WO2020113361A1

WO2020113361A1 - Functionalized black phosphorus/gold composite material and application thereof

Info

Publication number: WO2020113361A1
Application number: PCT/CN2018/118880
Authority: WO
Inventors: 吴立冬; 刘欢; 李晋成; 韩刚
Original assignee: 中国水产科学研究院
Priority date: 2018-12-03
Filing date: 2018-12-03
Publication date: 2020-06-11
Also published as: KR102623728B1; KR20210029813A

Abstract

An L-cysteine modified black phosphorus, comprising ionic bonding formed between -OH and -NH2. The modified black phosphorus is used for preparing an electrode and a sensor for detecting malachite green.

Description

Functionalized black phosphorus/gold composite material and its application

Technical field

The present application relates to the field of biosensors, in particular, to functionalized black phosphorus, functionalized black phosphorous gold nanocomposite, metal electrodes and biosensors including them, and applications of the biosensors.

Background technique

In 2014, a single layer and a few layers of black phosphorus (BP) were separated. Since then, the global heat of BP research has been detonated. At present, more than 400 publications on BP materials have been obtained. BP is a recognized new two-dimensional (2D) material with many advantages, such as high strength and hardness, tunable electronic, optical and catalytic properties. Recently, BP having a wide band nonlinear optical response characteristics, associated to a thickness of 0.3 to 2.0eV ⁹ from the band gap range, a strong in-plane anisotropy characteristics, high charge carrier mobility (around cm2 V ^-1 s ^{- 1} ) ¹² and the theoretical capacity of 2596mAh g ^-1 as the anode of Li/Na ion battery, so it has broad application prospects in the fields of optoelectronics, electronics, selective sensors, catalysis, biosensors, batteries, etc. Because of its biocompatibility, low cytotoxicity, and high charge carrier mobility, BP is a promising candidate for a biosensor platform. Although BP has attractive properties and is one of the most thermodynamically stable phosphorus allotropes, its degradation in oxygen-enriched and water environments is still quite serious, resulting in rapid loss of semiconductor properties. This limits the development of BP in electrochemical sensing.

The degradation of BP caused by oxygen and water, especially for thinner BP, will cause further deterioration due to exposure to light and high temperature humidity. The researchers explored many different methods to overcome the degradation of BP in oxygen and water environments, including encapsulation, non-covalent functionalization, and covalent chemical modification. The non-covalent functionalization of BP not only maintains its original honeycomb structure and good conductivity, but also improves its stability and dispersion in aqueous solution, providing it with good performance as a biosensing platform in aqueous solution Possibility. Traditional non-covalent functionalization has the problem of high cost of modifiers. In addition, it also has the disadvantage that BP is difficult to support with composite materials. The existing known non-covalent functional groups mainly rely on the NH ₂ support, but only play a role in passivating black phosphorus, but do not make it have more functional groups.

Summary of the invention

In response to various problems in the prior art, the present invention uses L-cysteine self-assembled membrane to passivate BP, so that the obtained BP can overcome the problem of instability under oxygen and humidity conditions. Au-S self-assembly connects functionalized BP to gold nanoparticles (AuNP), providing a binding site for further anchoring of aptamers. Functionalized BP and aptamers work together to improve the sensitivity and selectivity of biosensors.

The aptamer has a specific recognition area for selective detection of environmental analytes. An aptamer is a single-stranded DNA or RNA that has a high affinity for a target and is called a "chemical antibody." In addition, aptamers are easy to prepare, low cost, repeatable, and non-immunogenic, thus providing a promising option for the development of selective biosensors. By using functionalized BP as a biosensing platform, high sensitivity and low detection limit (DL) can be achieved.

In a promising application of the invention, functionalized BP as a biosensor material can be used to detect malachite green (MG). MG is a cationic triphenylmethane dye, which can be used as the detection target of the biosensor constructed by functional BP. Since 1936, although MG has adverse effects on the immune system and reproductive system, such as infertility and respiratory diseases, it is still widely used in commercial aquaculture to resist saprophytic bacteria. In order to ensure food safety, it is urgent MG needs to be screened quickly, selectively and sensitively.

According to one aspect of the present application, an L-cysteine-modified black phosphorus is provided. By forming an ionic bond between -OH and -NH ₂ , the black phosphorus combines with L-cysteine to form functionalized black phosphorus, and the functionalized black phosphorus has a uniform protective layer on the surface.

The present invention introduces a thiol group, thereby making the mounting of gold nanoparticles easier. Gold particles are currently the classic material for bio-immobilization, and the application range of black phosphorus materials has been expanded since then. In addition, cysteine is cheap and readily available, making it an ideal black phosphorus modifier.

L-cysteine with NH ₂ and SH groups was used for non-covalent coating of the BP surface at room temperature. According to the structure of BP and L-cysteine, L-cysteine is coated non-covalently through the electrostatic interaction between the NH _{2 of} L-cysteine and the lone pair of electrons of BP BP surface.

According to another aspect of the present application, an L-cysteine-black phosphorus-gold nanocomposite is provided. The nanocomposite includes L-cysteine-modified black phosphorus and gold nanoparticles, and the L-cysteine-modified black phosphorus and the gold nanoparticles are connected by Au-S self-assembly.

According to another aspect of the present application, a method for preparing L-cysteine-modified black phosphorus is provided. The method includes the steps of: placing a sheet-shaped substrate on which black phosphorus is placed in a container and sealing, an oil bath Heat to 110℃～140℃ and keep warm for 10-30 minutes; then contact L-cysteine with black phosphorus at 100℃～150℃ for not less than 5 minutes to obtain the L-cysteine Modified black phosphorus.

According to another aspect of the present application, a method for preparing L-cysteine-black phosphorus-gold nanocomposite is provided. The method includes the steps of preparing L-cysteine-modified black phosphorus, and: A solution containing the L-cysteine-modified black phosphorus is mixed with a solution in which gold nanoparticles are dispersed to obtain a mixture; the mixture is separated, and the resulting solid is washed to obtain the L-cysteine-black Phosphorus-gold nanocomposite; preferably, the mass ratio of L-cysteine-modified black phosphorus to gold nanoparticles in the mixture is 0.1 to 0.5: 0.5 to 5.

The gold nanoparticle-dispersed solution is prepared by a method including the following steps: mixing chloroauric acid and vitamin C with a mass of 0.2 to 1:20 to 60 in water.

In the step of preparing gold nanoparticles, a preferred embodiment is to mix 50 mL of 0.1-10 mg L ^-1 chloroauric acid and 1-10 mL of vitamin C (4 mg mL ^-1 ) for 2 hours.

In the step of mixing the functionalized black phosphorus solution with the gold nanoparticle solution, a preferred embodiment is to mix 50 mL of 0.1-0.5 mg mL ^-1 functionalized BP solution with 0.1-5 mg mL ^-1 gold nanoparticle solution, and quickly Stir for 10 minutes.

According to yet another aspect of the present application, a metal electrode is provided, the metal electrode comprising L-cysteine-modified black phosphorus or L-cysteine-black phosphorus-gold nanocomposite. Preferably, the metal electrode is a gold electrode.

In a preferred embodiment, the metal electrode further contains an aptamer solution, and the aptamer includes a specific recognition area to specifically bind to the analyte. The user can select the aptamer solution according to the needs of the biochemical properties of the analyte. For example, aptamers such as polychlorinated biphenyl aptamers and aflatoxin aptamers can be used in the biosensor constructed in the present invention.

According to yet another aspect of the present application, there is provided a method for preparing a metal electrode, the method comprising the steps of preparing an L-cysteine-black phosphorus-gold nanocomposite, and: applying a series of alumina powder to the metal electrode After polishing, it was cleaned with ethanol and deionized water ultrasonically; the metal electrode was electrochemically polished by potential scanning; the obtained L-cysteine-black phosphorus-gold nanocomposite was oscillated with a vortex meter and added to On the surface of the metal electrode.

In the electrochemical polishing step, a preferred embodiment is to perform electrochemical polishing from 0 to 1.7 V in the range of 0.1-5 mol L ^-1 H ₂ SO ₄ . The metal electrode may be gold, platinum, glassy carbon electrode and the like. Preferably, the metal electrode is a gold electrode.

In a preferred embodiment, the method further includes adding an aptamer solution to the metal electrode

In a preferred embodiment, the aptamer solution is a mercapto aptamer solution.

In a preferred embodiment, the sulfhydryl aptamer solution is selected from 5'-terminal thiol modified single-stranded DNA.

In a preferred embodiment, it is washed with deionized water to remove weakly adsorbed aptamers.

According to yet another aspect of the present application, there is provided a biosensor including or modified from L-cysteine-modified black phosphorus, L-cysteine-black phosphorus-gold nanocomposite At least one of the metal electrodes.

The biosensor according to the present invention can be used to detect malachite green.

The beneficial effects of the present invention include but are not limited to the following:

1) The present invention provides a black phosphorus modified with L-cysteine which is stable under water environment;

2) The present invention provides an L-cysteine-black phosphorus-gold nanocomposite, which provides a binding site for further anchoring of aptamers;

3) The present invention provides a method for preparing L-cysteine-modified black phosphorus which is stable in an aqueous environment;

4) This book provides a method for preparing L-cysteine-black phosphorus-gold nanocomposite;

5) The present invention provides a metal electrode for detecting environmental targets, and further provides a biosensor including such an electrode, which has low DL, high sensitivity and high specificity, and is expected to become a rapid on-site measurement of the object to be tested Tool of.

BRIEF DESCRIPTION

FIG. 1 shows a schematic diagram of the preparation process and detection mechanism of the BP-AuNP-Ap/Au biosensor. The BP and cysteine were immersed in DMSO, and then Au-S self-assembly was used to fix the gold nanoparticle particles on the BP surface. The BP-AuNP nanocomposite was transferred from the solution to the gold electrode surface, and then the thioaptamer solution was added to the BP-AuNP modified gold electrode. BP-AuNP-aptamer biosensor detects MG.

Figure 2 shows the results of atomic force microscopy (AFM): (A) BP, (B) BP in water for 12 hours, (C) BP functionalized with L-cysteine and (D) with L- Cysteine functionalized BP was in water for 12 hours. (E) Atomic force microscopy was used to characterize the thickness, position and height distribution of the same BP before and after coating L-cysteine as a protective layer. (F) Infrared spectra of L-cysteine and L-cysteine/BP.

Figure 3 shows the TEM image and SEM image results, where (A) is the TEM image of BP, (B) is the TEM image of gold nanoparticles and (C) is the TEM image of BP-AuNP nanocomposite, (D) Is the SEM image of BP, (E) is the SEM image of BP-AuNP nanocomposite.

Figure 4 shows the results of a cyclic voltammogram, where (A) is in 1 mol L ^-1 H ₂ SO ₄ , the bare gold electrode has a CV (20 times) from 0.3 to 1.55V, and (B) is a different gold electrode CV in 5 mmol L ^-1 [Fe(CN) ₆ ] ^3-/4- (a) bare gold electrode, (b) Ap/Au electrode, and (C)BP-AuNP-Ap/Au electrode.

Figure 5 shows a differential pulse voltammogram (DPV), where (A) the corresponding DPV of the biosensor at different concentrations of MG from 1pg L ^-1 to 10 μg L ^-1 , (B) the peak current of MG at different concentrations increase. The standard deviations are all less than 5%.

Fig. 6(A) shows the reproducibility of the BP-AuNP-Ap/Au biosensor, and Fig. 6(B) screens the MG and chloramphenicol (CP) by the BP-AuNP-Ap/Au biosensor, Screening of nitrofurans (NF), methyltestosterone (MTS) and eugenol (EG).

detailed description

The present application will be described in detail below in conjunction with examples, but the present application is not limited to these examples.

MG, methylene blue (MB), L-cysteine, dimethyl sulfoxide (DMSO), and other chemicals were purchased from Sigma Aldrich (USA). MG is dissolved in H ₂ O to obtain a series of standard solutions. The aptamer with the sequence 5'-SH-(CH ₂ ) ₆ -GGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCC-MB-3' (screened by SELEX method) was obtained from Takala Biotechnology Co., Ltd. (Dalian, China) and was prepared at 20 mmolL L ^-1 pH 8 Dissolve in the hydrochloric acid solution. Unless otherwise stated, 50 mmol L L ^-1 phosphate buffer (referred to as "PBS") was used as the electrolyte. Unless otherwise stated, the raw materials and catalysts in the examples of the present application are purchased through commercial channels.

Transmission electron microscope (TEM) and scanning electron microscope (SEM) images were obtained by JEM-2100 (Japan) and Zeiss Sigma 300 (Germany), respectively. Atomic force microscopy (AFM) was performed using peak force mode and silicon nitride cantilever tip (Bruker, USA). Fourier transform infrared spectroscopy (FTIR) was obtained by Nicolet 6700 (Thermo, USA). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) are operated by CHI660B Electrochemical Workstation (CHI Instruments). The three-electrode system includes a gold electrode used as a working electrode, an Ag/AgCl electrode used as a reference electrode, and a platinum wire used as an auxiliary electrode.

Example 1: Preparation of BP, L-cysteine-coated BP and BP-AuNP nanocomposite Preparation of L-cysteine-coated BP

The preparation process of BP can be divided into several steps, all of which are operated in acrylic glove boxes. The glove box needs to maintain a certain level of oxygen and water, which is necessary for the uniform oxidation and hydroxylation of the BP surface. The first step is to perform ultrasonic assisted stripping of BP in DMSO under a nitrogen atmosphere to remove dissolved oxygen molecules. After centrifugation to remove non-shedding particles, BP was collected from the supernatant. The second step is to transfer the BP onto the SiO ₂ /Si wafer immersed in the glass bottle. The glass bottle was then capped and placed in silicone oil. The glass bottle was heated to 130°C and incubated for 20 minutes. After this step, -OH groups are present on the BP surface. Subsequently, BP was transferred into DMSO with L-cysteine at 100°C for 20 minutes. In this way, the L-cysteine molecule binds to BP via the formation of an ionic bond between -OH and -NH ₂ . A uniform protective monolayer is formed on the BP surface. Obtained functionalized BP.

Preparation of BP-AuNP Nanocomposite

Preparation of gold nanoparticles and BP nanocomposites by self-assembly method: Preparation of gold nanoparticles in water by reduction of chloroauric acid by vitamin C. 50 mL of 0.1 mg L ^-1 chloroauric acid and 2.5 mL of vitamin C (4 mg mL ^-1 ) were mixed for 2 hours, and then 0.5 mL of sodium citrate (10 mg mL ^-1 ) was added to the solution to terminate the reaction. Gold nanoparticles were obtained. Then, 0.25 mg mL ^-1 functionalized BP solution (50 mL) was mixed with 0.5 mg mL ^-1 gold nanoparticle solution and stirred rapidly for 10 minutes. Through three centrifugations (1300g, 5 minutes), the bottom of the centrifuge bottle was washed and nanocomposite material G1# was obtained.

The preparation process of samples G2#～G5# is the same as that of sample G1#, except that the ratio of raw materials and reaction conditions are changed. The relationship between the sample number of the obtained samples and the ratio of raw materials and reaction conditions is shown in Table 1.

Table 1

Example 2: Preparation of BP-AuNP-Ap/Au electrode

The BP-AuNP-Ap/Au electrode was constructed by the following method: After the gold electrode was polished with a series of alumina powder (0.3 and 0.05 μm diameter), it was ultrasonically cleaned three times with ethanol and deionized water. Then, the gold electrode was electrochemically polished from 0 to 1.7 V in 1 mol L ^-1 H ₂ SO ₄ by potential scanning. The construction process is shown in Figure 1: The BP-AuNP nanomaterial (0.5 mg mL ^-1 ) solution was vortexed for 20 minutes and added to the gold electrode surface. Then, 8 μL, ¹ μmol L ^-1 thiol aptamer solution was added to the Au electrode at room temperature for 2 hours. All other electrodes are washed with deionized water to remove weakly adsorbed aptamers, and they have a similar preparation method as the BP-AuNP-Ap/Au electrode.

Example 3: Detection of MG by BP-AuNP-Ap/Au electrode

MG is classified as a Class II health hazard and can cause cancer. Select MG as the target compound and add 8 μL of MG solution to the PBS solution for 3 minutes to provide sufficient time for the aptamer to recognize MG. Then, the BP-AuNP-Ap/Au biosensor is used to monitor the change of the response signal generated by the MG through the DPV method.

Example 4: Characterization of BP, L-cysteine-coated BP and BP-AuNP nanocomposites

The long-term stability of functionalized BP was characterized using atomic force microscopy (AFM). As shown in FIG. 2A and FIG. 2B, after applying L-cysteine, the passivation of BP stably exists in the aqueous solution for about 2 weeks. But without coating L-cysteine, BP degraded in aqueous solution within 12 hours. (Figure 2C and Figure 2D). AFM was also used to characterize the functionalized BP and BP thickness. The sample G1# was used as a typical representative to observe the characterization of BP L-cysteine-coated BP and BP-AuNP nanocomposites. FIG. 2E shows that the coating thickness of L-cysteine is about 0.6 nm. This indicates that there is a uniform protective layer on the BP surface. In order to verify the L-cysteine immobilized on the surface of BP, their FTIR spectra were tested. It is noted that the FTIR spectrum of L-cysteine shows typical amino acid spectral characteristics. The 2550 cm ^-1 peak is the SH group of the L-cysteine molecule. When L-cysteine is bound to the BP surface, it is at 1390 (symmetrical stretching of -COOH), 1600 (asymmetric stretching of -COOH) and 3000-3500cm ^-1 (-NH ₃ ⁺ stretching) The absorption band becomes weak. This is most likely due to the fact that when L-cysteine is bound to the BP surface with the -OH group, their dipole moment changes. These results indicate the successful modification of L-cysteine on the surface of BP.

The structure and morphology of functionalized BP images were obtained using TEM and SEM instruments. Figure 3 shows representative TEM images of functionalized BP (A), gold nanoparticles (B), and BP-AuNP (C). Under the strong electron beams of TEM and SEM, the uncoated BP degrades within one minute, so the patterns of these uncoated BP cannot be captured by TEM and SEM. Figure 3A shows that high-quality functionalized BP is obtained by liquid stripping and site functionalization. FIG. 3B shows a TEM image of gold nanoparticles with a size of about 10 nm. Figure 3C is a BP-AuNP nanocomposite. The results show that through Au-S self-assembly, BP is connected to gold nanoparticles, and gold nanoparticles are dispersed on the surface of BP. As shown in FIG. 3C, the gold nanoparticles cover approximately 20% of the BP surface. The particle size of gold nanoparticles is about 10 nm. The structure of functionalized BP and BP-AuNP nanocomposites was confirmed by SEM. As shown in Figure 3D, high-quality functionalized BP has been prepared. Figure 3E shows gold nanoparticles dotted on the BP surface. Both SEM and TEM images indicate that BP-AuNP nanocomposite was prepared.

Sample G2# ~ sample G5# have similar structure and morphology as sample G1#.

Example 5: Fixing process of BP-AuNP-Ap/Au biosensor

BP has excellent carrier mobility (1000 cm ² /V·s), which is higher than that of molybdenum disulfide (MoS ₂ , 200 cm ² /V·s). BP plays a key role in the development of biosensors as a biosensor platform. In this study, CV was used to monitor the preparation process of Au electrodes. FIG. 4A shows that the repeated CV of the bare electrode in mol L ⁻¹ H ₂ SO ₄ is 0.2˜1.55V. The results showed that the electrode was cleaned by electrochemical corrosion in 1mol L ^-1 H ₂ SO ₄ . Fig. 4B shows the CV signals of the bare gold electrode, Ap/Au electrode, and BP-AuNP-Ap/Au electrode in 5 mmol L ^-1 [Fe(CN) ₆ ] ^3-/4- solution (curve A, curve B and curve C). The cathode and anode of the bare gold electrode peak separation of about 65mV, which indicates an electrochemical redox process is reversible single electron transfer process ^28, and a gold electrode has been cleaned by pickling. After the aptamer was fixed on the electrode, the response signal of the electrode (curve B) was significantly weakened. This is because the thiol aptamer covers the Au electrode surface through AU-S self-assembly. When the BP-AuNP nanocomposite and aptamer were immobilized on the Au electrode, the response signal was significantly enhanced. This is mainly due to the high conductivity of the BP-AuNP nanocomposite improving the electron mobility. The results showed that BP-AuNP was successfully fixed on the Au electrode.

Example 6: MG determination by BP-AuNP-Ap/Au electrode

MG is a cationic triphenylmethane dye that has been used in commercial aquaculture since 1936 to resist saprophytic bacteria. In recent years, due to its adverse effects on the immune and reproductive systems, infertility and respiratory diseases, it has attracted widespread attention. Based on the above reasons, a preferred embodiment of the present invention uses a BP-AuNP-Ap/Au biosensor to detect MG. DPV is one of the most sensitive electrochemical methods, which is used to monitor the concentration of MG by BP-AuNP-Ap/Au biosensor. The redox-active molecules MB electrochemical response signals for amplification, the redox potential of the low -0.25-0V, ³³ can be reduced potential coexistence interference. As shown in FIG. 5A, the response signal of this biosensor continuously increases with the addition of MG. The peak current of the BP-AuNP-Ap/Au biosensor showed a linear correlation with the change in MG concentration from ¹ pg L ^-1 to 10 μg L ^-1 (Figure 5B). The DL of the biosensor for MG is as low as 0.3 pg L ^-1 . Compared with the DL obtained by other reports, the DL of this biosensor (0.3 pg L ^-1 ) is significantly better than that of other biosensors (Table 2). This indicates that functionalized BP plays a key role in improving the performance of biosensors. Figure 5 shows a possible mechanism by which MG is recognized by the aptamer. Before the aptamer binds to MG, there is no helix or hairpin structure. The aptamer showed a random linear structure to keep the MG away from the electrode. Therefore, in the absence of MG, this biosensor has no obvious response signal. After the aptamer binds to MG, a hairpin structure is formed. The hairpin structure of the aptamer brings the MB close to the electrode and has an enhanced response signal. The high conductivity BP significantly improves the DL of the biosensor.

Table 2. Analysis performance comparison between BP-AuNP-Ap/Au biosensor and other methods for detecting MG. PPy means polypyrrole

In addition, the sensitivity of BP-AuNP-Ap/Au biosensor and Ap/Au biosensor are 95.1 μA cm ^-2 and 42.5 μA cm ^{-2, respectively} . The selectivity of the biosensor, that is, the ratio of the signal output to the output signal of the individual analyte to the output signal of the individual interfering substance, is determined by the coexisting interfering substances, such as chloramphenicol (CP) and nitrofuran (NF) methyl testosterone (MTS) and eugenol (EG). As shown in FIG. 6A, the BP-AuNP-Ap/Au biosensor screened 4 types of interferors with 10ng L ^-1 . Based on the excellent selectivity of the aptamer, the response signal of MG is the strongest signal among these chemicals. For analyte concentrations within the usable range, reproducibility can be roughly determined. Fig. 6B is the response signal of PP-AuNPs-Ap/Au biosensor to MG at 10ng L ^-1 for 8 times. This shows that under the output current, the functionalized BP with protective layer is very stable in the liquid, and it is not a disposable biosensor.

Example 7: Detection of breeding water samples by BP-AuNP-Ap/Au electrode

In this paper, BP-AuNP-Ap/Au biosensors were used to evaluate the cultured water samples in 7 provinces of China. BP-AuNP-Ap/Au biosensor showed that only three aquaculture water samples had MG, and their response signals increased by 33%, 24% and 16% respectively. As shown in Table 3, the differences between these values are 23.5%, 26.1%, and 8.3%, respectively. The results show that the biosensor results are consistent with the LC-MS results. The biosensor developed is an effective tool for selective determination of MG from coexisting aquaculture drugs, although it may not be very accurate.

Table 3. Comparison of MG detection using BP-AuNP-Ap/Au biosensor and LC-MS

^a difference = (biosensor _(value) -LC-MS _(value) )/LC-MS _(value) × 100%

A person skilled in the art can select a specific recognition region on the metal electrode according to the nature of the substance to be detected to specifically bind to the analyte. Therefore, the biosensor constructed by the present invention is not limited to detecting MG.

In this study, a simple and easy method for obtaining L-cysteine-coated BP was established. L-cysteine-coated BP can stably stay in water for two weeks. It overcomes the current shortcomings of BP. This is the first report that L-cysteine is used to functionalize BP and that functionalized BP is stably present in an aqueous solution. Using a stable BP as a biosensing platform, a new BP-AuNP-Ap/Au biosensor was constructed. This biosensor is used to detect MG quickly and selectively. It shows a concentration-dependent response to MG as well as low DL and high selectivity. It is a useful early warning tool for the rapid and selective detection of MG in aquaculture water from coexisting drugs.

The above are only a few examples of this application, and are not intended to limit this application in any way. Although this application is disclosed in the preferred embodiments as above, it is not intended to limit this application. Any person skilled in the art, Without departing from the scope of the technical solution of the present application, making slight changes or modifications using the technical content disclosed above is equivalent to an equivalent implementation case and belongs to the scope of the technical solution.

Claims

A black phosphorus modified with L-cysteine, characterized in that, by forming an ionic bond between -OH and -NH 2 , the black phosphorus combines with L-cysteine to form L-cysteine Modified black phosphorus, and the functionalized black phosphorus has a protective layer on the surface.
An L-cysteine-black phosphorus-gold nanocomposite, characterized in that the nanocomposite comprises gold nanoparticles and the L-cysteine-modified black phosphorus according to claim 1, The gold nanoparticles and the L-cysteine-modified black phosphorus are connected by Au-S self-assembly.
A method for preparing the L-cysteine-modified black phosphorus according to claim 1, wherein the method comprises at least the following steps:

Put the sheet substrate with black phosphorus in the container and seal it. Heat the oil bath to 110℃～140℃ and keep it warm for 10-30 minutes;

Then, the L-cysteine-modified black phosphorus is obtained by contacting L-cysteine with black phosphorus for not less than 5 minutes at 100°C to 150°C.
A method for preparing the L-cysteine-black phosphorus-gold nanocomposite according to claim 2, characterized in that the method comprises at least the method for preparing L-cysteine modified according to claim 3 Black phosphorus steps, and:

Mixing the solution containing the L-cysteine-modified black phosphorus with the gold nanoparticle-dispersed solution to obtain a mixture; separating the mixture and washing the obtained solid to obtain the L-cysteine- Black phosphorus-gold nanocomposite;

Preferably, the mass ratio of L-cysteine-modified black phosphorus to gold nanoparticles in the mixture is 0.1 to 0.5: 0.5 to 5.
The method according to claim 4, wherein the solution in which the gold nanoparticles are dispersed is prepared by a method including the following steps:

Mix chloroauric acid and vitamin C with a mass of 0.2～1:20～60 in water.
A metal electrode, characterized in that the metal electrode comprises the L-cysteine-modified black phosphorus according to claim 1, and the L-cysteine-black phosphorus-gold nanocomposite according to claim 2. At least one of

Preferably, the metal electrode is at least one of a gold electrode, a platinum electrode and a glassy carbon electrode.
The metal electrode according to claim 6, wherein the metal electrode further comprises an aptamer, and the aptamer includes a specific recognition region to specifically bind to the analyte.
The metal electrode according to claim 6, wherein the aptamer is a mercapto aptamer;

Preferably, the sulfhydryl aptamer is selected from single-stranded DNA modified with a 5′ terminal sulfhydryl group.
A biosensor, characterized in that the biosensor comprises the L-cysteine-modified black phosphorus as claimed in claim 1, and the L-cysteine-black phosphorus-gold nanocomposite as claimed in claim 2. Substances, the metal electrode according to any one of claims 6 to 8, the L-cysteine-modified black phosphorus prepared according to the method of claim 3, and the L-half prepared according to the method of claim 4 or 5. At least one of cystine-black phosphorus-gold nanocomposite.
The biosensor according to claim 9, wherein the biosensor is used to detect malachite green.