution is introduced into the microchannel, allowed to stand to introduce amino groups on the hydroxyl groups and then subjected to thermal annealing treatment, including: introducing a 3-aminopropyltriethoxysilane anhydrous ethanol solution into the microchannel, storing it at room temperature for 1 hour, washing it with ethanol and deionized water, and placing it in a rapid annealing furnace for annealing at 150°C for 30 minutes.

[0082] In step S104, an aldehyde introduction solution is introduced into the microchannel and allowed to stand to introduce aldehyde groups on the amino groups, including: introducing glutaraldehyde solution into the microchannel and sealing it in the dark at 37° C. for 45 minutes.

[0083] In step S105 , a probe molecule solution is introduced into the microchannel and allowed to stand to introduce the probe molecule onto the aldehyde group, including: introducing the probe molecule solution into the microchannel and incubating in a water bath at 37° C. for 45 minutes.

[0084] In some embodiments, in step S104, after the probe molecule solution is introduced into the microchannel and allowed to stand to introduce the probe molecules onto the aldehyde groups, steps S1041 to S1042 are further included:

[0085] Step S1041: introducing a phosphate buffer solution containing bovine serum albumin into the microchannel of the silicon nanowire field effect transistor biosensor and allowing the solution to stand for treatment.

[0086] Step S1042: Clean the microchannel with a phosphate buffer solution.

[0087] On the other hand, the present invention also provides a method for detecting kidney disease markers, referring to Figure 2 , including steps S201 to S203:

[0088] Step S201: A renal disease marker solution of a set concentration is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor. After the current stabilizes, the detected current is recorded as the first electrical characteristic data.

[0089] Step S202: Cleaning the renal disease marker sensor with a phosphate buffer solution.

[0090] Step S203: introducing a renal disease marker solution to be tested into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as the second electrical characteristic data after the current stabilizes.

[0091] Step S204: comparing the distribution relationship between the first electrical characteristic data and the second electrical characteristic data, determining whether the renal disease marker solution to be tested contains the renal disease marker and calculating the renal disease marker concentration.

[0092] Kidney disease marker sensors primarily use a label-free detection method to indicate biomolecule concentration by detecting the charge of biomolecules. Field-effect transistors (FETs) are electronic components whose channel conductivity can be controlled by an external electric field. Because their channel conductivity is also affected by the charge of biomolecules, they can be used in biosensors. Based on silicon nanowire field-effect transistor biosensors, kidney disease marker sensors sequentially introduce hydroxyl, amino, and aldehyde groups onto silicon nanowires, and covalently link the amino groups to probe molecules to capture and detect specific kidney disease markers. Therefore, in steps S201-S204 of this embodiment, a kidney disease marker solution of a set concentration is first tested to obtain corresponding electrical properties. In some embodiments, multiple kidney disease marker solutions of known concentrations can be tested to determine the corresponding relationship between kidney disease marker concentration and electrical properties. Then, the corresponding electrical property data is obtained by testing the kidney disease marker solution to be tested, and the concentration of the kidney disease marker solution to be tested is calculated based on this corresponding relationship. In some embodiments, in step S201, a renal disease marker solution of a set concentration is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor. After the current stabilizes, the detected current is recorded as the first electrical characteristic data. Figure 3 , including steps S2011 to S2014:

[0093] Step S2011: a phosphate buffer solution is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor. After the current stabilizes, the detected current is recorded as the initial current characteristic data.

[0094] Step S2012: Cleaning the renal disease marker sensor with a phosphate buffer solution.

[0095] Step S2013: introducing a renal disease marker solution of a first set concentration into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as the first set concentration current characteristic data after the current stabilizes.

[0096] Step S2014: Clean the renal disease marker sensor with a phosphate buffer solution.

[0097] Step S2015: introducing a renal disease marker solution of a second set concentration into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as the second set concentration current characteristic data after the current stabilizes.

[0098] Step S2016: obtaining distribution characteristics of detection current corresponding to kidney disease marker solutions with different concentrations according to the initial current characteristic data, the first set concentration current characteristic data, and the second set concentration current characteristic data.

[0099] In steps S2011-S2016, referring to the description of step S201, this embodiment performs detection at renal marker concentrations of 0, a first set concentration, and a second set concentration. The electrical characteristics detected by the renal marker sensor at different renal marker concentrations are analyzed. Specifically, the relationship between the current distribution characteristics and the renal marker concentration is analyzed. Based on this current distribution characteristic, the renal marker concentration in the renal marker solution to be tested is calculated and evaluated. Specifically, the renal marker detection method described in this embodiment first applies a set voltage to a renal marker solution of known concentration, generating a detection current. As shown in Figure 6(c), the probe molecules in the renal marker sensor are capable of capturing the renal marker to be detected. After applying the set voltage to the renal marker sensor, a stable detection current is generated. The magnitude of the detection current has a specific distribution relationship with the renal marker concentration, typically exhibiting a linear relationship.

[0100] The present invention will be described below in conjunction with a specific embodiment:

[0101] This embodiment provides a kidney disease marker sensor, such as Figure 4 , modify its surface and introduce probe molecules, such as Figure 5 and 6.

[0102] The test liquid is added to a silicon nanowire field-effect transistor biosensor covalently linked to a probe molecule, wherein a kidney disease marker is capable of specifically binding to the probe molecule. A hydroxyl group is introduced onto the surface of the silicon nanowire field-effect transistor biosensor; an amino group is introduced at the outer end of the hydroxyl group; an aldehyde group is introduced at the outer end of the amino group; and a probe molecule is introduced at the outer end of the aldehyde group, thereby obtaining a silicon nanowire sensor covalently linked to the probe molecule. The silicon nanowire field-effect transistor biosensor is a silicon nanowire field-effect transistor, the kidney disease marker is a human serum cystatin C (Cys-C) antigen, and the probe molecule is a Cys-C antibody.

[0103] The step of introducing hydroxyl groups on the surface of the silicon nanowire field effect transistor biosensor comprises: introducing hydroxyl groups by cleaning the nanosensor with oxygen plasma.

[0104] After introducing hydroxyl groups on the surface of the silicon nanowire field effect transistor biosensor, the method further includes: bonding microchannels to the surface of the silicon nanowire field effect transistor biosensor treated with oxygen plasma, including: aligning and bonding the microchannels treated with oxygen plasma gun to the sensor surface.

[0105] The invention also introduces an amino group at the outer end of the hydroxyl group, comprising: introducing a 3-aminopropyltriethoxysilane anhydrous ethanol solution into a microchannel of a silicon nanowire field effect transistor biosensor into which the hydroxyl group has been introduced, allowing the solution to stand to introduce the amino group, and placing the solution in a rapid thermal annealing furnace to ensure that the introduced amino group is more firmly introduced.

[0106] Introducing an aldehyde group at the outer end of the amino group comprises: introducing a glutaraldehyde solution into a microfluidic channel of a silicon nanowire field effect transistor biosensor having the amino group introduced therein, and allowing the solution to stand to introduce the aldehyde group.

[0107] The probe molecule is introduced at the outer end of the aldehyde group, comprising introducing a probe molecule solution into a microfluidic channel of a silicon nanowire field effect transistor biosensor having the aldehyde group introduced therein and allowing the solution to stand to introduce the probe molecule.

[0108] After introducing the probe molecule at the outer end of the aldehyde group, the method also includes: introducing a phosphate buffer solution containing bovine serum albumin into the silicon nanowire field effect transistor biosensor, allowing it to stand to block unreacted aldehyde groups on the surface of the silicon nanowire field effect transistor biosensor, and washing with a phosphate buffer solution.

[0109] For the kidney disease marker sensor in this example, as shown in Figure 7(a), the current flowing through the silicon nanowire biosensor gradually decreases as the concentration of the Cys-C solution increases. This result is consistent with the fact that the Cys-C protein has an isoelectric point of 9.3 and is positively charged in PBS solution. As shown in Figure 7(b), the current values ​​are averaged and linearly fitted, showing that the current decreases linearly with increasing concentration.

[0110] As shown in Figure 8(a), when 1 ng / ml retinol binding protein solution (RBP solution) and 1 ng / ml Cys-C solution were introduced, the current flowing through the silicon nanowires remained basically unchanged, so the unmodified kidney disease marker sensor could not detect Cys-C.

[0111] As shown in Figure 8(b), after the introduction of a 1 ng / ml retinol binding protein solution (RBP solution), the current flowing through the silicon nanowires remained essentially unchanged. However, when a 1 ng / ml Cys-C solution was introduced, the current flowing through the nanowires decreased significantly. The modified kidney disease marker sensor in this example only reacted to the Cys-C protein solution.

[0112] Exemplarily, a process for preparing a renal disease marker sensor is provided, comprising:

[0113] Preparation before retouching:

[0114] Prepare or obtain a silicon nanowire field effect transistor having a structure such as Figure 1 A PDMS (polydimethylsiloxane) microchannel of appropriate size was prepared on the silicon nanowire field-effect transistor. This channel was prepared with 1× PBS (phosphate buffered saline), 5% APTES (3-aminopropyltriethoxysilane) solution, 3% glutaraldehyde solution, 0.05% BSA (bovine serum albumin) solution, and Cys-C antibody solution. Electrical properties of the silicon nanowire field-effect transistor were measured before the experiment.

[0115] During the modification process, the silicon wafer and microfluidics are first cleaned with ethanol and deionized water, and the moisture is blown away with nitrogen to remove contamination caused by storage and transportation. The cleaned silicon wafer and microfluidics are then treated with O2 plasma to produce more silanol groups on the surface of the silicon nanowires, and to form a bond that can adhere to the surface of the silicon wafer and the microfluidic channel. The microfluidic channel is then bonded to the sensor surface. This step creates a biosensor with a silicon nanowire field-effect transistor with a microfluidic channel. In order to achieve specific detection of Cys-C, we next need to use silanization to modify the Cys-C antibody on the surface of the silicon nanowire. The specific operation steps are as follows: ethanol and deionized water are injected into the microchannel to clean the detection channel, and this is repeated three times; then 5% APTES solution is injected into the microchannel to completely immerse the silicon nanowires for 1 hour; the soaked silicon nanowire biosensor device is washed three times with ethanol and deionized water and blown dry with nitrogen; annealing is performed at 150°C for 30 minutes; 3% glutaraldehyde solution is passed into the microchannel and sealed in a dark place at 37°C for 45 minutes; then the prepared Cys-C antibody solution is passed into the microchannel and sealed in a dark place at 37°C for 45 minutes to allow the Cys-C antibody to fully modify the surface of the silicon nanowires; the modified device is washed with 1×PBS; the washed silicon nanowire biosensor device is passed into the channel with 0.05% BSA solution and stored in a plastic seal at 4°C for 2 hours to allow the aldehyde groups of the unbound antibodies to bind to bovine serum albumin; it is removed and washed three times with PBS solution, and then an appropriate amount of PBS solution is passed into the channel to prepare for the test.

[0116] During the testing process, refer to Figure 5 As shown in Figure 6, after the specific probe molecules are modified on the surface of the silicon nanowire field-effect transistor biosensor, PBS buffer (phosphate buffer) is passed into the microchannel, a fixed voltage is applied to the sensor, and after the current passing through the silicon nanowire field-effect transistor biosensor stabilizes, the current is recorded as the first electrical characteristic data.

[0117] The test liquid is passed through the microchannel of the silicon nanowire field-effect transistor biosensor and incubated for 20 minutes. After the kidney disease marker molecules specifically bind to the corresponding antibodies modified on the surface of the silicon nanowire field-effect transistor biosensor, PBS buffer is passed into the sensor and a fixed voltage is applied to the silicon nanowire field-effect transistor biosensor. After a stable current passes through, the current is recorded as the second electrical characteristic data.

[0118] By comparing the second electrical characteristic data with the first electrical characteristic data, it is determined whether the liquid to be tested contains the kidney disease marker and the concentration of the kidney disease marker.

[0119] In another embodiment, a method for measuring first electrical characteristic data includes: passing PBS buffer into a microchannel of a silicon nanowire field effect transistor biosensor connected to a probe molecule, applying a fixed voltage, and measuring initial electrical characteristic data; passing a solution containing a first known concentration of a kidney disease marker into the microchannel of the silicon nanowire field effect transistor biosensor connected to the probe molecule, waiting for 20 minutes, and re-measuring the electrical characteristic data under a fixed voltage; cleaning the silicon nanowire field effect transistor biosensor to which the kidney disease marker solution of the first known concentration was added; adding a solution containing a second known concentration of a kidney disease marker to the cleaned silicon nanowire field effect transistor biosensor, and re-measuring the electrical characteristic data again under a fixed voltage; obtaining first electrical characteristic data based on the initial electrical characteristic data, the re-measured electrical characteristic data, and the re-measured electrical characteristic data to present the distribution characteristics of the detection current corresponding to kidney disease marker solutions of different concentrations.

[0120] Specifically, Figure 2 FIG. 1 is a flow chart of a biomolecule detection method according to an embodiment of the present invention. Figure 2 As shown, the detection method may include the following steps:

[0121] A solution of set concentration was introduced into the microchannel of the prepared silicon nanowire biosensor and allowed to stand for 20 minutes. Then, a fixed voltage was applied to measure the first electrical characteristic data.

[0122] The test liquid was introduced into the microchannel of the prepared silicon nanowire biosensor and allowed to stand for 20 minutes. Then, a fixed voltage was applied to measure the second electrical characteristic data.

[0123] The second electrical characteristic data is compared with the first electrical characteristic data. By comparing the current magnitudes, it can be determined whether the test fluid contains a specific kidney disease marker, and by linear comparison, the concentration of the marker can be determined.

[0124] Further, Figure 3 FIG. 1 is a flow chart of the method for testing the first electrical characteristic data in the biomolecule detection method of this embodiment. Figure 3 As shown, the test method of the electrical characteristic data includes the following steps:

[0125] An appropriate amount of 1×PBS solution was introduced into the microchannel of the silicon nanowire biosensor connected to the probe molecule, and a fixed voltage was applied to measure the initial electrical characteristics data.

[0126] A sufficient amount of a first known concentration of a kidney disease marker solution is then introduced into the microchannel and allowed to stand for 20 minutes to allow the kidney disease marker to fully and specifically bind to the probe molecule. An appropriate amount of 1× PBS solution is then introduced, and a fixed voltage is applied to measure the electrical characteristics corresponding to the first concentration.

[0127] A sufficient amount of a second known concentration of kidney disease marker solution was then introduced into the microchannel. The solution was allowed to stand for 20 minutes to allow the kidney disease marker to fully bind to the probe molecule. An appropriate amount of 1× PBS solution was then introduced, and a fixed voltage was applied to measure the electrical characteristics corresponding to the second concentration.

[0128] The initial electrical characteristic data, the electrical characteristic data corresponding to the first concentration, and the electrical characteristic data corresponding to the second concentration are sorted to obtain first electrical characteristic data in preparation for detection.

[0129] In summary, the described renal disease marker sensor, sensor preparation method, and renal disease marker detection method utilizes a renal disease marker sensor targeting a specific renal disease marker by sequentially introducing hydroxyl, amino, and aldehyde groups into the microfluidic channel on the surface of a silicon nanowire field-effect transistor biosensor to covalently link probe molecules. After the probe molecules capture the renal disease marker, the sensor applies a set voltage and records the detected current after the current stabilizes. Based on the monitored currents of a renal disease marker solution with a known set concentration and the renal disease marker solution to be tested, the presence of the renal disease marker in the tested solution is determined and the renal disease marker concentration is calculated. This simplifies the detection process and improves detection accuracy.

[0130] It should also be noted that the exemplary embodiments described herein describe methods or systems based on a series of steps or devices. However, the present invention is not limited to the order of the steps described above. In other words, the steps may be performed in the order described in the embodiments, or in a different order, or several steps may be performed simultaneously.

[0131] In the present invention, features described and / or illustrated for one embodiment may be used in the same or similar manner in one or more other embodiments, and / or combined with or replace features of other embodiments.

[0132] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for detecting kidney disease markers, characterized in that: include: A renal disease marker solution of a set concentration is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor, and after the current is stabilized, the detection current is recorded as the first electrical characteristic data; Cleaning the renal disease marker sensor with a phosphate buffer solution; Passing a renal disease marker solution to be tested into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as the second electrical characteristic data after the current stabilizes; Comparing the distribution relationship between the first electrical characteristic data and the second electrical characteristic data, determining whether the renal disease marker solution to be tested contains the renal disease marker and calculating the renal disease marker concentration; The kidney disease marker sensor comprises a silicon nanowire field effect transistor biosensor, wherein a silicon nanowire in a microchannel on the surface of the silicon nanowire field effect transistor biosensor is provided with continuously introduced hydroxyl groups, amino groups and aldehyde groups, wherein the aldehyde groups are covalently linked to probe molecules, wherein the probe molecules are obtained by modifying the silicon nanowires with a human serum cystatin C antibody solution, and consuming the aldehyde groups of unbound antibodies with a 0.05% concentration of bovine serum protein solution, and using human serum cystatin C antigen as a kidney disease marker; the silicon nanowire field effect transistor biosensor uses a single crystal silicon layer as a substrate, wherein silicon dioxide is provided on the substrate as an insulating layer, wherein silicon nanowires are provided on the insulating layer, wherein metal films are deposited at both ends of the silicon nanowires as source and drain electrodes, and polydimethylsiloxane is filled between the source electrode, the drain electrode and the silicon nanowires as a microchannel; The preparation method of the kidney disease marker sensor comprises: A silicon nanowire field effect transistor biosensor is obtained, wherein the silicon nanowire field effect transistor biosensor uses a single crystal silicon layer as a substrate, a silicon dioxide layer is provided on the substrate as an insulating layer, a silicon nanowire is provided on the insulating layer, a metal film is deposited at both ends of the silicon nanowire as a source and a drain, and polydimethylsiloxane is filled between the source, the drain and the silicon nanowire as a microchannel; Plasma cleaning the microchannel of the silicon nanowire field effect transistor biosensor by oxygen plasma treatment to bond hydroxyl groups to the silicon nanowires; Passing a 3-aminopropyltriethoxysilane anhydrous ethanol solution into the microchannel, allowing it to stand to introduce amino groups onto the hydroxyl groups and performing a thermal annealing treatment; Passing glutaraldehyde solution into the microfluidic channel and allowing it to stand to introduce aldehyde groups on the amino groups; A human serum cystatin C antibody buffer is introduced into the microfluidic channel, and the microfluidic channel is allowed to stand to introduce a probe molecule on the aldehyde group, wherein the probe molecule is an antibody for capturing a kidney disease marker, and a 0.05% concentration of bovine serum albumin solution is introduced to consume the aldehyde group of the unbound antibody to obtain the kidney disease marker sensor; Plasma cleaning the microchannel of the silicon nanowire field effect transistor biosensor by oxygen plasma treatment, bonding hydroxyl groups on the silicon nanowires, including: using an oxygen plasma cleaner to perform oxygen plasma treatment on the silicon nanowire field effect transistor biosensor for 2 minutes under the conditions of a power of 15 W and a flow rate of 100 sccm; using an oxygen plasma gun to treat the surface of the microchannel for 20 seconds, aligning and bonding the microchannel with the channel of the silicon nanowire field effect transistor biosensor; After the probe molecule solution is introduced into the microchannel and allowed to stand to introduce the probe molecule onto the aldehyde group, the method further includes: introducing a phosphate buffer solution containing bovine serum albumin into the microchannel of the silicon nanowire field effect transistor biosensor and allowing the solution to stand; and cleaning the microchannel with a phosphate buffer solution.

2. The method for detecting kidney disease markers according to claim 1, characterized in that: A renal disease marker solution of a set concentration is introduced into the microchannel of the renal disease marker sensor, and a set voltage is applied to the renal disease marker sensor, and after the current is stabilized, the detection current is recorded as the first electrical characteristic data, including: Passing a phosphate buffer solution into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as initial current characteristic data after the current stabilizes; Cleaning the renal disease marker sensor with a phosphate buffer solution; Passing a renal disease marker solution of a first set concentration into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as the current characteristic data of the first set concentration after the current stabilizes; Cleaning the renal disease marker sensor with a phosphate buffer solution; Passing a renal disease marker solution of a second set concentration into the microchannel of the renal disease marker sensor, applying a set voltage to the renal disease marker sensor, and recording the detected current as the current characteristic data of the second set concentration after the current stabilizes; Obtaining distribution characteristics of detection currents corresponding to kidney disease marker solutions of different concentrations according to the initial current characteristic data, the first set concentration current characteristic data, and the second set concentration current characteristic data; A 3-aminopropyltriethoxysilane anhydrous ethanol solution is introduced into the microchannel, and the solution is allowed to stand to introduce amino groups on the hydroxyl groups and perform a thermal annealing treatment, comprising: introducing the 3-aminopropyltriethoxysilane anhydrous ethanol solution into the microchannel, and after being stored at room temperature for 1 hour, the solution is washed with ethanol and deionized water, and the solution is placed in a rapid annealing furnace, and annealed at 150° C. for 30 minutes; Passing a glutaraldehyde solution into the microchannel and allowing it to stand to introduce an aldehyde group on the amino group, comprising: passing the glutaraldehyde solution into the microchannel and sealing it in a dark environment at 37° C. for 45 minutes; A human serum cystatin C antibody buffer is introduced into the microfluidic channel and allowed to stand to introduce a probe molecule onto the aldehyde group, comprising: introducing the probe molecule solution into the microfluidic channel and culturing in a water bath at 37° C. for 45 minutes.

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