CN116713044A - Fluorescent detection chip based on vertical graphene and nano gold, preparation method thereof and application thereof in AD marker detection - Google Patents

Fluorescent detection chip based on vertical graphene and nano gold, preparation method thereof and application thereof in AD marker detection Download PDF

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CN116713044A
CN116713044A CN202310413627.9A CN202310413627A CN116713044A CN 116713044 A CN116713044 A CN 116713044A CN 202310413627 A CN202310413627 A CN 202310413627A CN 116713044 A CN116713044 A CN 116713044A
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graphene
fluorescent
vertical graphene
detection chip
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CN116713044B (en
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刘一镳
刘醒云
刘琼
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LONGGANG DISTRICT CENTRAL HOSPITAL OF SHENZHEN
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Abstract

The invention discloses a fluorescent detection chip based on vertical graphene and nano gold, a preparation method thereof and application thereof in AD marker detection, belonging to the technical field of biological detection, wherein the preparation process of the fluorescent detection chip is as follows: growing vertical graphene on the surface of a substrate by a CVD method, wherein the height of the vertical graphene is 0.5-5 mu m, etching the graphene into an origin array with the diameter of 0.5-5 mm by a laser etching method, taking the graphene as a working electrode, ag/AgCl as a reference electrode, taking 1-15 mmol/L chloroauric acid solution as electrolyte, and electrodepositing gold on the surface of the graphene by a constant voltage method for 50-1000 s, wherein the electric potential is applied to the surface of the graphene during electrodeposition, wherein the electric potential is-0.1-1.8V.

Description

Fluorescent detection chip based on vertical graphene and nano gold, preparation method thereof and application thereof in AD marker detection
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a fluorescent detection chip based on vertical graphene and nano gold, a preparation method thereof and application thereof in AD marker detection.
Background
Alzheimer's disease is a neurodegenerative disease with very long duration, and at present, the AD diagnosis method mainly comprises the steps of cognitive scale scoring, neuroimaging examination, cerebrospinal fluid molecular marker content detection and the like, wherein the change of the molecular marker content in cerebrospinal fluid can directly reflect the nerve injury condition of an AD patient, and the AD diagnosis can be found at the early stage of the disease, so that the AD diagnosis is greatly advanced. However, cerebrospinal fluid is difficult to obtain, is highly invasive and is prone to cause adverse reactions, thereby limiting its application in screening of the general population. Compared with cerebrospinal fluid, blood is easy to obtain, has small injury, and gradually has the tendency of replacing the cerebrospinal fluid.
The AD blood marker detection method applied to the market at present is mainly a single-molecule immune array technology from Quantix corporation of America, which needs expensive instruments, and one index of a single sample needs to cost thousands of yuan, which is far higher than the consumption level of the common people.
Disclosure of Invention
The invention aims to provide a fluorescence detection chip based on vertical graphene and nano gold, a preparation method thereof and application thereof in AD marker detection.
Based on the above purpose, the invention adopts the following technical scheme:
a preparation method of a fluorescent detection chip based on vertical graphene and nano gold comprises the following steps:
growing vertical graphene on the surface of a substrate by a CVD method, wherein the height of the vertical graphene is 0.5-5 mu m, etching the graphene into an origin array with the diameter of 0.5-5 mm by a laser etching method, taking the graphene as a working electrode, ag/AgCl as a reference electrode, taking 1-15 mmol/L chloroauric acid solution as electrolyte, and electrodepositing gold on the surface of the graphene by a constant voltage method for 50-1000 s, wherein the electric potential is applied to the surface of the graphene during electrodeposition, wherein the electric potential is-0.1-1.8V.
Further, the growth process of the vertical graphene is as follows: and (3) placing the substrate into a vapor deposition furnace, wherein methane gas is used as a raw material, the flow rate of the methane gas is 5mL/min, the deposition temperature is 750-850 ℃, and the pressure is less than or equal to 10Pa.
Further, the substrate is a silicon wafer, glass or ceramic.
Further, the concentration of the chloroauric acid solution is 10mmol/L, and the electrodeposition potential is-0.6V.
The fluorescence detection chip based on the vertical graphene and the nano gold is prepared by the preparation method.
The fluorescent detection chip based on the vertical graphene and the nano gold is applied to AD marker detection.
Further, firstly, dripping an aqueous solution of sulfhydryl PEG on the surface of a fluorescence detection chip, standing overnight at the temperature of 200-10000,4 ℃, washing off unbound PEG by using PBS, then dripping a first antibody of Abeta for reaction for 1 hour at the temperature of 37 ℃, then washing off excessive antibody by using PBS, dripping Abeta protein, incubating for 1 hour at the temperature of 37 ℃, washing off excessive Abeta protein by using PBS, finally dripping a second antibody marked by fluorescent dye activated ester IR-800-NHS, incubating for 1 hour at the temperature of 37 ℃, washing off excessive antibody by using PBS, and reading fluorescent signals by using a chip reader after airing.
Further, the preparation process of the fluorescent dye activated ester IR-800-NHS labeled secondary antibody is as follows: incubating fluorescent dye activated ester IR-800-NHS and secondary antibody for 1h at 37 ℃ according to a molar ratio of 2-5:1 to obtain the fluorescent dye activated ester IR-800-NHS.
Further, the concentration of the aqueous solution of sulfhydryl PEG and the secondary antibody of the fluorescent dye activated ester IR-800-NHS label is 10mM, the concentration of the primary antibody of Abeta is 10 mug/mL, and the volumes of the aqueous solution of sulfhydryl PEG, abeta protein, the primary antibody of Abeta and the secondary antibody of the fluorescent dye activated ester IR-800-NHS label are equal.
According to the invention, the high-flux detection chip with fluorescence enhancement performance based on the vertical graphene and the nano gold is prepared, the surface gold plating amount of the vertical graphene is found to be different, the fluorescence enhancement performance of the surface is different, and the chip is used for ultrasensitive detection of the Alzheimer disease marker Abeta, and the detection limit can reach 0.1pg/mL.
Drawings
FIG. 1 is an SEM image of VG@Au prepared at different deposition times, a) 50s, b) 100s, c) 500s, d) 1000s;
FIG. 2 is a graph of VG@Au real objects prepared by electrodeposition time of 500 s;
FIG. 3 is a graph showing the fluorescence enhancement effect of fluorescent dye activated ester IR-800-NHS labeled secondary antibody and VG@Au prepared by different deposition times;
FIG. 4 shows the results of detecting different concentrations of Abeta protein by VG@Au prepared by electrodeposition time of 500 s.
Detailed Description
The technical scheme of the present invention will be further described in detail with reference to the accompanying drawings and examples, but the scope of the present invention is not limited thereto. The concentration of PBS used in the examples described below was 10mM and the pH was 7.35.
Example 1
A preparation method of a fluorescent detection chip based on vertical graphene and nano gold comprises the following steps:
(1) Preparing vertical graphene, namely growing the vertical graphene on the surface of the ceramic by using a CVD method, wherein the process is as follows: placing a ceramic substrate into a vapor deposition furnace, and obtaining vertical graphene with the height of 1 μm by taking methane gas as a raw material, wherein the flow rate of the methane gas is 5mL/min, the deposition temperature is 800 ℃, the pressure is less than or equal to 10Pa, and the growth time is 30 minutes;
(2) Etching graphene into an origin array with the diameter of 2mm by adopting a laser etching method;
(3) Graphene is used as a working electrode, ag/AgCl is used as a reference electrode, 10mmol/L chloroauric acid solution is used as electrolyte, the applied potential is-0.6V, gold is electrodeposited on the surface of the graphene by a constant voltage method, and the deposition time is respectively 50s, 100s, 500s and 1000s, so that fluorescent detection chips based on vertical graphene and nano gold with different morphologies can be obtained and are marked as VG@Au.
SEM images of vg@au with different electrodeposition times are shown in fig. 1. As can be seen from fig. 1, the longer the electrodeposited gold time, the greater the mass fraction of surface gold.
An example of electrodeposited gold 500s is shown in FIG. 2.
In order to further detect the detection effect of the different electrodeposition times, the fluorescent dye activated ester IR-800-NHS labeled secondary antibody is prepared by the following specific preparation process: adding fluorescent dye activated ester IR-800-NHS and a secondary antibody (D3D 2N mouse monoclonal antibody of cell signaling company, wherein a recognition site is an amino end, the molecular weight of the antibody is calculated according to 15 ten thousand) into 1mLPBS according to a molar ratio of 2:1, incubating for 1h at 37 ℃, separating by a chromatographic column (PBS buffer solution with pH value of 7.35 is used as an eluent), and removing excessive fluorescent molecules to obtain the fluorescent dye activated ester IR-800-NHS marked secondary antibody.
The fluorescent dye activated ester IR-800-NHS labeled secondary antibody is dissolved in PBS buffer solution with pH=7.35 to prepare solution with the concentration of 10 mug/mL, 3 mug/mL of fluorescent dye activated ester IR-800-NHS labeled secondary antibody solution is dripped on the surface of VG@Au, after standing for 1 hour, the surface superfluous molecules are washed off by PBS, after airing, fluorescence is tested, the excitation wavelength is 785nm, and as shown in the result of figure 3, the fluorescence intensity is brightest when electrodepositing gold for 500 seconds, which indicates that VG@Au has the best fluorescence enhancement effect when electrodepositing gold for 500 seconds.
Aβ was detected with VG@Au electrodeposited with gold 500 s: firstly, 3. Mu.L of 10mM of aqueous solution of sulfhydryl PEG with molecular weight of 2000 and standing overnight at 4 ℃ is dripped on the surface of VG@Au, and after the unbound PEG is washed off by PBS, 3. Mu.L of 10. Mu.g/mL Abeta (U.S. R)&The MM26R monoclonal antibody of D company, the recognition site is carboxyl end, when in use, diluted to 10 mug/mL with PBS), reacted for 1 hour at 37 ℃, then washed with PBS to remove excessive antibody, then 3 mug of Abeta protein with the concentration of 0.1,1,10,100,1000pg/mL (when in use, configured to the corresponding concentration with PBS) is respectively dripped into 5 holes, incubated for 1 hour at 37 ℃, then washed with PBS to remove excessive protein, finally dripped with 3 mug of 10mM of fluorescent dye activated ester IR-800-NHS labeled secondary antibody, incubated for 1 hour at 37 ℃, then washed with PBS to remove excessive antibody, and after airing, fluorescent signals are read by a chip reader, the result is shown in figure 4. As can be seen from FIG. 4, VG@Au already produced stronger fluorescence with A.beta.protein at a concentration of 0.1pg/mL. Drawing a standard curve by using fluorescence intensities corresponding to different concentrations to obtain a linear equation：y=17.861x+152.86(R 2 =0.99), the theoretical detection limit can be calculated to be 0.018 pg/mL.
In conclusion, the VG@Au-based high-flux fluorescence detection chip is used for ultrasensitive detection of the AD blood marker Abeta, the detection limit can reach 0.1pg/mL, and the linear range is 0.1-1000pg/mL.
Finally, it should be noted that: the above examples are provided for illustrating the technical solution of the present invention and are not to be construed as limiting the present invention, and it should be understood by those skilled in the art that any equivalent or obvious modification of the embodiments of the present invention without changing the performance or use thereof without departing from the spirit of the present invention is intended to be included in the scope of the present invention as claimed.

Claims (8)

1. The preparation method of the fluorescent detection chip based on the vertical graphene and the nano gold is characterized by comprising the following steps of:
growing vertical graphene on the surface of a substrate by a CVD method, wherein the height of the vertical graphene is 0.5-5 mu m, etching the graphene into an origin array with the diameter of 0.5-5 mm by a laser etching method, taking the graphene as a working electrode, ag/AgCl as a reference electrode, taking 1-15 mmol/L chloroauric acid solution as electrolyte, and electrodepositing gold on the surface of the graphene by a constant voltage method for 50-1000 s, wherein the electric potential is applied to the surface of the graphene during electrodeposition, wherein the electric potential is-0.1-1.8V.
2. The method for preparing the fluorescence detection chip based on the vertical graphene and the nano gold according to claim 1, wherein the growth process of the vertical graphene is as follows: and (3) placing the substrate into a vapor deposition furnace, wherein methane gas is used as a raw material, the flow rate of the methane gas is 5mL/min, the deposition temperature is 750-850 ℃, and the pressure is less than or equal to 10Pa.
3. The method for manufacturing a fluorescence detection chip based on vertical graphene and nanogold according to claim 1 or 2, wherein the substrate is a silicon wafer, glass or ceramic.
4. The method for preparing the fluorescent detection chip based on the vertical graphene and the nano-gold according to claim 1, wherein the concentration of the chloroauric acid solution is 10mmol/L, and the electrodeposition potential is-0.6V.
5. A fluorescence detection chip based on vertical graphene and nanogold manufactured by the manufacturing method according to any one of claims 1 to 4.
6. The application of the fluorescence detection chip based on the vertical graphene and the nano-gold in AD marker detection.
7. The method according to claim 6, wherein the fluorescent detection chip is first coated with an aqueous solution of thiol PEG having a molecular weight of 200-10000,4 ℃and allowed to stand overnight, the unbound PEG is washed off with PBS, then the first antibody to Abeta is coated with Abeta at 37℃and reacted for 1 hour, then the excess antibody is washed off with PBS, then Abeta protein is coated with Abeta protein, incubated at 37℃for 1 hour, then the excess Abeta protein is washed off with PBS, finally the fluorescent dye activated ester IR-800-NHS labeled secondary antibody is coated with Abeta protein, incubated at 37℃for 1 hour, then the excess antibody is washed off with PBS, and after air drying, the fluorescent signal is read by a chip reader.
8. The use according to claim 7, wherein the fluorescent dye activated ester IR-800-NHS labeled secondary antibody is prepared as follows: and incubating the IR800 fluorescent molecules and the secondary antibodies in PBS for 1h at 37 ℃ according to the molar ratio of 2-5:1, thus obtaining the fluorescent antibody.
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Citations (10)

* Cited by examiner, † Cited by third party
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