CN110018207A - Biomolecule detection method and device - Google Patents

Abstract

The invention provides a biomolecule detection method and a device, wherein the method comprises the following steps: adding liquid to be detected to a nano sensor which is covalently connected with probe molecules, wherein the probe molecules can be specifically combined with a biomolecule; measuring first electrical characteristic data of the nanosensor added with the liquid to be measured under the condition that a fixed bias voltage is applied to the nanosensor added with the liquid to be measured; the method comprises the steps of comparing first electrical characteristic data and second electrical characteristic data to determine whether a liquid to be measured contains biomolecules, wherein the second electrical characteristic data is obtained by adding a liquid containing biomolecules with set concentration to a nano sensor to which probe molecules are covalently connected in advance and measuring the nano sensor under fixed bias. The content of the biological molecules is detected by the detection method, the target molecules can be free from marking in the detection process, and the rapid real-time detection can be realized to determine whether the biological molecules are contained or the concentration content of the biological molecules is measured.

Description

Biomolecule detection method and device

technical field

The present invention relates to the field of biomedicine, in particular to a method and device for detecting biomolecules.

Background technique

Malignant tumor is a kind of disease characterized by uncontrolled abnormal cell proliferation, which is difficult to cure and endangers human health, and has become a major problem faced by the medical field. Major diseases such as malignant tumors have caused a heavy burden on society and families. However, if the vast majority of tumors can be diagnosed in a timely and efficient manner, early detection and early treatment of tumors can be achieved, and the survival rate of patients can be significantly improved. Therefore, the detection of tumor molecular markers for early diagnosis and prognosis evaluation of tumors has always been a hot spot in the field of laboratory medicine.

Interleukin-6 (IL-6) is a multifunctional cytokine known as a regulator of immune and inflammatory responses. Its overexpression is associated with a variety of cancers including head and neck squamous cell carcinoma (HNSCC), colorectal cancer, and gastrointestinal cancer. In healthy people, IL-6 levels are less than 6 pg/mL, while in patient serum, IL-6 levels are 20 pg/mL or higher. Therefore, molecular detection of IL-6 is of great significance for the early detection and diagnosis of various diseases such as head and neck cancer.

At present, the conventional detection methods for IL-6 include enzyme-linked immunosorbent assay, fluorescence immunoassay and chemiluminescence immunoassay. However, these detection methods are complicated in the pre-processing of the samples, and need to label the analyte during the detection process, and the experimental operation process is cumbersome. The most important thing is that its detection time is long, and it cannot achieve fast real-time detection. Therefore, it is of great significance to develop a new method for biomolecular detection with high sensitivity, good selectivity, label-free and rapid real-time detection.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a biomolecule detection method and device to rapidly detect the concentration of biomolecules.

In order to achieve the above object, the present invention adopts the following scheme:

According to an aspect of the embodiments of the present invention, a biomolecule detection method includes: adding a liquid to be tested to a nanosensor covalently linked with a probe molecule, wherein the probe molecule can specifically bind to a biomolecule ;

Under the condition of applying a fixed bias voltage to the nanosensor after adding the liquid to be measured, measure the first electrical characteristic data of the nanosensor after adding the liquid to be measured;

By comparing the first electrical characteristic data with a second electrical characteristic data, it is determined whether the liquid to be tested contains the biomolecule, wherein the second electrical characteristic data is obtained by preliminarily adding all the biomolecules containing the set concentration. The liquid of the biomolecule is added to the nanosensor to which the probe molecule is covalently attached and the nanosensor is measured under the fixed bias.

In some embodiments of the present invention, a method for surface modification of the nanosensor with covalently linked probe molecules, comprising: introducing a hydroxyl group on the surface of the nanosensor; introducing an amino group at the outer end of the hydroxyl group; and adding an amino group to the amino group An aldehyde group is introduced at the outer end of the aldehyde group; the probe molecule is introduced into the outer end of the aldehyde group to obtain a nanosensor covalently connected with the probe molecule.

In some embodiments of the present invention, the method for measuring and obtaining the second electrical characteristic data includes: applying a fixed bias voltage to the nanosensor connected with the probe molecule to measure initial electrical characteristic data; A solution of the biomolecule with a known concentration is added to the nanosensor connected with the probe molecule, and the electrical characteristic data is re-measured under the fixed bias voltage; solution of the nanosensor; adding a solution containing a second known concentration of the biomolecules to the cleaned nanosensor, and re-measurement of the electrical characteristic data under the fixed bias voltage; according to the initial electrical characteristic The data, the re-measured electrical characteristic data, and the re-measured electrical characteristic data again obtain the second electrical characteristic data.

In some embodiments of the present invention, the nanosensor is a silicon nanowire field effect transistor.

In some embodiments of the present invention, the biomolecule is an IL-6 antigen, and the probe molecule is an IL-6 antibody.

In some embodiments of the present invention, both the first electrical characteristic data and the second electrical characteristic data are characteristic curves of current changing with time.

In some embodiments of the present invention, introducing hydroxyl groups on the surface of the nanosensor includes: introducing hydroxyl groups by cleaning the nanosensor with oxygen plasma.

In some embodiments of the present invention, introducing an amino group at the outer end of the hydroxyl group includes: immersing the nanosensor into which the hydroxyl group is introduced into an anhydrous ethanol solution of 3-aminopropyltriethoxysilane to introduce amino.

In some embodiments of the present invention, introducing an aldehyde group at the outer end of the amino group includes: immersing the amino group-introduced nanosensor in a glutaraldehyde solution to introduce an aldehyde group.

In some embodiments of the present invention, after introducing the probe molecule at the outer end of the aldehyde group, the method further includes: soaking the nanosensor in a phosphate buffer solution containing bovine serum albumin to block all the probes. Unreacted aldehyde groups on the nanosensor surface were removed and washed with the phosphate buffer solution.

According to another aspect of the embodiments of the present invention, a biomolecule detection device includes: a nanosensor covalently linked with a probe molecule;

Wherein, the probe molecule can specifically bind to a biomolecule; if the liquid to be tested is added to the nanosensor to which the probe molecule is covalently connected, and the added test liquid is measured under a fixed bias voltage After measuring the liquid, the nanosensor covalently connected with the probe molecule can obtain the first electrical characteristic data; The nanosensor covalently connected with the probe molecule, and measuring the nanosensor under the fixed bias voltage, can obtain second electrical characteristic data; wherein, by comparing the first electrical characteristic data and the The second electrical characteristic data can determine whether the biomolecule is contained in the liquid to be tested.

The biomolecule detection method and device of the present invention can obtain beneficial effects at least including: detecting the content of biomolecules by the above-mentioned detection method, the target molecule can be exempted from labeling during the detection process, and rapid real-time detection can be realized to determine whether Contain the biomolecule or measure its concentration.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts. In the attached image:

1 is a schematic diagram of a biomolecule detection method according to some embodiments of the present invention;

2 is a schematic diagram of a method for measuring and obtaining second electrical characteristic data according to some embodiments of the present invention;

3 is a graph showing the response results of a nanosensor applying a fixed bias voltage under different concentrations of biomolecule solutions in some embodiments of the present invention, and a graph of the current passing through the nanosensor as a function of time;

4 is a flow chart of a surface modification method for obtaining a nanosensor covalently linked with a probe molecule according to some embodiments of the present invention;

5 is a schematic structural diagram of some embodiments of the present invention, wherein the nanosensor can use a silicon nanowire field effect transistor;

6 is a schematic diagram of the principle of using a silicon nanowire field effect transistor to fabricate a nanosensor in some embodiments of the present invention;

7 is a schematic structural diagram of the combination of biomolecules and nanosensors in some embodiments of the present invention;

FIG. 8 is a comparative schematic diagram of the conductance changes of nanosensors before and after binding to biomolecules in some embodiments of the present invention;

FIG. 9 is a schematic diagram of the molecular structure of the modified silicon nanowire field effect transistor surface and the binding of IL-6 antigen and antibody according to some embodiments of the present invention.

Detailed ways

In order to make the purposes, technical solutions and advantages of the embodiments of the present invention more clearly understood, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Here, the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but not to limit the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, the same reference numbers represent the same or similar parts, or the same or similar steps.

In the prior art, conventional detection methods for biomolecules such as IL-6 include enzyme-linked immunosorbent assay, fluorescence immunoassay, and chemiluminescence immunoassay. It is long and cannot achieve fast real-time detection. In view of this, the present invention provides a biomolecule detection method, which can overcome or make up for the defects of the existing detection method that the object to be tested needs to be labeled and the detection time is long.

FIG. 1 is a flowchart of a method for detecting biomolecules according to some embodiments of the present invention. As shown in Figure 1, the detection method may include the following steps:

S1: adding the liquid to be tested to the nanosensor covalently linked with the probe molecule, wherein the probe molecule can specifically bind to a biomolecule;

S2: under the condition of applying a fixed bias voltage to the nanosensor after adding the liquid to be measured, measure the first electrical characteristic data of the nanosensor after adding the liquid to be measured;

S3: Determine whether the liquid to be tested contains biomolecules, or determine the concentration of the biomolecules by comparing the first electrical characteristic data with a second electrical characteristic data. Wherein, the second electrical characteristic data is obtained in advance by adding a liquid containing a biomolecule of a predetermined concentration to the nanosensor to which the probe molecule is covalently linked, and measuring the nanosensor under a fixed bias voltage.

In the above step S1, the biomolecule can be a certain antigen, the probe molecule can be a corresponding antibody, for example, the biomolecule can be IL-6 antigen, and the probe molecule can be IL-6 Antibody. In this case, the nanosensor can be covalently linked with the probe molecule IL-6 antibody, and the IL-6 antibody can specifically bind to the IL-6 antigen, resulting in the change of the conductance of the nanosensor.

The nanosensor may be a nanofield effect transistor, wherein the structure of the nanomaterial may be a nanowire, a nanosheet or the like. For example, the nanosensor may be a nanowire field effect transistor. The components of the nanomaterials may be various semiconductor materials, such as silicon, and in this case, the nanosensor may be a silicon nanosensor.

In the above step S2, when the nanosensor is a nanofield effect transistor, the liquid to be tested added to the nanosensor can form a liquid gate. This fixed bias can be applied on the backside of the nanosensor, ie the substrate. The size of the fixed bias voltage can be determined according to the specific conditions of the nanosensor.

In the above step S3, the first electrical characteristic data and the second electrical characteristic data are generally the same type of electrical characteristic data, or, the first electrical characteristic data can be obtained by converting the type of data to which the second electrical characteristic data belongs. The data of the type to which the characteristic data belongs, so that the two can be compared. For example, both the first electrical characteristic data and the second electrical characteristic data may be characteristic curves of current changing with time, may be characteristic curves of voltage, capacitance, etc. changing with time, or may be characteristics of current changing with voltage curve. Generally, the nanosensor used for measuring the second electrical characteristic data should be the same or similar in structure to the nanosensor used for measuring the first electrical characteristic data, so that more accurate detection results can be obtained by data comparison.

In this embodiment, the biomolecule content is detected by the above detection method, the target molecule can be exempted from labeling during the detection process, and rapid real-time detection can be realized to determine whether the biomolecule is contained or its concentration content can be measured.

In some embodiments, before the above-mentioned step S1, a nanosensor with covalently connected probe molecules can be obtained by surface modification in advance, wherein, the surface modification method for obtaining the nanosensor with covalently connected with probe molecules may include:

S41: Introducing hydroxyl groups on the surface of the nanosensor;

S42: introduce an amino group at the outer end of the hydroxyl group;

S43: introduce an aldehyde group at the outer end of the amino group;

S44: introducing the probe molecule at the outer end of the aldehyde group to obtain a nanosensor covalently linked with the probe molecule.

In the above-mentioned step S41, hydroxyl groups can be introduced into the nanosensor by cleaning the nanosensor with oxygen plasma. Specifically, a pre-prepared nanosensor (eg, silicon nanowire field effect transistor) can be cleaned with oxygen plasma for a period of time. Over time, hydroxyl groups are introduced on the surface of the nanosensor (eg, on silicon atoms).

In the above step S42, the amino group can be introduced based on the previously introduced hydroxyl group by soaking the nanosensor into which the hydroxyl group is introduced in 3-aminopropyltriethoxysilane absolute ethanol solution for a period of time.

In the above-mentioned step S43, an aldehyde group may be introduced based on the previously introduced amino group by soaking the amino group-introduced nanosensor in a glutaraldehyde solution for a period of time.

In some embodiments, a method for obtaining the second electrical characteristic data may be obtained by measurement as described below. As shown in FIG. 2 , the method may include:

S51: apply a fixed bias voltage to the nanosensor connected with the probe molecule, and measure the initial electrical characteristic data;

S52: adding a solution containing a biomolecule of a first known concentration to the nanosensor connected with the probe molecule, and re-measure the electrical characteristic data under a fixed bias voltage;

S53: cleaning the solution nanosensor to which the first known concentration of biomolecules is added;

S54: adding a solution containing a second known concentration of biomolecules to the cleaned nanosensor, and measuring the electrical characteristic data again under a fixed bias voltage;

S55: Obtain the second electrical characteristic data according to the initial electrical characteristic data, the re-measured electrical characteristic data, and the re-measured electrical characteristic data.

In the above step S51, the initial electrical characteristic data may refer to the electrical characteristic data of the nanosensor itself to which the probe molecules are connected. For example, a fixed voltage may be applied to the back of the nanosensor (ie, the substrate), and then the source-drain measurement may be performed. The electrical property data of the nanosensor connected with the probe molecule can be obtained by comparing the electrical conductivity between them.

In the above steps S52 and S54, if deionized water is added to the nanosensor in the above step S51, the nanosensor can be dried before adding a solution with a certain concentration of biomolecules, so that the concentration of the solution used is accurate. When adding a nanosensor with a solution of a certain concentration of biomolecules, the liquid gate of the nanosensor can be formed by using the liquid of this concentration.

In the above-mentioned step S55, for example, a plurality of electrical characteristic curves can be formed by using the electrical characteristic data of the nanosensor connected with the probe molecule and the electrical characteristic data at two concentrations. The position of the regions in the plurality of electrical characteristic curves is used to determine whether the liquid to be tested contains the detected biomolecules and the corresponding concentration.

In the above embodiments, the same unused nanosensors can also be employed when measuring solutions containing a second known concentration of biomolecules. The first known concentration and the second known concentration can change in an equal gradient, and the obtained second electrical characteristic data includes multiple sets of electrical characteristic data of gradient concentrations. The smaller the gradient, the more detailed the obtained second electrical characteristic, and the result of data comparison. Also more precise.

By measuring the second electrical characteristic data according to the above method, the electrical characteristic data of the nanosensor in the biomolecule solution of each gradient concentration under the same bias voltage, for example, the relationship between current and time can be obtained. Based on this, the electrical characteristic data of the nanosensor in the biomolecule solution of unknown concentration can be compared, and the specific concentration value of the biomolecule solution of unknown concentration can be confirmed according to the corresponding relationship.

In this embodiment, the second electrical characteristic data may include data corresponding to a plurality of biomolecule solutions with different concentrations, and a more accurate biomolecule concentration range can be obtained by comparing the data with the first electrical characteristic data.

Based on the same inventive concept as the above-mentioned biomolecule detection method, the embodiment of the present invention further provides a biomolecule detection device, and the specific implementation of the device can be implemented with reference to the specific embodiment of the above-mentioned method, so the repetition will not be repeated. In some embodiments, the biomolecule detection device may include: a nanosensor covalently linked with a probe molecule; wherein, the probe molecule can specifically bind to a biomolecule; if the liquid to be detected is added to the covalently The first electrical characteristic data can be obtained by measuring the nanosensor covalently connected with the probe molecule after adding the liquid to be tested under a fixed bias voltage. A liquid containing a set concentration of the biomolecules is added to the covalently attached probe molecule nanosensor, and the nanosensor is measured under the fixed bias , second electrical characteristic data can be obtained; wherein, by comparing the first electrical characteristic data and the second electrical characteristic data, it can be determined whether the liquid to be tested contains the biomolecule. For example, the above-mentioned biomolecule can be an IL-6 antigen, the probe molecule can be an IL-6 antibody, and the nanosensor can be a silicon nanowire field effect transistor. The nanosensors covalently linked with probe molecules can include the following molecular structures:

Wherein, the double bond at the upper end of the above molecular structure is used to connect the probe molecules, and the three single bonds at the lower end are used to connect the atoms of the material of the nanosensor.

FIG. 3 is a graph showing the response results of a nanosensor with a fixed bias applied under different concentrations of biomolecule solutions in some embodiments of the present invention, and a graph of the current passing through the nanosensor as a function of time. As shown in Figure 3, a graph of the current versus time was obtained for solutions of biomolecules at 1, 3 and 5 mg/ml concentrations of the nanosensor at the same bias voltage.

In some embodiments of the present invention, in order to facilitate the understanding of the method and principle of measuring the second electrical characteristic data of the present invention, taking a nanosensor as an example, a set of specific data is used to explain the steps of measuring the second electrical characteristic data below. , but the numerical values listed are not limited, and can be appropriately adjusted as needed.

For the detection of biomolecules, such as IL-6, the present invention adopts the following scheme, and the specific equipment can use the semiconductor parameter analyzer 1500 and the pipette, and the specific operation steps are as follows:

A: First, apply a fixed bias voltage to the nanosensor, and scan the relationship between the current and time of the nanosensor (I-T curve);

B: After the current is stable, use a pipette to absorb a certain amount of IL-6 antigen of a certain concentration, drop it on the area to be measured of the nanosensor, and observe the current change;

C: After the current is stable, use a pipette to absorb a certain amount of ultrapure water to wash the antigen in the area to be tested, so that antigens of other concentrations can be detected in the same area to be measured by the nanosensor.

D: After the area to be tested is cleaned and dried, repeat steps AB to test other concentrations of IL-6 antigen in turn, and finally obtain a curve of the relationship between concentration gradient and current (as shown in Figure 3).

As shown in Figure 3, under a certain fixed bias voltage, the current value of the nanosensor does not change much in the first 40 seconds of the solution of IL-6 with different concentrations, and the solution concentration has little effect on the current value of the nanosensor; From 40 to 60 seconds, the antigen-antibody binds. Due to the different binding numbers of the labeled molecules, the conductance of the nanosensor changes, and the current value of the nanosensor increases and tends to rise; after 60 seconds, the current value tends to be stable. and the current change curve, the concentration of the solution to be tested can be determined. In addition, the higher the concentration of IL-6 in the solution, the more upward the curve of the current versus time of the nanosensor.

The whole testing process of biomolecules such as IL-6 in the present invention only takes 30-60s, and no other labeling signals are required during the testing process, so that real-time label-free detection can be realized, and it has ultra-high sensitivity. At the same time, this nanosensor will not destroy the activity of biomolecules and has the advantage of non-invasiveness.

In some embodiments of the present invention, the nanosensor may be a silicon nanowire field effect transistor. The combination of Field Effect Transistor (FET, Field Effect Transistor) biosensors and nanomaterials is one of the most valuable biosensors in recent years. It has the characteristics of signal amplification. At the same time, in addition to the excellent characteristics of nanomaterials, one-dimensional silicon nanowire devices also have other advantages, such as: the size is comparable to that of biological entities, good biocompatibility, large specific surface area, and easy to immobilize more probe molecules , it is also compatible with the current CMOS (Complementary Metal-Oxide-Semiconductor) manufacturing process, easy to integrate and mass-produce, and has a natural oxide layer on the surface, easy to modify biological groups, can be Greatly improve the sensitivity of biosensors.

As shown in Figure 5 and Figure 6, the structure of the silicon nanowire field effect transistor is similar to that of the FET. It consists of three electrodes: source S, drain D and gate. A wire (silicon nanowire) is connected to form a conductive channel, which is used as a sensing element of the device. As shown in Figure 6, under a buffer, the solution to be tested is used as a liquid gate to modulate the conductance.

The detection principle is shown in Figure 7. Before the detection, the probe molecule used to identify the target molecule to be detected needs to be modified on the surface of the nanosensor. During the detection process, the charged target molecule, that is, the biological molecule to be detected will be detected. Binds directly to the probe molecule, causing a change in the conductance of the nanosensor. As shown in Figure 8, Figure 8(a) is a curve of the conductance of the nanosensor unbound to the target molecule with time, and Figure 8(b) is a schematic diagram of the conductance change after the nanosensor is bound to the target molecule (target).

In some embodiments of the present invention, the nanosensor adopts a silicon nanowire field effect transistor, and after the p-type silicon nanowire modified with the probe molecule is specifically bound to the negatively charged target molecule, the silicon nanowire is used for conducting electricity. The number of hole carriers decreases, resulting in a decrease in its conductance, and the properties and concentration of the analyte are reflected by the change of the electrical signal. The electrical signal can be a current or a voltage signal.

In some embodiments of the present invention, the nanosensor can be fabricated by using a Fin FET (Fin Field-Effect Transistor) process, which is not only efficient, but also can greatly reduce the size of silicon nanowires. While improving the sensitivity of the device, the size of the sensor is also reduced, which is beneficial to integration and portability. In the detection of biomolecules, in order to improve the sensitivity and selectivity, the surface of the silicon nanowires can be modified to facilitate the coupling of more probe molecules.

In some embodiments of the present invention, as shown in FIG. 4 , the following surface modification methods for obtaining nanosensors covalently linked with probe molecules can be used, including: introducing hydroxyl groups (-OH) on the surface of the nanosensors; An amino group (-NH ₂ ) is introduced into the outer end of the hydroxyl group; an aldehyde group (-CHO) is introduced into the outer end of the amino group; the probe molecule is introduced into the outer end of the aldehyde group to obtain a covalently connected probe Molecular nanosensors. This method can couple more probe molecules on the surface of the nanosensor to improve the sensitivity and selectivity of detecting target molecules.

In some embodiments of the present invention, introducing hydroxyl groups on the surface of the nanosensor includes introducing hydroxyl groups by cleaning the nanosensor with oxygen plasma.

In some embodiments of the present invention, introducing an amino group at the outer end of the hydroxyl group includes immersing the nanosensor into which the hydroxyl group has been introduced into an anhydrous ethanol solution of 3-aminopropyltriethoxysilane to introduce an amino group .

In some embodiments of the present invention, introducing an aldehyde group at the outer end of the amino group includes introducing the aldehyde group by soaking the amino group-introduced nanosensor in a glutaraldehyde solution.

In some embodiments, the method of the above embodiment, after introducing the probe molecule at the outer end of the aldehyde group, may further include: soaking the nanosensor in a phosphate buffer solution containing bovine serum albumin , to block unreacted aldehyde groups on the nanosensor surface, and washed with the phosphate buffer solution.

In some embodiments of the present invention, cleaning can be performed before and after the step of introducing each group into the silicon nanowire field effect transistor.

In order to facilitate the understanding of the principle of the nanosensor fabrication method of the present invention, taking a silicon nanowire field effect transistor as an example, a set of specific data is used to explain the fabrication process steps of the nanosensor, as well as the structure and working principle of the nanosensor. The value of is not limited and can be adjusted appropriately according to needs.

In order to detect IL-6, the following scheme was adopted for the surface modification of silicon nanowires of silicon nanowire field effect transistors, and the specific steps are as follows:

A: First, soak the silicon nanowire field effect transistors in acetone, ethanol and deionized water respectively, wash with a shaker for 10 minutes, and blow dry with nitrogen for use.

B: Then, the surface of the silicon nanowire field effect transistor was cleaned with oxygen plasma for 2 minutes, the power was 150W, and the oxygen flow rate was 100sccm. After cleaning, more hydroxyl groups (-OH) are formed on the surface of the silicon nanowires, and then silanol ends (Si-OH) are formed.

C: In order to introduce amino groups (-NH ₂ ) on the surface of silicon nanowire field effect transistors, the device was placed in 2% v/v (volume ratio) of 3-aminopropyltriethoxysilane (APTES) with structural formula (I). ) in anhydrous ethanol solution for 40min. Then rinsed with absolute ethanol for 3 times and heated at 120 °C for 30 min to remove unbound APTES and absolute ethanol on the surface.

D: Next, the silicon nanowire field effect transistor with surface ammonia functionalization was immersed in a 2.5% glutaraldehyde solution with structural formula (II) for 1 h at room temperature, and the terminal aldehyde surface was introduced, namely the aldehyde group (-CHO) ), then rinsed thoroughly with deionized water to remove excess glutaraldehyde.

E: After surface functionalization, the IL-6 antibody was dropped on the surface of the silicon nanowire field effect transistor, and incubated at 37°C for 1 h under humid conditions, so that the antibody was covalently linked to the surface of the silicon nanowire to obtain a nanosensor. The molecular structure of the sensor can be shown in Figure 9, which also shows the molecular binding structure of the IL-6 antigen and antibody.

F: Finally, in order to prevent the non-specific binding of other proteins in the detection step, the nanosensor with IL-6 antibody immobilized was placed in a solution containing 5% w/v (weight to volume) BSA (Albumin from bovine serum, bovine serum albumin). ) in PBS buffer for 5 min to block unreacted aldehyde groups on the surface of silicon nanowires, as shown in Figure 9, and then washed with the same buffer for 3 min.

Before detection, the treated nanosensors were placed in PBS buffer and stored at 4°C for later use. PBS is a phosphate buffered saline (phosphate buffer saline), which is generally used as a solvent to dissolve the protective reagent.

According to the biomolecule detection method of the present invention, the entire test process for biomolecules such as IL-6 only takes 30-60s, and no other label signals are required during the test process, so that real-time label-free detection can be realized, and it has ultra-high sensitivity. At the same time, this nanosensor will not destroy the activity of biomolecules and has the advantage of non-invasiveness.

In the description of this specification, reference to the description of the terms "one embodiment", "one specific embodiment", "some embodiments", "for example", "example", "specific example" or "some examples" etc. means A particular feature, structure, material, or characteristic described in connection with this embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. The sequence of steps involved in each embodiment is used to schematically illustrate the implementation of the present invention, and the sequence of steps therein is not limited, and can be appropriately adjusted as required.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above-mentioned specific embodiments are only specific embodiments of the present invention, and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. a kind of biomolecule detecting method characterized by comprising

Testing liquid, which is added to, to be covalently attached has on the nano-sensor of probe molecule, wherein the probe molecule can be with One biomolecule is specifically bound；

In the case where applying a fixed-bias transistor circuit to the nano-sensor after the addition testing liquid, described in measurement addition First electrology characteristic data of the nano-sensor after testing liquid；

By comparing the first electrology characteristic data and one second electrology characteristic data, determine in the testing liquid whether contain There is the biomolecule, wherein the second electrology characteristic data are pre- first pass through the biology point comprising setting concentration The liquid of son, which is added to, to be covalently attached the nano-sensor for having probe molecule and passes under the fixed-bias transistor circuit to this nanometer What sensor measured.

2. biomolecule detecting method as described in claim 1, which is characterized in that obtaining the covalent linkage has probe molecule Nano-sensor surface modification method, comprising:

Hydroxyl is introduced on the surface of nano-sensor；

Amino is introduced in the outer end of the hydroxyl；

Aldehyde radical is introduced in the outer end of the amino；

The probe molecule is introduced in the outer end of the aldehyde radical, obtains being covalently attached the nano-sensor for having probe molecule.

3. biomolecule detecting method as described in claim 1, which is characterized in that the measurement of the second electrology characteristic data Obtain method, comprising:

Apply fixed-bias transistor circuit in the nano-sensor for being connected with probe molecule, measures initial electrology characteristic data；

The solution of the biomolecule comprising the first known concentration is added to the nanosensor for being connected with probe molecule On device, electrology characteristic data are measured again under the fixed-bias transistor circuit；

Nano-sensor described in the solution of the biomolecule of cleaning the first known concentration of addition；

The solution of the biomolecule comprising the second known concentration is added on the nano-sensor after cleaning, in institute It states and measures electrology characteristic data under fixed-bias transistor circuit again again；

According to initial electrology characteristic data, again the electrology characteristic data measured and the electrology characteristic data measured again again obtain To the second electrology characteristic data.

4. biomolecule detecting method as claimed in claim 2, which is characterized in that the nano-sensor is silicon nanowires field Effect transistor.

5. biomolecule detecting method as claimed in claim 2, which is characterized in that the biomolecule is IL-6 antigen, institute Stating probe molecule is IL-6 antibody.

6. biomolecule detecting method as claimed in claim 3, which is characterized in that the first electrology characteristic data and described Second electrology characteristic data are the characteristic curve that electric current changes over time.

7. biomolecule detecting method as claimed in claim 2, which is characterized in that introduce hydroxyl on the surface of nano-sensor Base, comprising:

Hydroxyl is introduced by cleaning the nano-sensor with oxygen plasma.

8. biomolecule detecting method as claimed in claim 2, which is characterized in that

Amino is introduced in the outer end of the hydroxyl, comprising:

It is impregnated by the way that the nano-sensor for being introduced into hydroxyl to be placed in 3- aminopropyl triethoxysilane ethanol solution, To introduce amino；

Aldehyde radical is introduced in the outer end of the amino, comprising:

By the way that the nano-sensor for being introduced into amino to be soaked in glutaraldehyde solution, to introduce aldehyde radical.

9. biomolecule detecting method as claimed in claim 2, which is characterized in that introduce the spy in the outer end of the aldehyde radical After needle molecule, further includes:

The nano-sensor is placed in the phosphate buffer solution containing bovine serum albumin(BSA) and is impregnated, to block the nanometer to pass The unreacted aldehyde radical of sensor surfaces, and washed with the phosphate buffer solution.

10. a kind of biomolecule detection devices characterized by comprising be covalently attached the nano-sensor for having probe molecule；

Wherein, the probe molecule can be specifically bound with a biomolecule；If testing liquid is added to described total Valence is connected on the nano-sensor of probe molecule, and described total after the measurement addition testing liquid under a fixed-bias transistor circuit Valence is connected with the nano-sensor of probe molecule, can obtain the first electrology characteristic data；If by comprising described in setting concentration The liquid of biomolecule is added to the nanosensor for being covalently attached and having the covalent linkage of probe molecule to have probe molecule Device, and the nano-sensor is measured under the fixed-bias transistor circuit, the second electrology characteristic data can be obtained；Wherein, lead to It crosses the first electrology characteristic data and the second electrology characteristic data can determine in the testing liquid whether contain There is the biomolecule.