CN109991217A - A kind of detection A β1-42The colorimetric bio sensor of oligomer - Google Patents

A kind of detection A β1-42The colorimetric bio sensor of oligomer Download PDF

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CN109991217A
CN109991217A CN201910195531.3A CN201910195531A CN109991217A CN 109991217 A CN109991217 A CN 109991217A CN 201910195531 A CN201910195531 A CN 201910195531A CN 109991217 A CN109991217 A CN 109991217A
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black phosphorus
oligomer
composite material
aptamer
detection
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CN109991217B (en
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孙莉萍
潘伟萍
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Xiamen University
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    • 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
    • G01N21/17—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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  • General Physics & Mathematics (AREA)
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Abstract

The invention discloses a kind of detection A β1‑42The colorimetric bio sensor of oligomer, including black phosphorus alkene nanogold composite material, gold-plated silicon wafer and with A β1‑42Oligomer specific bond and 5 ' the terminal modified aptamers for having SH, aptamer are connected with gold-plated silicon wafer and black phosphorus alkene nanogold composite material respectively by SH, and aptamer and A β1‑42Oligomer specific bond；Black phosphorus alkene nanogold composite material is catalyzed reaction of the 4- nitrophenol by sodium borohydride reduction for 4-aminophenol, and the effect through the catalysis of ultraviolet-visible spectral detection, realizes to the A β with aptamer specific bond1‑42The detection of oligomer.The present invention can be under ultraviolet-visible spectrum to A β1‑42Oligomer carries out the detection of specificity.

Description

A kind of detection A β1-42The colorimetric bio sensor of oligomer
Technical field
The invention belongs to A β1-42A kind of oligomer detection technique field, and in particular to detection A beta oligomers1-42Colorimetric it is raw Object sensor.
Background technique
Alzheimer's disease (AD) patient's body A β1-42Oligomer (A β O) concentration is 70 times higher than normal person, therefore A β1-42It is few Aggressiveness be AD core biomarker (The Journal of Physical Chemistry C, 2017,121 (36): 20007-20015).A kind of use there is no to detect A β with black phosphorus alkene nanocomposite at present1-42The colorimetric bio of oligomer senses Device.
Summary of the invention
The purpose of the present invention is to provide a kind of detection A β1-42The colorimetric bio sensor of oligomer.
Another object of the present invention is to provide the applications of black phosphorus alkene nanogold composite material.
The principle of the present invention is as follows:
As shown in Figure 1, black phosphorus alkene nanogold composite material (BP-Au) is to 4- nitrophenol (4-NP) by sodium borohydride reduction There is strong catalytic action for the reaction of 4-aminophenol (4-AP), on the basis of 4-NP reduction reaction generates color change, system Standby BP-Au bio-sensing can carry out specific detection to A β O by colorimetric method.Sulfydryl A β O aptamer (SH-A β O Aptamer, TTT the TTTTTT TGC CTG TGG TGT TGG GGC GGG TGC SH of G, modification, 5 ' C6, SEQ ID NO:01) on the one hand it can be combined with A β O, on the other hand due to the strong interaction between golden sulfide linkage, probe can It combines closely with gold-plated silicon wafer and BP-Au.Gold-plated silicon wafer/A β O/BP-Au sandwich knot can be constructed on this basis Structure.It will be first connected on gold-plated silicon wafer with mercapto-modified A β O aptamer, then successively add A β O and BP- on silicon wafer Au forms stable sandwich structure, the sensor being prepared is added in 4-NP solution after pure water rinsing carry out it is ultraviolet Visible spectrum scanning monitors its catalytic performance effect, realizes the specific detection to A β O.
Technical scheme is as follows:
A kind of detection A β1-42The colorimetric bio sensor of oligomer, it is characterised in that including black phosphorus alkene nanogold composite wood Material, gold-plated silicon wafer and with A β1-42Oligomer specific bond and 5 ' the terminal modified aptamers for having SH, aptamer pass through SH It is connected respectively with gold-plated silicon wafer and black phosphorus alkene nanogold composite material, and aptamer and A β1-42Oligomer specific bond；It is black Phosphorus alkene nanogold composite material is catalyzed reaction of the 4- nitrophenol by sodium borohydride reduction for 4-aminophenol, and through UV, visible light The effect of the light spectral detection catalysis is realized to the A β with aptamer specific bond1-42The detection of oligomer.
In a preferred embodiment of the invention, the sequence of the aptamer is as shown in SEQ ID NO:01.
It is further preferred that the preparation method of the black phosphorus alkene nanogold composite material includes: to be removed using ultrasonic liquid-phase Method prepares few layer black phosphorus from black phosphorus, then lacks layer black phosphorus and gold chloride as raw material using this, is made described black using direct synthesis technique Phosphorus alkene nanogold composite material.
Another technical solution of the invention is as follows:
Black phosphorus alkene nanogold composite material is for detecting A β1-42Application in the colorimetric bio sensor of oligomer.
It is further preferred that further including gold-plated silicon wafer and A β1-42Oligomer specific bond and 5 ' the terminal modified nucleic acid for having SH Aptamers specific bond；It is 4-aminophenol that black phosphorus alkene nanogold composite material, which is catalyzed 4- nitrophenol by sodium borohydride reduction, Reaction, and the effect through the catalysis of ultraviolet-visible spectral detection are realized to the A β with aptamer specific bond1-42Oligomerization The detection of body.
Still more preferably, the sequence of the aptamer is as shown in SEQ ID NO:01.
Still further preferably, the preparation method of the black phosphorus alkene nanogold composite material includes: to be shelled using ultrasonic liquid-phase Few layer black phosphorus is prepared from black phosphorus from method, then layer black phosphorus and gold chloride are lacked as raw material using this, is made described using direct synthesis technique Black phosphorus alkene nanogold composite material.
The beneficial effects of the present invention are: the present invention can be under ultraviolet-visible spectrum to A β1-42Oligomer carries out special The detection of property.
Detailed description of the invention
Fig. 1 is the principle of the present invention schematic diagram.
Fig. 2 is the scanning electron microscope (SEM) photograph of the FL-BP and BP-Au in the embodiment of the present invention 1.
Fig. 3 is the BP-Au optics picture and uv-vis spectra in the embodiment of the present invention 1.
Fig. 4 is the linear relationship chart of the catalytic reaction rate and A β O concentration in the embodiment of the present invention 2.
Fig. 5 is the BP-Au in the embodiment of the present invention 3 for A β1-42Monomer, A β1-42Oligomer, A β1-42The selection of corpus fibrosum Property result figure.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment combination attached drawing.
The preparation of 1 black phosphorus alkene nanogold composite material (BP-Au) of embodiment
(1) preparation of few layer black phosphorus (FL-BP) uses ultrasonic liquid-phase stripping method: 50mL is added in precise 40mL ultrapure water Centrifuge tube leads to 30min in Ar gas, weighs 20mg black phosphorus and is added in centrifuge tube, black phosphorus mixed liquor is put into cell crushing instrument and is surpassed Sound 5h；Black phosphorus suspension after ultrasound is centrifuged 10min on high speed freezing centrifuge with the revolving speed of 2000rpm；It is considerable after centrifugation Supernatant and bottom precipitation are observed, the supernatant of black phosphorus solution is pipetted in new centrifuge tube；The centrifuge tube of supernatant will be filled Continue to be put on high speed freezing centrifuge and be centrifuged, revolving speed is set as 10000rpm, time 20min；It can be observed after centrifugation Supernatant is poured out reservation precipitating, finally obtains FL-BP, encased centrifuge tube using masking foil by apparent supernatant and precipitating, It is placed in 4 DEG C of refrigerators and saves backup.
(2) 0.1g HAuCl is accurately weighed4·3H2O is added into the ultrapure water of 8mL, and shaking up fills it for a period of time Divide dissolution, is transferred the solution into 10mL volumetric flask later, then be settled to 10mL with ultrapure water, solution is finally transferred to 10mL Vial in, vial is encased using masking foil, 25mM gold chloride standard solution (HAuCl can be obtained4).In 1.5mL Be diluted in centrifuge tube concentration be 10mM chlorauric acid solution, encased with masking foil, be placed in 4 DEG C of refrigerators save it is standby With.
(3) accurately weighing 1mL concentration is the FL-BP of 0.4mg/mL in 1.5mL centrifuge tube；50 are added into centrifuge tube μ L concentration is the HAuCl of 10mM4；Mixed solution is put into ultrasound 5min in ultrasonic machine；Mixed liquor is taken out after ultrasound Static 10min at room temperature, finally obtains BP-Au.
The characterization of FL-BP and BP-Au: using scanning electron microscope (Scannig electronic microscopy, SEM) pattern of FL-BP and BP-Au are characterized, as a result as shown in Figure 2, it can be seen that nanogold is distributed in lamella black phosphorus On；Using visible light and ultraviolet-visible absorption spectroscopy (UV-Vis Absorption Spectrum, UV-vis) to FL-BP and The structure of BP-Au is characterized, as a result as shown in Figure 3, it can be seen that BP-Au is purple, and BP-Au and AuNPs have at 525nm One apparent absorption peak, shows FL-BP and HAuCl4Nanogold is generated after mixing, the appearance of absorption peak is because of nanometer Fitting has surface plasma body resonant vibration peak.
The preparation of 2 BP-Au colorimetric bio sensor of embodiment
(1) 5 ' the mercapto-modified A β O aptamer TTT TTT TTT TGC CTG TGG in end that compound concentration is 10nM TGT TGG GGC GGG TGC G (SEQ ID NO:01) solution.
(2) the A β O solution of various concentration, concentration setting are as follows: 0.1nM, 1nM, 10nM, 100nM, 1 μM are prepared.Take six platings Gold silicon piece number 1-6 toward the aptamer solution that 20 μ L concentration are 10nM is added dropwise on gilding, educates 60min respectively.
(3) 40 μ L aptamer solution and the 80 freshly prepared BP-Au of μ L are added toward 1.5mL centrifuge tube, cultivate 60min 5min is centrifuged with the revolving speed of 8000rpm afterwards.
(4) it is added dropwise the A β O solution of 10 μ L various concentrations after the completion of cultivating respectively into No. 1-5 gold-plated silicon wafer, concentration is from big To small sequence, 10 μ L PBS solutions are added dropwise into No. 6 silicon wafers, cultivate 60min.
(5) mixed liquor of aptamer and BP-Au that 25 μ L are prepared is added dropwise into gold-plated silicon wafer respectively, continues to cultivate 60min。
(6) mixed solution of 4-NP and sodium borohydride are prepared；UV-vis spectral scan is carried out to 4-NP solution, silicon wafer is used Pure water is added in 4-NP after being rinsed, and acquires the UV-vis spectrum of different time points.
It as a result, will not as shown in figure 4, A β O concentration different sensors are also different the catalytic performance that 4-NP reacts It is unified for the unit of mol/L with the numerical value of A β O concentration, is taken the reactive kinetics parameters for taking negative corresponding again after logarithm K does figure, it is found that when A β O concentration is between 0.1nM to 1 μM, the two meets linear relationship.
3 A β of embodiment1-42The detection of oligomer:
(1) the A β that concentration is 100nM is prepared respectively1-42Monomer, oligomer, corpus fibrosum.
(2) 10 μ LA β are added dropwise into silicon wafer after the completion of being incubated for respectively for gold-plated silicon wafer and aptamer1-42Monomer, oligomerization Body, fiber liquid solution cultivate 60min.
(3) mixed liquor of aptamers and BP-Au that 25 μ L are prepared is added dropwise toward gold-plated silicon wafer, cultivates 60min.
(4) gold-plated silicon wafer is cleaned with pure water, UV-vis spectral scan is carried out to 4-NP.
As a result it is fitted to obtain reactive kinetics parameters k as shown in figure 5, doing first order kinetics to reaction at 400nm.Such as figure Shown in 5A, when the A β O of 100nM is added, there is catalytic action, the kinetic parameter k=of reaction for 4-NP reaction 1.05x10-5s-1, when addition sample be 100nM A β1-42When monomer or corpus fibrosum, reactive kinetics parameters k is minimum, phase A β O than same concentration can almost ignore (Fig. 5 B).The result shows that utilizing A β1-42The sandwich structure that monomer, corpus fibrosum construct It is being destroyed after pure water rinsing, no catalytic action is being reacted for 4-NP, BP-Au biosensor is capable of the inspection of specificity Survey A β O.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e., Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Sequence table
<110>Xiamen University
<120>a kind of colorimetric bio sensor for detecting A beta oligomers
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> DNA
<213> Artifical Sequence
<400> 1
tttttttttt gcctgtggtg ttggggcggg tgcg 34

Claims (7)

1. a kind of detection A β1-42The colorimetric bio sensor of oligomer, it is characterised in that: including black phosphorus alkene nanogold composite material, Gold-plated silicon wafer and and AB1-42Oligomer specific bond and 5 ' the terminal modified aptamers for having SH, aptamer pass through SH points It is not connected with gold-plated silicon wafer and black phosphorus alkene nanogold composite material, and aptamer and A β1-42Oligomer specific bond；Black phosphorus Alkene nanogold composite material is catalyzed reaction of the 4- nitrophenol by sodium borohydride reduction for 4-aminophenol, and through ultraviolet-visible The effect of the spectral detection catalysis is realized to the A β with aptamer specific bond1-42The detection of oligomer.
2. colorimetric bio sensor as described in claim 1, it is characterised in that: the sequence of the aptamer such as SEQ ID Shown in NO:01.
3. colorimetric bio sensor as claimed in claim 1 or 2, it is characterised in that: the black phosphorus alkene nanogold composite material Preparation method include: to prepare few layer black phosphorus from black phosphorus using ultrasonic liquid-phase stripping method, then layer black phosphorus and gold chloride are lacked with this For raw material, the black phosphorus alkene nanogold composite material is made using direct synthesis technique.
4. black phosphorus alkene nanogold composite material is for detecting A β1-42Application in the colorimetric bio sensor of oligomer.
5. application as described in claim 1, it is characterised in that: further include gold-plated silicon wafer and A β1-42Oligomer specific bond and 5 ' The terminal modified aptamer specific bond for having SH；Black phosphorus alkene nanogold composite material be catalyzed 4- nitrophenol by sodium borohydride also Originally be the reaction of 4-aminophenol, and the effect through the catalysis of ultraviolet-visible spectral detection, realize to aptamer spy The A β of different combination1-42The detection of oligomer.
6. application as claimed in claim 5, it is characterised in that: the sequence of the aptamer such as SEQ ID NO:01 institute Show.
7. the application as described in any claim in claim 4 to 6, it is characterised in that: the black phosphorus alkene nanogold is compound The preparation method of material includes: to prepare few layer black phosphorus from black phosphorus using ultrasonic liquid-phase stripping method, then lack layer black phosphorus and chlorine with this Auric acid is raw material, and the black phosphorus alkene nanogold composite material is made using direct synthesis technique.
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