WO2021128956A1

WO2021128956A1 - Nanowire biosensor, preparation method therefor and application thereof

Info

Publication number: WO2021128956A1
Application number: PCT/CN2020/114822
Authority: WO
Inventors: 王晗; 孙凯
Original assignee: 清华大学
Priority date: 2019-12-26
Filing date: 2020-09-11
Publication date: 2021-07-01
Also published as: CN111175347A; CN111175347B

Abstract

A nanowire biosensor, a preparation method therefor and an application thereof, which relate to the technical field of biological testing. The method comprises preparing a substrate (1), preparing a signal collection electrode (4) and a metal nanowire (6) on the substrate (1), and connecting a probe (8) on the metal nanowire (6). In the preparation method for the nanowire biosensor, as long as a corresponding single-stranded nucleic acid probe is designed according to a target to be detected, the target nucleic acid may be detected. At the same time, by means of an electrically heated nucleic acid solution chain and in-situ release technology, the continuous and dynamic detection of the target nucleic acid can be implemented in situ, thereby grasping changes in the concentration of target nucleic acid molecules.

Description

Nanowire biosensor and its preparation method and application

Technical field

The invention relates to a nanowire biosensor, a preparation method and application thereof, and belongs to the technical field of biological testing.

Background technique

The real-time detection of human biochemical indicators can provide a wealth of diagnostically valuable health information for medical clinical practice, such as multi-dimensional and multi-level information such as human metabolites, cytokines, and biomarkers. Among them, the detection of biomarkers is especially useful for early tumors. Screening and prognosis are of great significance. At present, biomarkers are mainly obtained by large-scale medical testing instruments or in vitro diagnostic equipment through in vitro analysis of blood, tissue fluid, urine, feces and other body fluids and excreta. It is difficult to achieve real-time detection, and there is a huge gap between the need for continuous and dynamic health information of the human body. . Therefore, it is of great significance to detect the dynamic trend of biomarkers with high sensitivity and real-time.

The currently used high-sensitivity nucleic acid detection method is an electrical biosensor supported by field effect tube (FET) technology. Although the graphene FET field effect tube sensor has the ability to detect single nucleotide polymorphism (SNP), it can only achieve a single detection of a perfectly matched nucleic acid fragment without other steps, and cannot achieve the same nucleic acid fragment. Repeated measurements for many times can neither detect the dynamic change trend of the target segment with one sensor.

Summary of the invention

In one aspect, the present invention provides a nanowire biosensor. The nanowire biosensor includes a substrate and a signal collection electrode and nanowires formed on the substrate. The signal collection electrode is connected to an external circuit for signal acquisition. The nanowire is used as a sensing unit to identify and detect after surface modification. Biological matter.

In another aspect, the present invention provides a method for preparing a nanowire biosensor. The method includes the following steps: preparing a substrate, preparing signal collection electrodes and metal nanowires on the substrate, and connecting probes on the metal nanowires.

In another aspect, a nanowire biosensor is provided. The nanowire biosensor includes a substrate, a signal collection electrode and a metal nanowire formed on the substrate, and a probe formed on the metal nanowire.

In another aspect, a biosensor is provided. The biosensor includes a sample exposure area and nanowires, and at least a part of the nanowires in the sample exposure area is addressable by the sample.

In another aspect, an application of the aforementioned nanowire biosensor for detecting biomarkers is provided. The application includes the following steps: pass the buffer into the nanowire biosensor, collect the first voltage-current curve, the conductivity curve and the impedance spectrum curve through the signal acquisition electrode; pass the probe complementary to the nanowire biosensor The target sequence collects the second voltage-current curve, conductivity curve and impedance spectrum curve through the signal acquisition electrode; applies voltage to the signal acquisition electrode to release the target sequence from the nanowire, and acquires the third voltage-current curve through the signal acquisition electrode , Conductivity curve and impedance spectrum curve; combine the first voltage-current curve, conductivity curve and impedance spectrum curve, the second voltage-current curve, conductivity curve and impedance spectrum curve, and the third voltage-current curve, conductivity The curve is compared with the impedance spectrum curve to achieve nucleic acid detection; repeat the above steps to perform multiple repeated detections of the target nucleic acid sequence.

In another aspect, an application of the aforementioned nanowire biosensor for detecting biomarkers is provided. The application includes the following steps: put the nanowire biosensor into the solution, collect the first voltage-current curve, the conductivity curve and the impedance spectrum curve through the signal acquisition electrode; according to the set time interval, the signal acquisition electrode collects more A voltage-current curve, conductivity curve and impedance spectrum curve, record multiple voltage-current curves, conductivity curve and impedance spectrum curve, by comparing multiple curves, realize the detection of the target sequence; apply voltage to the signal acquisition electrode , To release the target sequence from the metal nanowire; repeat the above steps to perform multiple repeated detections of the target sequence.

Description of the drawings

Fig. 1 is a flow chart of a method for preparing a nanowire biosensor according to the present invention.

Figure 2 is a schematic diagram of the structure of the nanowire biosensor prepared by the method of the present invention.

Fig. 3 is a top view of the nanowire biosensor shown in Fig. 2.

FIG. 4 is a schematic diagram of the structure of the drainage block in the nanowire biosensor shown in FIG. 2.

Fig. 5 is a schematic diagram of the second structure of the nanowire biosensor of the present invention.

Fig. 6 is a curve of impedance, conductivity and time when the nanowire biosensor prepared by the present invention is not modified.

Fig. 7 is a curve of impedance, conductivity and time of the nanowire biosensor prepared by the present invention after modification.

Fig. 8 is the impedance change curve of the nanowire biosensor prepared by the present invention after each operation.

Fig. 9 is a change curve of the conductivity of the nanowire biosensor prepared by the present invention after each operation.

Detailed ways

The invention relates to a nanowire biosensor and a preparation method and application thereof. The nanowire biosensor is prepared simply and quickly by using mature microprocessing technology, and the nanowire biosensor is modified to make it have the function of detecting nucleic acid biomarkers. At the same time, the electric heating characteristics of the nanowires are used to melt and release the captured nucleic acid biomarkers in situ, so as to realize the continuous and dynamic detection of the nucleic acid biomarkers.

In one aspect, as shown in Figures 2-5, the present invention provides a nanoelectrode biosensor. The nanoelectrode biosensor includes a substrate 1 and a signal collection electrode 4 and a nanowire 6 formed on the substrate. The signal collection electrode 4 is connected to an external circuit for obtaining signals. The nanowire 6 is used as a sensing unit after surface modification. Used to identify and detect biological substances.

In some embodiments, the signal collection electrode 4 and the nanowire 6 are both made of a metal material, and each includes a metal sensing layer and a metal connecting layer, the metal sensing layer includes at least one of gold and platinum, and the metal connecting layer At least one of chromium and titanium is included.

In some embodiments, the substrate 1 is made of rigid or flexible material.

In some embodiments, the nano-electrode biosensor further includes an insulating layer formed on the substrate 1, wherein the signal collecting electrode 4 and the nanowire 6 are formed on the insulating layer. Specifically, the electrical conductivity of the insulating layer is greater than 45.2×10 ^-6 S/m.

In some embodiments, after surface modification, the nanowire 6 is used to identify and capture biological materials, the biological materials include at least one of extracellular vesicles, cells, bacteria, and viruses such as nucleic acids, proteins, and exosomes.

In some embodiments, the signal acquired by the signal acquisition electrode 4 is an electrical signal, and the electrical signal includes at least one of electric potential, electrical conductivity, electrical impedance, and electrical impedance spectrum.

In some embodiments, by applying a signal to the signal collection electrode 4, the surface temperature of the nanowire 6 can be controlled, and the captured biological substance can be released in situ.

In another aspect, as shown in FIG. 1, the present invention provides a method for preparing a nanowire biosensor. The method includes the following steps S101 to S103.

In step S101, the substrate 1 is prepared.

In some embodiments, the substrate material is repeatedly rinsed with acetone and isopropanol successively, and the surface is dried with nitrogen. The substrate material includes silicon wafer, glass, organic polymer material or flexible material, etc., preferably silicon wafer.

In some embodiments, the dried substrate is placed in an oxidation furnace or a plasma-enhanced chemical vapor deposition (PECVD) coating equipment, and an insulating layer is grown on the surface of the substrate to obtain a substrate 1, as shown in FIGS. 2-5. The thickness of the insulating layer is 10 to 100,000 nanometers, preferably 100 to 1,000 nanometers, and more preferably 500 nanometers. The substrate and the insulating layer constitute the base 1.

After the insulating layer is formed, the substrate 1 is repeatedly rinsed three times with acetone and isopropanol, the surface is dried with nitrogen, and then placed on a hot plate at 100-120°C, such as 120°C, for 1-2 minutes to make the substrate 1 complete. dry.

In step S102, the signal collection electrode 4 and the metal nanowire 6 are prepared on the substrate 1.

In some embodiments, the step of preparing signal collection electrodes and metal nanowires on the substrate includes using electron beam exposure to form nanowire grooves on the surface of the substrate, using magnetron sputtering to prepare metal nanowires in the nanowire grooves, and using light The signal acquisition electrode groove is prepared by engraving on the surface of the substrate, and the signal acquisition electrode is formed in the signal acquisition electrode groove by magnetron sputtering.

In an example, the dried substrate 1 is placed on a homogenizer, and electron beam glue is spin-coated on the surface of the substrate 1, and a 300-400 nanometer thick electron beam glue thin layer is formed on the surface of the substrate 1, preferably electron beam The glue is polymethyl methacrylate.

In one example, the electron beam exposure method is adopted, for example, the acceleration voltage is 30 keV, the exposure time is 1 minute, and the exposure width is 50-500 nanometers at the center position and the alignment mark position of the substrate coated with electron beam glue. Preferably 200 nanometers, with a length of 50-1000 microns, preferably 200 microns of nanowire recesses and alignment mark recesses. Put the electron beam exposed substrate into the electron beam developer to remove the electron beam glue in the exposed part. The surface of the nanowire groove and the alignment mark groove are cleaned with deionized water and dried with nitrogen.

In some embodiments, using magnetron sputtering to prepare metal nanowires in the nanowire groove includes sputtering a first metal connection layer in the nanowire groove and sputtering a first metal sensing layer on the first metal connection layer, The first metal sensing layer includes at least one of gold and platinum, the first metal connecting layer includes at least one of chromium and titanium, the thickness of the first metal connecting layer is 5 to 20000 nanometers, and the first metal sensing layer The thickness of the layer is 5 to 30000 nanometers.

In one example, a magnetron sputtering method is used to sputter a 5-20 nanometer thick metal connection layer, such as 5 nanometers of metallic chromium, on a substrate with a nanowire groove and an alignment mark groove on the surface, and then sputter it. A metal sensing layer with a thickness of 5 to 300 nm, such as a metal gold with a thickness of 100 nm, is prepared on the surface of the substrate 1 to obtain a metal nanowire 6 and an alignment mark 9. Use a combination of acetone and ultrasound to clean, peel off the electron beam glue on the unexposed part of the substrate surface from the metal, rinse with deionized water, blow dry with nitrogen, and keep it on a hot plate at 100-120℃, preferably at 120℃. Seconds, completely dry.

In one example, the substrate 1 with the metal nanowires 6 and the alignment marks 9 is placed on a homogenizer, and photoresist (the photoresist is negative) is spin-coated to form a thickness of 1-2 microns on the surface of the substrate 1 , Preferably a thin layer of photoresist with a thickness of 1.3-1.5 microns. Preferably, 4340 photoresist is used, first spin coating at 100 revolutions per minute for 10 seconds, and then spin coating at 4000 revolutions per minute for 40 seconds.

In one example, a substrate with spin-on photoresist is placed on a photolithography machine. Using a plate-making method (for example, using a direct-write lithography device) on the chromium plate, a mask chromium plate with alignment marks and signal collection electrodes is prepared. Place the mask chrome plate on the substrate 1 coated with photoresist, align the alignment marks 9 on the substrate 1 with the alignment marks on the mask chromium plate, and make the substrate 1 and the mask chrome plate adhere to each other, using light The engraving method is to perform photoetching from the top of the mask chromium plate, and then put it into the developer to remove the unexposed part of the photoresist, prepare a signal collection electrode groove on the surface of the substrate 1, and clean it with deionized water and nitrogen Blow dry.

In some embodiments, using magnetron sputtering to form the signal collecting electrode in the signal collecting electrode groove includes sputtering the second metal connecting layer in the signal collecting electrode groove and sputtering the second metal sensor on the second metal connecting layer. The second metal sensing layer includes at least one of gold and platinum, the second metal connecting layer includes at least one of chromium and titanium, the thickness of the second metal connecting layer is 5 to 20000 nanometers, and the second metal The thickness of the sensing layer is 5 to 30000 nanometers.

In one example, the magnetron sputtering method is used to perform magnetron sputtering on the surface of the substrate with signal collection electrode grooves, first sputtering a 5-20 nanometer thick metal connection layer, such as a 5 nanometer thick metal chromium, and then Sputtering a metal sensing layer with a thickness of 5 to 300 nanometers, such as metal gold with a thickness of 100 nanometers. A method of combining acetone and ultrasound is used for cleaning, the exposed part of the photoresist is peeled off from the metal, rinsed with deionized water, and dried with nitrogen, and the signal collection electrode 4 and the metal nanowire 6 are prepared on the substrate 1.

In step S103, a probe is connected to the metal nanowire.

In some embodiments, step S103 includes preparing the drainage block 2, as shown in FIG. 4.

In some embodiments, the step of preparing the drainage block includes forming a first through hole and a second through hole in the drainage block, and forming a channel at the bottom of the drainage block, so that the width of the channel is equal to the length of the metal nanowire, and the channel is equal to the length of the metal nanowire. The first through hole communicates with the second through hole, the length of the channel is 1 to 50 mm, preferably 4-6 mm, more preferably 5 mm, and the depth of the channel is 10 to 500 micrometers, preferably 50-200 micrometers, more preferably 50 micrometers . As shown in Figure 4, for example, the first through hole 3 and the second through hole 5 are processed in the drainage block 2, and the bottom of the drainage block 1 is processed with a groove, so that the width of the groove is equal to the length of the metal nanowire. The length of the groove is 1-50 mm, preferably 4-6 mm, more preferably 5 mm, and the depth of the groove is 10-500 micrometers, preferably 50-200 micrometers, more preferably 50 micrometers, so that the groove becomes the first through hole 3 The bottom channel 7 between and the second through hole 5.

In some embodiments, the bottom of the drainage block 2 is relatively fixed to the substrate 1 so that the length direction of the bottom channel 7 of the drainage block 2 and the length direction of the nanowire 6 are perpendicular to each other, and the nanowire 6 is completely covered.

Figure 6 shows the nanowire biosensor prepared through the above steps, after filling the first through hole 3, the bottom channel 7 and the second through hole 5 with a phosphate buffered saline solution, the impedance measured by the two signal collection electrodes 4 , The curve of conductivity and time.

In some embodiments, step S103 further includes using the first through hole, the second through hole, and the channel to connect the probe to the metal nanowire through physical adsorption or thiol self-assembly.

In one example, using the first through hole, the second through hole and the channel to connect the probe on the metal nanowire through physical adsorption or thiol self-assembly includes the following steps:

Into the first through hole 3 of the drainage block 2 is passed a biotin-modified bovine serum albumin solution dissolved in a phosphate buffered saline solution. The mass volume concentration of the biotin-modified bovine serum albumin solution is 200 μg/ml, so that the biotin The modified bovine serum albumin solution fills the first through hole 3, the bottom channel 7 and the second through hole 5, so that the biotin-modified bovine serum albumin solution stays in the bottom channel 7 at room temperature for 2 hours, and flows to the first part of the drainage block 2. A phosphate buffered saline solution is passed through the through hole 3, so that the biotin-modified bovine serum protein solution flows out from the second through hole 5;

Into the first through hole 3 of the drainage block 2, a streptavidin solution dissolved in phosphate buffered saline solution is passed, and the mass volume concentration of the streptavidin solution dissolved in phosphate buffered saline solution is 100 μg/ml, Fill the first through hole 3, the bottom channel 7 and the second through hole 5 with the streptavidin solution dissolved in the phosphate buffered saline solution, and the streptavidin solution dissolved in the phosphate buffered saline solution is in the bottom channel 7 37 Stay at ℃ for 1 hour, and pass the phosphate buffered saline solution into the first through hole 3 of the drainage block 2, so that the streptavidin solution dissolved in the phosphate buffered saline solution flows out of the second through hole 5 of the drainage block 2 ；

Pass the biotin-modified probe solution in phosphate buffered saline solution into the first through hole 3 of the drainage block 2, the molar concentration of the probe solution is 1 micromol/ml, so that the probe solution fills the first through hole 3. The bottom channel 7 and the second through hole 5, stay at room temperature for 1 hour, and pour the phosphate buffered saline solution into the first through hole 3 of the drainage block 2 to make the probe solution pass from the second through hole of the drainage block 2. 5, when the probe 8 is connected to the nanowire 6, a nanowire biosensor is obtained.

Figure 7 shows the nanowire biosensor modified through the above steps. After filling the first through hole 3, the bottom channel 7 and the second through hole 5 with a phosphate buffered saline solution, the impedance and conductance measured by the two signal collection electrodes 4 The curve of rate and time.

In another example, using the first through hole, the second through hole and the channel to connect the probe to the metal nanowire through physical adsorption or thiol self-assembly includes the following steps:

The 11-mercaptoundecanoic acid solution dissolved in pure ethanol is passed into the first through hole 3 of the drainage block 2, and the molar concentration of the 11-mercaptoundecanoic acid solution dissolved in pure ethanol is 1 mmol/L. Make the 11-mercaptoundecanoic acid solution dissolved in pure ethanol fill the first through hole 3, the bottom channel 7 and the second through hole 5. The 11-mercaptoundecanoic acid solution dissolved in pure ethanol is in the bottom channel 7 at room temperature Stay for 1 hour, and pass pure ethanol into the first through hole 3 of the drainage block 2, so that 11-mercaptoundecanoic acid dissolved in pure ethanol flows out of the second through hole 5 of the drainage block 2;

Into the first through hole 3 of the drainage block 2, a solution of 2-(N-morpholine)ethanesulfonic acid with a molar concentration of 50 mmol/L and pH=5.0 was passed, and the solution contained a molar concentration of 100 mmol/L. Liters of N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS ) To fill the first through hole 3, the bottom channel 7 and the second through hole 5 with the solution, and stay in the bottom channel 7 at room temperature for 30 minutes;

Into the first through hole 3 of the drainage block 2, a streptavidin solution dissolved in phosphate buffered saline solution is passed, and the mass volume concentration of the streptavidin solution dissolved in phosphate buffered saline solution is 100 μg/ml, Make the solution fill the first through hole 3, the bottom channel 7 and the second through hole 5, stay in the bottom channel 7 at room temperature for 1 hour, and pass the phosphate buffered salt solution into the first through hole 3 of the drainage block 2 to make The streptavidin solution dissolved in the phosphate buffered saline solution flows out from the second through hole 5 of the drainage block 2;

A glycine solution dissolved in deionized water is passed into the first through hole 3 of the drainage block 2, and the molar concentration of the glycine solution dissolved in deionized water is 1 mol/L, so that the solution fills the first through hole 3 and the bottom The channel 7 and the second through hole 5, stay in the bottom channel 7 at room temperature for 20 minutes, and pass deionized water into the first through hole 3 of the drainage block 2, so that the glycine solution dissolved in the deionized water is discharged from the drainage block 2. Flows out of the second through hole 5;

Pass the biotin-modified probe solution in phosphate buffered saline solution into the first through hole 3 of the drainage block 2, the molar concentration of the probe solution is 1 micromol/ml, so that the probe solution fills the first through hole 3. The bottom channel 7 and the second through hole 5, the probe solution stays in the bottom channel 7 at room temperature for 1 hour, and the phosphate buffered saline solution is passed into the first through hole 3 of the drainage block 2 to make the probe solution drain from The second through hole 5 of the block 2 flows out. At this time, the probe 8 is connected to the nanowire 6 to obtain a nanowire biosensor.

In other embodiments, the step of connecting the probe to the metal nanowire includes placing the wire on the signal collecting electrode, covering the signal collecting electrode with the wire on the insulator, and exposing the nanowire to the outside, and by physical adsorption or Thiol self-assembly connects probes on metal nanowires.

For example, as shown in FIG. 5, two wires 11 are placed on the signal collection electrode 4 respectively, and the signal collection electrode 4 on which the wires are placed is covered with an insulating member 10, so that the nanowire 6 is exposed to the outside to form a detection unit.

In one example, using wires to connect probes on metal nanowires through physical adsorption or thiol self-assembly includes the following steps:

Put the detection unit into the phosphate buffered saline solution with biotin-modified bovine serum albumin. The mass volume concentration of the phosphate-buffered saline solution with biotin-modified bovine serum albumin is 200 micrograms/ml, and let stand at room temperature 2 After hours, take out the detection unit and rinse with phosphate buffered saline solution to remove the unreacted biotin-modified bovine serum albumin on the surface of the detection unit;

Put the detection unit into the phosphate buffer salt solution dissolved with streptavidin. The mass volume concentration of the phosphate buffer salt solution dissolved with streptavidin is 100 μg/ml, and let stand at 37°C for 1 hour. Take out the detection unit and rinse with phosphate buffered saline solution to remove unreacted streptavidin on the surface of the detection unit;

Put the detection unit into the phosphate buffered saline solution with the biotin-modified probe, the molar concentration of the solution is 1 micromol/ml, and let it stand at 37°C for one hour. Connect the exposed nanowires on the surface of the detection unit The probe is taken out of the detection unit, rinsed with phosphate buffered saline solution, and the unreacted probe on the surface of the detection unit is removed. At this time, the probe is connected to the nanowire to obtain a nanowire biosensor.

In another example, using wires to connect probes on metal nanowires through physical adsorption or thiol self-assembly includes the following steps:

Put the detection unit into a pure ethanol solution containing 11-mercaptoundecanoic acid, the molar concentration of the pure ethanol solution containing 11-mercaptoundecanoic acid is 1 mmol/L, and let it stand at room temperature for 1 hour. Take out the detection unit, rinse the detection unit with pure ethanol, and remove the unreacted 11-mercaptoundecanoic acid on the surface of the detection unit;

Put the detection unit into 2-(N-morpholine)ethanesulfonic acid (MES) with a molar concentration of 50 mmol/L and pH=5.0, which contains N-ethyl- with a molar concentration of 100 mmol/L. N'-(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) with a molar concentration of 50 mmol/L, stand at room temperature for 30 Minutes, take out the detection unit;

Quickly put the detection unit into the phosphate buffered saline solution dissolved with streptavidin, the mass volume concentration of the solution is 100 μg/ml, and let stand at room temperature for 1 hour, take out the detection unit, and rinse with the phosphate buffered saline solution for detection Unit to remove unreacted streptavidin on the surface of the detection unit;

Put the detection unit in deionized water with a molar concentration of 1 mol/L with glycine dissolved in it, let it stand for 20 minutes at room temperature, take out the detection unit, rinse the detection unit with deionized water, and remove unreacted glycine on the surface of the detection unit;

Put the detection unit in a phosphate buffered saline solution with a biotin-modified probe dissolved in a molar concentration of 1 micromol/ml, and let it stand at 37°C for one hour. Connect the probe to the exposed nanowire on the surface of the detection unit. Take out the detection unit, rinse the detection unit with a phosphate buffered saline solution, remove unreacted probes on the surface of the detection unit, and obtain a nanowire biosensor.

In the preparation method of the nanowire biosensor of the present invention, the corresponding single-stranded nucleic acid probe is designed according to the target to be detected, and the target nucleic acid can be detected. At the same time, through electric heating nucleic acid melting and in-situ release technology, continuous and dynamic detection of target nucleic acid can be realized in situ, so as to grasp the change of the concentration of target nucleic acid molecules. The nanowire biosensor prepared by the method of the present invention can conveniently and quickly prepare the required nanowire biosensor, and is used to realize the melting and in-situ release of the biomarker simply and quickly. It can be used for the early diagnosis and prognosis of the tested person according to the changes in the concentration of specific biomarkers for the biomarkers of a certain disease. It can be widely used in early tumor screening and prognosis monitoring, single-cell research, and embryos in assisted reproduction. Cultivation and development and other fields.

In another aspect, the present invention provides a nanowire biosensor prepared using the above method. As shown in FIGS. 2 to 5, the nanowire biosensor includes a substrate, a signal collection electrode 4 and a metal nanowire 6 formed on the substrate, and a probe 8 formed on the metal nanowire 6.

In some embodiments, the material of the substrate includes at least one of silicon wafer, glass, organic polymer material or flexible material, preferably silicon wafer.

In some embodiments, the nanowire biosensor further includes an insulating layer formed on the substrate, wherein the signal collection electrode and the nanowire are formed on the insulating layer, and the thickness of the insulating layer is 10 to 100,000 nanometers. The substrate and the insulating layer constitute the base 1.

In some embodiments, the metal nanowire 6 includes a first metal connection layer formed on the substrate 1 and a first metal sensing layer formed on the first metal connection layer. The first metal sensing layer includes gold and platinum. The first metal connection layer includes at least one of chromium and titanium, the thickness of the first metal connection layer is 5 to 20000 nanometers, and the thickness of the first metal sensing layer is 5 to 30000 nanometers.

In some embodiments, the signal collection electrode 4 includes a second metal connection layer formed on the substrate 1 and a second metal sensing layer formed on the second metal connection layer. The second metal sensing layer includes gold and platinum. The second metal connection layer includes at least one of chromium and titanium, the thickness of the second metal connection layer is 5 to 20000 nanometers, and the thickness of the second metal sensing layer is 5 to 30000 nanometers.

In some embodiments, as shown in FIGS. 2 to 4, the nanowire biosensor further includes a drainage block 2 formed on the signal collection electrode 4 and the metal nanowire 6, wherein the bottom of the drainage block 2 is fixedly connected to the substrate 1. In some embodiments, the drainage block 2 is formed with a first through hole 3 and a second through hole 5, and a groove is formed at the bottom of the drainage block 2, and the width of the groove is equal to the length of the metal nanowire 6, and the width of the groove is equal to that of the metal nanowire 6. The length is 1-50 mm, preferably 4-6 mm, more preferably 5 mm, the depth of the groove is 10-500 micrometers, preferably 50-200 micrometers, more preferably 50 micrometers, and the groove becomes the first through hole 3 and the first through hole 3 The bottom channel 7 between the two through holes 5. In some embodiments, the length direction of the bottom channel 7 of the drainage block 2 and the length direction of the metal nanowire 6 are perpendicular to each other, and completely cover the metal nanowire 6.

In some embodiments, as shown in FIG. 5, the nanowire biosensor further includes a wire 11 formed on the signal collection electrode 4 and an insulating member 10 formed on the signal collection electrode 4 on which the wire 11 is placed. At least a part of the metal nanowire 6 is not covered by the insulating member 10. Optionally, two wires 11 are respectively placed on the signal collection electrode 4, the insulating member 10 covers the signal collection electrode 4 on which the wire 11 is placed, and the nanowire 6 is exposed to the outside to form a detection unit.

In some embodiments, the nanowires are metal nanowires. In some embodiments, the nanowire is an unmodified nanowire.

In some embodiments, the biosensor further includes a probe that is bound to the nanowire. In some embodiments, the probe is bound to the nanowire by non-specific binding. In some embodiments, the probe is bound to the nanowire by means of specific binding. In some embodiments, the probe is fixed on the nanowire by a chemical bond, and the chemical bond includes at least one of _{-SH, -OH, -COOH, and -NH 2.} In some embodiments, the probe is composed of specific biomolecules, and the biomolecules include at least one of DNA, DNA fragments, antigens, antibodies, proteins, and enzymes.

In some embodiments, the electrical properties of the nanowire make it sensitive to chemical changes on the surface of the nanowire. In some embodiments, the binding of the probe to the analyte in the sample can cause a detectable change in the electrical properties of the nanowire surface.

In some embodiments, microchannels are defined in the sample exposure area. In some embodiments, the smallest lateral dimension of the microchannel is less than 50 mm, preferably less than 4 mm. In some embodiments, the lateral dimension of the microchannel is the same as the longitudinal dimension of the nanowire.

In some embodiments, the sample exposure area is configured to receive a liquid sample.

In some embodiments, the biosensor further includes a substrate, and the nanowires are fixed on the substrate.

In some embodiments, the biosensor further includes a detector for detecting the characteristics of the nanowire. In some embodiments, the in-situ release of the object to be measured is achieved by applying a voltage to the detector. In some embodiments, the voltage applied to the detector does not cause the surface temperature of the nanowire to be higher than 95 degrees Celsius.

In another aspect, an application of the aforementioned nanowire biosensor for detecting biomarkers is provided. The application includes the following steps:

Pass a buffer solution (such as a phosphate buffered saline solution) into the nanowire biosensor, and collect the first voltage-current curve, conductivity curve, and impedance spectrum curve through the signal acquisition electrode;

Pass in the target sequence complementary to the probe in the nanowire biosensor, and collect the second voltage-current curve, conductivity curve and impedance spectrum curve through the signal acquisition electrode;

Apply voltage to the signal acquisition electrode to release the target sequence from the nanowire, and collect the third voltage-current curve, conductivity curve and impedance spectrum curve through the signal acquisition electrode;

Compare the first voltage-current curve, conductivity curve, and impedance spectrum curve, the second voltage-current curve, conductivity curve, and impedance spectrum curve, and the third voltage-current curve, conductivity curve, and impedance spectrum curve to achieve Nucleic acid amplification testing;

Repeat the above steps to perform multiple repeated detections of the target nucleic acid sequence.

In one example, the application of a nanowire biosensor for detecting biomarkers includes the following steps:

(I) Pour phosphate buffered saline solution (PBS) into the first through hole 3 of the drainage block 2 in the nanowire biosensor, so that the phosphate buffered saline solution fills the first through hole 3, the bottom channel 7 and the second through hole 5 , Using an electrochemical workstation to connect the first voltage-current curve, conductivity curve and impedance spectrum curve of the signal acquisition electrode 4 on the signal acquisition nanowire biosensor;

(II) Pass the target sequence complementary to the probe in the nanowire biosensor into the first through hole 3 of the drainage block 2, and after standing for 10 minutes, collect the second voltage of the signal collection electrode 4 on the nanowire biosensor -Current curve, conductivity curve and impedance spectrum curve;

(III) Apply a DC voltage of 0.5-1.5 volts, preferably 0.8-1.4 volts, such as 1 volt to the signal collection electrode 4 on the nanowire biosensor, and the application time is 10-120 seconds, preferably 60-90 seconds. The present invention In one embodiment, 1.2 volts are used for 60 seconds to release the target sequence from the nanowire 6 and flow out from the second through hole 5 of the drainage block 2 to collect the third voltage-current curve of the signal acquisition electrode 4, Conductivity curve and impedance spectrum curve. When the nanowire biosensor is energized to release the target nucleic acid sequence, conditions such as ultrasound, mechanical oscillation, and fluid shock can be applied to assist the release of the target sequence;

(IV) Pass a target sequence complementary to the probe in the nanowire biosensor into the first through hole 3 of the drainage block 2, and after standing for 10 minutes, collect the fourth voltage of the signal collection electrode 4 on the nanowire biosensor -Current curve, conductivity curve and impedance spectrum curve;

(V) The above-mentioned first voltage-current curve, conductivity curve and impedance spectrum curve, the second voltage-current curve, conductivity curve and impedance spectrum curve, the third voltage-current curve, conductivity curve and impedance spectrum curve and The fourth voltage-current curve, conductivity curve and impedance spectrum curve are compared to realize nucleic acid detection;

(VI) Repeat the above steps (I) to (V) to perform multiple repeated detections of the target nucleic acid sequence.

FIG. 8 shows the change curve of the impedance measured by the two signal collection electrodes 4 after each modification operation of the nanowire biosensor and each operation (I)-(III) described above. Figure 9 shows the change curve of the conductivity measured by the two signal acquisition electrodes 4 of the nanowire biosensor prepared by the first method after each modification operation and each step (I)-(III) above . In Figures 8 and 9, 1 represents the data measured by the two signal collection electrodes 4 in the initial state of the nanowire biosensor, and 2 represents the nanowire biosensor modified with biotin modified bovine serum albumin and collected by two signals The data measured by

electrode

4, 3 represents the data measured by the two signal acquisition electrodes 4 after the nanowire biosensor is modified with streptavidin, and 4 represents the nanowire biosensor modified by the probe modified with biotin. The data measured by the two

signal acquisition electrodes

4, 5 represents the data measured by the two signal acquisition electrodes 4 after passing 1 femtomolar target sequence into the nanowire biosensor and letting it stand for 15 minutes. Data, 6 represents the data measured by the two signal acquisition electrodes 4 after passing 1 picomolar target sequence into the nanowire biosensor and letting it stand for 15 minutes, after passing the phosphate buffered saline solution, 7 represents the data measured by the two signal acquisition electrodes 4 Electrode 4 is applied with a DC voltage of 1.1 volts, and the application time is 1 minute. The data measured by electrode 4 is collected by two signals. 8 represents passing 1 picomolar target sequence into the nanowire biosensor and letting it stand for 15 minutes. After the phosphate buffered saline solution, the data measured by the electrodes 4 are collected through two signals.

Put the nanowire biosensor into a solution (for example, a solution containing biological samples such as cell culture media, human body fluids, etc.), and collect the first voltage-current curve, conductivity curve, and impedance spectrum curve through the signal acquisition electrode;

According to the set time interval, collect multiple voltage-current curves, conductivity curves and impedance spectrum curves through the signal acquisition electrode, record multiple voltage-current curves, conductivity curves and impedance spectrum curves, and compare multiple curves , To achieve the detection of the target sequence;

Apply voltage to the signal acquisition electrode to release the target sequence from the metal nanowire;

Repeat the above steps to perform multiple repeated detections of the target sequence.

Put the nanowire biosensor into the cell culture medium, and collect the first voltage-current curve, the conductivity curve and the impedance spectrum curve of the signal acquisition electrode 4 on the nanowire biosensor;

According to the set time interval, collect multiple voltage-current curves, conductivity curves, and impedance spectrum curves of the signal collection electrode on the nanowire biosensor, record multiple voltage-current curves, conductivity curves, and impedance spectrum curves. Compare multiple curves to achieve the detection of target nucleic acid sequence;

Apply a DC voltage of 0.5-1.5 volts, preferably 0.8-1.4 volts, such as 1 volt, to the signal collection electrode on the nanowire biosensor, and the application time is 10-120 seconds, preferably 60-90 seconds, so that the target sequence is removed from the nanowire On release

The principle of the method of the present invention is that the nucleic acid melting method in the prior art includes heating up, changing the pH value and using helicase, etc. In the present invention, in order to realize the melting of nucleic acid without adding additional reagents, heating up is the best solution. Metal nanowires meet the above requirements, so metal nanowires are selected as the core component of the sensor. The preparation of metal nanowires adopts a double-layer method, the lower layer is a metal connection layer, the commonly used metals are chromium, titanium, etc., the upper layer is a metal sensing layer, and the commonly used metals are gold, platinum, etc. The above-mentioned metals have excellent physical and chemical properties, do not chemically react with most chemicals, and have good stability. Gold and platinum are excellent candidates for nanoelectrodes in electrochemical applications, which can be applied to pressure sensors, DNA detection sensors, etc. At the same time, metal nanowires can provide high current density, high signal-to-noise ratio and low double-electron-layer capacitance, making them very suitable for making sensors.

Due to the excellent electrical conductivity and thermal conductivity of metal nanowires, its width is nanometer level and its length is micrometer level. When a fixed voltage is applied to both ends of the metal nanowire for a certain period of time, a certain temperature can be generated on the surface of the metal nanowire. To melt and release the captured biomarkers in situ, at the same time, it is necessary to ensure that the modification on the surface of the metal nanowire will not be destroyed.

The modification system used in the present invention is based on the combination of avidin and biotin. According to literature research, the bond energy of adenine and thymine is 8kJ/mol or 1.9kcal/mol, and the bond energy of guanine and cytosine is 13kJ/mol or 3.1 kcal/mol. The affinity constant of avidin bound to biotin is one million times that of ordinary antigen-antibody reaction. The dissociation constant of the complex formed by the two is very small, and it will not be denatured at 100°C, showing irreversible reactivity. , Alkali, denaturant, proteolytic enzyme and organic solvent do not affect its binding.

Therefore, when the metal nanowire generates a lot of heat by applying electrical pulse signals to the nanowire, the stability of the combination of avidin and biotin is much higher than that of the DNA double-stranded reaction with the antigen-antibody. Heating of the wire surface realizes the melting and in-situ release of biomarkers. It is used for dynamic real-time detection of biomarker content to reflect the patient's physical health.

The nanowire biosensor and its preparation method and application provided by the present invention have the following advantages:

The preparation method of the nanowire biosensor provided by the present invention has the advantages of convenience, rapidity and low price. The corresponding single-stranded nucleic acid probe is designed according to the detected target, and the target nucleic acid can be detected after the sensor is modified. Then use the electric heating characteristics of the nanowire to realize nucleic acid melting and in-situ release technology, which can realize continuous and dynamic detection of target nucleic acid in situ, so as to grasp the concentration change of target nucleic acid molecules. Compared with the traditional melting method, changing the pH value, using DNA helicase, heating, etc., the nanowire biosensor prepared by the method of the present invention does not need to add any additional reagents, large auxiliary equipment, etc., and is convenient for miniaturization and portability. The preparation of nanowires adopts a double-layer structure of a metal connection layer and a metal sensing layer. The commonly used metals for the metal connection layer are chromium, titanium, etc., which can effectively improve the adhesion between the metal sensing layer and the substrate. The metal sensing layer commonly used metal is Gold, platinum and other materials with stable chemical properties and excellent electrical properties. Using this sensor, through the detection of specific biomarkers, the concentration changes can be used for early diagnosis and prognosis of the detected person. It can be widely used in early tumor screening and prognostic monitoring, single cell research, and assisted reproduction. In the field of embryo culture and development, the sensor is extremely small in size, high in detection sensitivity, and has the potential to develop into wearable and implantable devices.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and purpose of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

A nanowire biosensor, including:

Substrate

Signal collection electrodes and nanowires formed on the substrate, wherein the signal collection electrodes are connected to an external circuit for acquiring signals, and the nanowires are used as sensing units to identify and detect biological substances after surface modification .
The nanowire biosensor according to claim 1, wherein the signal collection electrode and the nanowire are both made of a metal material, and each includes a metal sensing layer and a metal connection layer, and the metal sensing layer includes gold. , At least one of platinum, and the metal connection layer includes at least one of chromium and titanium.
The nanowire biosensor according to claim 1, wherein the substrate is made of rigid or flexible material.
The nanowire biosensor according to claim 1, further comprising:

An insulating layer, the insulating layer is formed on the substrate, and the signal collection electrode and the nanowire are formed on the insulating layer.
The nanowire biosensor according to claim 1, wherein the nanowire is used to identify and capture biological substances after surface modification, and the biological substances include extracellular vesicles, cells, bacteria, such as nucleic acids, proteins, and exosomes. , At least one of the viruses.
The nanowire biosensor according to claim 1, wherein the signal obtained by the signal collecting electrode is an electric signal, and the electric signal includes at least one of electric potential, conductivity, electrical impedance, and electrical impedance spectrum.
The nanowire biosensor according to claim 5, wherein by applying a signal to the signal collection electrode, the surface temperature of the nanowire can be controlled, and the captured biological substance can be released in situ.
A method for preparing a nanowire biosensor includes the following steps:

Prepare the substrate;

Preparing signal collection electrodes and metal nanowires on the substrate; and

A probe is connected to the metal nanowire.
8. The method for preparing a nanowire biosensor according to claim 8, wherein the substrate material comprises at least one of silicon wafer, glass, organic polymer material or flexible material.
The method for preparing a nanowire biosensor according to claim 9, wherein the substrate material comprises silicon wafer.
The method for preparing a nanowire biosensor according to claim 8, further comprising the following steps:

An insulating layer is formed on the substrate, wherein the signal collection electrode and the nanowire are formed on the insulating layer, the thickness of the insulating layer is 10 to 100000 nanometers, the substrate and the insulating layer Form the base.
The method for preparing a nanowire biosensor according to claim 11, wherein the step of preparing signal collection electrodes and metal nanowires on the substrate comprises:

Using electron beam exposure to form nanowire grooves on the surface of the substrate;

Using magnetron sputtering to prepare the metal nanowire in the nanowire groove;

Preparing signal collection electrode grooves on the surface of the substrate by photolithography;

Magnetron sputtering is used to form the signal collection electrode in the signal collection electrode groove.
The method for preparing a nanowire biosensor according to claim 12, wherein using magnetron sputtering to prepare the metal nanowire in the nanowire groove comprises:

Sputtering a first metal connection layer in the nanowire groove;

Sputtering a first metal sensing layer on the first metal connection layer,

The first metal sensing layer includes at least one of gold and platinum, the first metal connecting layer includes at least one of chromium and titanium, and the thickness of the first metal connecting layer is 5 to 20000 nanometers. The thickness of the first metal sensing layer is 5 to 30000 nanometers.
12. The method for preparing a nanowire biosensor according to claim 12, wherein forming the signal collection electrode in the signal collection electrode groove by magnetron sputtering comprises:

Sputtering a second metal connection layer in the signal collection electrode groove;

Sputtering a second metal sensing layer on the second metal connection layer,

The second metal sensing layer includes at least one of gold and platinum, the second metal connecting layer includes at least one of chromium and titanium, and the thickness of the second metal connecting layer is 5 to 20000 nanometers. The thickness of the second metal sensing layer is 5 to 30000 nanometers.
The method for preparing a nanowire biosensor according to claim 11, further comprising the following steps:

Prepare drainage block.
The method for preparing a nanowire biosensor according to claim 15, wherein the step of preparing the drainage block comprises:

A first through hole and a second through hole are formed in the drainage block, and a channel is formed at the bottom of the drainage block, so that the width of the channel is equal to the length of the metal nanowire, and the channel is The first through hole communicates with the second through hole, the length of the channel is 1 to 50 millimeters, and the depth of the channel is 10 to 500 microns.
The method for preparing a nanowire biosensor according to claim 16, wherein the step of preparing the drainage block further comprises:

The bottom of the drainage block and the base are relatively fixed.
The method for preparing a nanowire biosensor according to claim 17, wherein the step of relatively fixing the bottom of the drainage block and the substrate comprises:

The length direction of the channel of the drainage block and the length direction of the metal nanowire are perpendicular to each other, and the metal nanowire is completely covered.
The method for preparing a nanowire biosensor according to claim 16, wherein the step of connecting a probe to the metal nanowire comprises:

The first through hole, the second through hole and the channel are used to connect probes on the metal nanowire through physical adsorption or thiol self-assembly.
8. The method for preparing a nanowire biosensor according to claim 8, wherein the step of connecting a probe to the metal nanowire comprises:

Placing a wire on the signal collecting electrode, covering the signal collecting electrode on which the wire is placed with an insulating member, so that the nanowire is exposed to the outside;

The probes are connected to the metal nanowires through physical adsorption or thiol self-assembly.
A nanowire biosensor, including:

Substrate

Signal collection electrodes and metal nanowires formed on the substrate; and

A probe formed on the metal nanowire.
The nanowire biosensor according to claim 21, wherein the substrate material includes at least one of silicon wafer, glass, organic polymer material or flexible material.
The nanowire biosensor according to claim 21, further comprising:

An insulating layer formed on the substrate, wherein the signal collection electrode and the nanowire are formed on the insulating layer, the thickness of the insulating layer is 10 to 100000 nanometers, the substrate and the insulating layer The layer constitutes the substrate.
The nanowire biosensor according to claim 23, wherein the metal nanowire comprises a first metal connection layer formed on a substrate and the first metal sensing layer formed on the first metal connection layer, the The first metal sensing layer includes at least one of gold and platinum, the first metal connecting layer includes at least one of chromium and titanium, and the thickness of the first metal connecting layer is 5 to 20000 nanometers. The thickness of the first metal sensing layer is 5 to 30000 nanometers.
The nanowire biosensor according to claim 23, wherein the signal collection electrode comprises a second metal connection layer formed on a substrate and a second metal sensing layer formed on the second metal connection layer, the The second metal sensing layer includes at least one of gold and platinum, the second metal connecting layer includes at least one of chromium and titanium, and the thickness of the second metal connecting layer is 5 to 20000 nanometers. The thickness of the second metal sensing layer is 5 to 30000 nanometers.
The nanowire biosensor according to claim 23, further comprising:

A drainage block formed on the signal collection electrode and the metal nanowire, wherein the bottom of the drainage block is fixedly connected with the substrate.
The nanowire biosensor according to claim 26, wherein a first through hole and a second through hole are formed in the drainage block, a channel is formed at the bottom of the drainage block, and the width of the channel is the same as that of the metal nanometer. The lengths of the wires are equal, the channel communicates with the first through hole and the second through hole, the length of the channel is 1 to 50 millimeters, and the depth of the channel is 10 to 500 microns.
The nanowire biosensor according to claim 27, wherein the length direction of the channel of the drainage block and the length direction of the metal nanowire are perpendicular to each other, and completely cover the metal nanowire.
The nanowire biosensor according to claim 21, further comprising:

A wire formed on the signal collection electrode; and

An insulating material layer formed on the signal collecting electrode where the wire is placed.
A biosensor includes a sample exposure area and nanowires, in which at least a part of the nanowires can be addressed by the sample.
The biosensor according to claim 30, wherein the nanowire is a metal nanowire.
The biosensor according to claim 30, wherein the nanowire is an unmodified nanowire.
The biosensor according to claim 30, further comprising:

Probe, which is bound to the nanowire.
The biosensor according to claim 33, wherein the probe is bound to the nanowire by non-specific binding.
The biosensor according to claim 33, wherein the probe is bound to the nanowire by means of specific binding.
The biosensor according to claim 33, wherein the probe is fixed on the nanowire by a chemical bond, the chemical bond comprising at least one of -SH, -OH, -COOH, and -NH 2.
The biosensor according to claim 33, wherein the probe is composed of a specific biomolecule, and the biomolecule includes at least one of DNA, DNA fragments, antigens, antibodies, proteins, and enzymes.
The biosensor according to claim 30, wherein the nanowire is sensitive to chemical changes on the surface of the nanowire due to the electrical properties of the nanowire.
The biosensor according to claim 33, wherein the combination of the probe and the analyte in the sample will cause a detectable change in the electrical properties of the nanowire surface.
The biosensor according to claim 30, wherein a microchannel is defined in the sample exposure area.
The biosensor according to claim 40, wherein the smallest lateral dimension of the microchannel is less than 50 mm.
The biosensor according to claim 41, wherein the smallest lateral dimension of the microchannel is less than 4 mm.
The biosensor according to claim 40, wherein the lateral dimension of the microchannel is the same as the longitudinal dimension of the nanowire.
The biosensor of claim 30, wherein the sample exposure area is configured to receive a liquid sample.
The biosensor according to claim 30, further comprising:

The substrate, the nanowire is fixed on the substrate.
The biosensor according to claim 30, further comprising:

The detector is used to detect the characteristics of the nanowire.
The biosensor according to claim 46, wherein the in-situ release of the object to be measured is achieved by applying a voltage to the detector.
The biosensor according to claim 47, wherein the voltage applied to the detector does not cause the surface temperature of the nanowire to be higher than 95 degrees Celsius.
The application of the nanowire biosensor according to any one of claims 21 to 28 for detecting biomarkers, comprising the following steps:

Pass a buffer into the nanowire biosensor, and collect a first voltage-current curve, a conductivity curve, and an impedance spectrum curve through the signal acquisition electrode;

A target sequence complementary to the probe in the nanowire biosensor is passed in, and a second voltage-current curve, a conductivity curve, and an impedance spectrum curve are collected through the signal collection electrode;

Applying a voltage to the signal collection electrode to release the target sequence from the nanowire, and collecting a third voltage-current curve, a conductivity curve, and an impedance spectrum curve through the signal collection electrode;

Compare the first voltage-current curve, conductivity curve, and impedance spectrum curve, the second voltage-current curve, conductivity curve, and impedance spectrum curve, and the third voltage-current curve, conductivity curve, and impedance spectrum curve , Realize nucleic acid detection;

Repeat the above steps to perform multiple repeated detections of the target nucleic acid sequence.
The application of the nanowire biosensor for detecting biomarkers according to claim 29, comprising the following steps:

Putting the nanowire biosensor into a solution, and collecting a first voltage-current curve, a conductivity curve, and an impedance spectrum curve through the signal collecting electrode;

According to the set time interval, multiple voltage-current curves, conductivity curves, and impedance spectrum curves are collected through the signal acquisition electrode, and multiple voltage-current curves, conductivity curves, and impedance spectrum curves are recorded. Make comparisons to achieve the detection of the target sequence;

Applying a voltage to the signal collection electrode to release the target sequence from the metal nanowire;

Repeat the above steps to perform multiple repeated detections of the target sequence.