CN109991217B - Detect A β1-42Colorimetric biosensors for oligomers - Google Patents

Detect A β1-42Colorimetric biosensors for oligomers Download PDF

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CN109991217B
CN109991217B CN201910195531.3A CN201910195531A CN109991217B CN 109991217 B CN109991217 B CN 109991217B CN 201910195531 A CN201910195531 A CN 201910195531A CN 109991217 B CN109991217 B CN 109991217B
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black phosphorus
aptamer
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gold
oligomer
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CN109991217A (en
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孙莉萍
潘伟萍
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Xiamen University
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G01N21/3103—Atomic absorption analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour

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Abstract

The invention discloses a detection A β1‑42Colorimetric biosensor for oligomers comprising black phosphorus alkene nanogold composite, gold-plated silicon chip and A β1‑42The oligomer is specifically combined with aptamer, the 5' end of the aptamer is modified with SH, the aptamer is respectively connected with a gold-plated silicon wafer and a black phosphorus alkene nano gold composite material through SH, and the aptamer and A β1‑42Oligomer-specific binding; black phosphorus alkene nanogoldThe composite material catalyzes the reaction of reducing 4-nitrophenol into 4-aminophenol by sodium borohydride, and the catalytic effect is detected by ultraviolet visible light spectrum, thus realizing the A β specifically combined with the aptamer1‑42Detection of oligomers the present invention enables the detection of A β in the UV-visible spectrum1‑42The oligomers were tested for specificity.

Description

Detect A β1-42Colorimetric biosensors for oligomers
Technical Field
The invention belongs to A β1-42The technical field of oligomer detection, in particular to a method for detecting A β oligomer1-42The colorimetric biosensor of (1).
Background
Alzheimer's Disease (AD) patients in A β1-42Oligomer (A β O) concentrations were 70-fold higher than in normal humans, thus A β1-42Oligomers are The core biomarkers of AD (The Journal of Physical Chemistry C, 2017, 121 (36): 20007-1-42Colorimetric biosensors of oligomers.
Disclosure of Invention
The invention aims to provide a detection A β1-42Colorimetric biosensors of oligomers.
The invention also aims to provide application of the black phosphorus alkene nano gold composite material.
The principle of the invention is as follows:
as shown in FIG. 1, the black phosphorus alkene nanometer gold composite material (BP-Au) has strong catalytic action on the reaction of reducing 4-nitrophenol (4-NP) into 4-aminophenol (4-AP) by sodium borohydride, on the basis of color change generated by the 4-NP reduction reaction, the prepared BP-Au biosensing can carry out specificity detection on A β O by a colorimetric method, on one hand, a mercapto A β O nucleic acid aptamer (SH-A β aptamer, TTT TTTTTT TGC CTG TGG TGT TGG GGC GGG TGC G, modification, 5' SH C6, SEQ ID NO: 01) can be combined with A β O, on the other hand, a probe can be tightly combined with a gold-plated silicon wafer and BP-Au due to strong interaction between gold and sulfur bonds, a sandwich structure of the gold-plated silicon wafer/A β O/BP-Au can be constructed on the basis of the principle, the A β O nucleic acid aptamer with mercapto modification is connected to the silicon wafer, then A β O and the BP-Au-plated silicon wafer are sequentially added to form a stable sandwich structure, the Au-plated sandwich structure is prepared, and the prepared by adding a washing solution of the Au-plated sandwich structure to realize visible light spectrum detection on the NP 26.
The technical scheme of the invention is as follows:
detect A β1-42The colorimetric biosensor of oligomer is characterized by comprising a black phosphorus alkene nano-gold composite material, a gold-plated silicon chip and A β1-42The oligomer is specifically combined with aptamer, the 5' end of the aptamer is modified with SH, the aptamer is respectively connected with a gold-plated silicon wafer and a black phosphorus alkene nano gold composite material through SH, and the aptamer and A β1-42Specifically combining oligomers, catalyzing the reaction of reducing 4-nitrophenol into 4-aminophenol by sodium borohydride by the black phosphorus alkene nano gold composite material, detecting the catalysis effect by ultraviolet visible light spectrum, and realizing the A β specifically combined with the aptamer1-42And (4) detecting oligomers.
In a preferred embodiment of the invention, the sequence of the aptamer is as set forth in SEQ ID NO: 01, shown in the figure.
Further preferably, the preparation method of the black phosphorus alkene nano gold composite material comprises the following steps: preparing a few-layer black phosphorus from black phosphorus by adopting an ultrasonic liquid phase stripping method, and preparing the black phosphorus alkene nano gold composite material by adopting a direct synthesis method by taking the few-layer black phosphorus and chloroauric acid as raw materials.
The other technical scheme of the invention is as follows:
application of black phosphorus alkene nano-gold composite material in detection of A β1-42Use of an oligomer in a colorimetric biosensor.
Further preferably, the silicon chip comprises a gold-plated silicon chip and A β1-42The oligomer is specifically combined with the aptamer, the 5' end of the oligomer is modified with SH, the black phosphorus alkene nano-gold composite material catalyzes the reaction that 4-nitrophenol is reduced into 4-aminophenol by sodium borohydride, and the catalytic effect is detected by ultraviolet visible light spectrum, so that A β specifically combined with the aptamer is realized1-42And (4) detecting oligomers.
Still more preferably, the sequence of the aptamer is as shown in SEQ ID NO: 01, shown in the figure.
Still further preferably, the preparation method of the black phosphorus alkene nano gold composite material comprises the following steps: preparing a few-layer black phosphorus from black phosphorus by adopting an ultrasonic liquid phase stripping method, and preparing the black phosphorus alkene nano gold composite material by adopting a direct synthesis method by taking the few-layer black phosphorus and chloroauric acid as raw materials.
The ultraviolet-visible light spectrum control agent has the beneficial effects that the ultraviolet-visible light spectrum control agent can control A β under the ultraviolet-visible light spectrum1-42The oligomers were tested for specificity.
Drawings
Fig. 1 is a schematic diagram of the principle of the present invention.
FIG. 2 is the scanning electron microscope images of FL-BP and BP-Au in example 1 of the present invention.
FIG. 3 shows an optical image of BP-Au and UV-visible spectrum in example 1 of the present invention.
FIG. 4 is a linear plot of the catalytic rate of the reaction versus the concentration of A β O in example 2 of the present invention.
FIG. 5 shows BP-Au vs. A β in example 3 of the present invention1-42Monomer, A β1-42Oligomer, A β1-42And (4) a selectivity result graph of the fiber body.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1 preparation of Black Phosphorenes Nanogold composite (BP-Au)
(1) The preparation of the few-layer black phosphorus (FL-BP) adopts an ultrasonic liquid phase stripping method: accurately weighing 40mL of ultrapure water, adding the ultrapure water into a 50mL centrifugal tube, introducing the ultrapure water into Ar gas for 30min, weighing 20mg of black phosphorus, adding the black phosphorus into the centrifugal tube, and putting the black phosphorus mixed solution into a cell disruption instrument for ultrasonic treatment for 5 h; centrifuging the black phosphorus suspension subjected to ultrasonic treatment on a high-speed refrigerated centrifuge at the rotating speed of 2000rpm for 10 min; after centrifugation, supernatant and bottom sediment can be observed, and the supernatant of the black phosphorus solution is transferred into a new centrifugal tube; continuously placing the centrifuge tube containing the supernatant into a high-speed refrigerated centrifuge for centrifuging, wherein the rotating speed is set to 10000rpm, and the time is 20 min; and after the centrifugation is finished, obvious supernatant and precipitate can be observed, the supernatant is poured out and kept to precipitate, FL-BP is finally obtained, the centrifugal tube is wrapped by tin foil paper, and the FL-BP is kept in a refrigerator at 4 ℃ for later use.
(2) Accurately weigh 0.1g of HAuCl4·3H2O, adding the solution into 8mL of ultrapure water, shaking for a period of time to fully dissolve the solution, transferring the solution into a 10mL volumetric flask, then adding the ultrapure water to the constant volume of 10mL, finally transferring the solution into a 10mL glass bottle, and wrapping the glass bottle by using tinfoil paper to obtain the 25mM chloroauric acid standard solution (HAuCl)4). The solution was diluted to a 10mM chloroauric acid solution in a 1.5mL centrifuge tube, wrapped with tinfoil paper, and stored in a refrigerator at 4 ℃ until use.
(3) Accurately weighing 1mL of FL-BP with the concentration of 0.4mg/mL in a 1.5mL centrifuge tube; add 50. mu.L of 10mM HAuCl to the centrifuge tube4(ii) a Putting the mixed solution into an ultrasonic machine for ultrasonic treatment for 5 min; and (4) taking out the mixed solution after the ultrasonic treatment is finished, standing at the room temperature for 10min, and finally obtaining the BP-Au.
Characterization of FL-BP and BP-Au: the morphology of FL-BP and BP-Au was characterized by Scanning Electron Microscopy (SEM), and as a result, as shown in FIG. 2, it can be seen that nanogold is distributed on the black phosphorus layer; the structures of FL-BP and BP-Au are characterized by visible light and ultraviolet visible Absorption Spectrum (UV-Vis), and the result is shown in FIG. 3, wherein BP-Au is purple, and the BP-Au and AuNPs have obvious Absorption peaks at 525nm, which indicates FL-BP and HAuCl4After mixing, nanogold was generated, and the absorption peak occurred because nanogold had a surface plasmon resonance peak.
EXAMPLE 2 preparation of BP-Au colorimetric biosensor
(1) A5' thiol-modified A β O aptamer TTT TTT TTT TGC CTG TGGTGT TGG GGC GGG TGC G (SEQ ID NO: 01) solution was prepared at a concentration of 10 nM.
(2) Preparing A β O solutions with different concentrations, setting the concentrations at 0.1nM, 1nM, 10nM, 100nM and 1 μ M, taking six gold-plated silicon wafers numbered 1-6, respectively dropping 20 μ L of aptamer solution with 10nM concentration on the gold-plated surface, and incubating for 60 min.
(3) Add 40. mu.L of the aptamer solution and 80. mu.L of freshly prepared BP-Au to a 1.5mL centrifuge tube, incubate for 60min, and centrifuge at 8000rpm for 5 min.
(4) After the incubation is finished, 10 mu L of A β O solutions with different concentrations are respectively dripped into No. 1-5 gold-plated silicon wafers, the concentrations are sorted from large to small, 10 mu L of PBS solution is dripped into No. 6 silicon wafers, and the incubation is carried out for 60 min.
(5) And respectively dripping 25 mu L of the prepared mixed solution of the aptamer and the BP-Au into the gold-plated silicon wafer, and continuously culturing for 60 min.
(6) Preparing a mixed solution of 4-NP and sodium borohydride; and (3) carrying out UV-vis spectrum scanning on the 4-NP solution, washing the silicon wafer by pure water, adding the silicon wafer into the 4-NP, and collecting UV-vis spectra at different time points.
The results are shown in FIG. 4, the catalytic performance of the sensors with different A β O concentrations for the 4-NP reaction is also different, the numerical values of the different A β O concentrations are unified into units of mol/L, the logarithm of the numerical values is taken, the negative number of the numerical values is taken, and the reaction kinetic parameter k corresponding to the negative number is plotted, and the results show that the two satisfy the linear relationship when the A β O concentration is between 0.1nM and 1 μ M.
Example 3A β1-42Detection of oligomers:
(1) a β was prepared at a concentration of 100nM each1-42Monomers, oligomers, and fibers.
(2) After the gold-plated silicon chip is incubated with the aptamer, respectively dripping 10 mu LA β into the silicon chip1-42Monomer, oligomer, and fiber solution, and incubating for 60 min.
(3) 25 mu L of the prepared aptamer and BP-Au mixed solution is dripped into the gold-plated silicon wafer, and the mixture is cultured for 60 min.
(4) The gold-plated silicon wafer was cleaned with pure water and subjected to UV-vis spectral scanning of 4-NP.
The results are shown in figure 5, and the reaction was first order kinetic fit at 400nM to obtain the kinetic parameter k, which is 1.05x10 as shown in figure 5A, and which is catalytic for the 4-NP reaction when 100nM of a β O is added-5s-1When the sample was added 100nM of A β1-42The reaction kinetic parameter k is very small for the monomer or the fibrous body and is almost negligible compared to the same concentration of A β O (FIG. 5B)1-42The sandwich structure constructed by the monomer and the fibrous body is destroyed after pure water washing, no catalytic action is generated on the 4-NP reaction, and the BP-Au biosensor can specifically detect A β O.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Sequence listing
<110> university of mansion
<120> a colorimetric biosensor for detecting A β oligomer
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>34
<212>DNA
<213>Artifical Sequence
<400>1
tttttttttt gcctgtggtg ttggggcggg tgcg 34

Claims (6)

1. Detect A β1-42The colorimetric biosensor of oligomer is characterized by comprising a black phosphorus alkene nano-gold composite material, a gold-plated silicon chip and A β1-42The oligomer is specifically combined with aptamer, the 5' end of the aptamer is modified with SH, the aptamer is respectively connected with a gold-plated silicon wafer and a black phosphorus alkene nano gold composite material through SH, and the aptamer and A β1-42Specifically combining oligomers, catalyzing the reaction of reducing 4-nitrophenol into 4-aminophenol by sodium borohydride by the black phosphorus alkene nano gold composite material, detecting the catalysis effect by ultraviolet visible light spectrum, and realizing the A β specifically combined with the aptamer1-42And (4) detecting oligomers.
2. The colorimetric biosensor of claim 1, wherein: the sequence of the aptamer is shown as SEQ ID NO: 01, shown in the figure.
3. The colorimetric biosensor of claim 1 or 2, wherein: the preparation method of the black phosphorus alkene nano gold composite material comprises the following steps: preparing a few-layer black phosphorus from black phosphorus by adopting an ultrasonic liquid phase stripping method, and preparing the black phosphorus alkene nano gold composite material by adopting a direct synthesis method by taking the few-layer black phosphorus and chloroauric acid as raw materials.
4. Application of black phosphorus alkene nano-gold composite material in detection of A β1-42The application of the oligomer colorimetric biosensor is characterized by also comprising a gold-plated silicon wafer and A β1-42The oligomer is specifically combined with the aptamer, the 5' end of the oligomer is modified with SH, the black phosphorus alkene nano-gold composite material catalyzes the reaction that 4-nitrophenol is reduced into 4-aminophenol by sodium borohydride, and the catalytic effect is detected by ultraviolet visible light spectrum, so that A β specifically combined with the aptamer is realized1-42And (4) detecting oligomers.
5. The use of claim 4, wherein: the sequence of the aptamer is shown as SEQ ID NO: 01, shown in the figure.
6. Use according to claim 4 or 5, characterized in that: the preparation method of the black phosphorus alkene nano gold composite material comprises the following steps: preparing a few-layer black phosphorus from black phosphorus by adopting an ultrasonic liquid phase stripping method, and preparing the black phosphorus alkene nano gold composite material by adopting a direct synthesis method by taking the few-layer black phosphorus and chloroauric acid as raw materials.
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