CN111239219B - Novel composite material for detecting beta-amyloid protein and preparation method and application thereof - Google Patents

Novel composite material for detecting beta-amyloid protein and preparation method and application thereof Download PDF

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CN111239219B
CN111239219B CN202010050552.9A CN202010050552A CN111239219B CN 111239219 B CN111239219 B CN 111239219B CN 202010050552 A CN202010050552 A CN 202010050552A CN 111239219 B CN111239219 B CN 111239219B
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amyloid
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杨晓燕
秦海新
张梦杰
刘树峰
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Beijing Zhongkeyi Microbiology Technology Co ltd
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Abstract

The invention discloses a preparation method of a novel composite material for detecting beta-amyloid protein, which is prepared by mixing g-C 3 N 4 Mixing the suspension with Hemin solution, adding ammonia water and hydrazine hydrate under high speed stirring, rapidly stirring, maintaining in 60 deg.C water bath for 4 hr, placing in 4 deg.C refrigerator overnight, centrifuging, washing, and dispersing in secondary deionized water to obtain g-C 3 N 4 -a Heme suspension. The novel composite material for detecting amyloid peptide can be used for constructing a biosensor for detecting beta-amyloid. The sensor is beneficial to the combination of heme and amyloid polypeptide, realizes the detection of amyloid behaviors, has better specificity on amyloid, and can quickly and sensitively realize the early detection of amyloid.

Description

Novel composite material for detecting beta-amyloid protein and preparation method and application thereof
Technical Field
The invention belongs to the field of detection of amyloid, and particularly relates to a novel composite material for detecting beta-amyloid, so that early detection of the amyloid is realized.
Background
Alzheimer's disease is an aging-related neurodegenerative disease, mainly found in the elderly, and is clinically characterized by memory impairment, aphasia, disorientation, cognitive impairment, etc., and gradually worsens with the passage of time. (Jack, C.R.; knoopman, D.S.; jagust, W.J.; shaw, L.M.; aisen, P.S.; weiner, M.W.; petersen, R.C.; trojanowski, J.Q.Lancet neurol.2010,9 (1), 119-128.). The pathogenesis of alzheimer's disease is mainly the amyloid β (a β) hypothesis and the Tau protein hypothesis. The former suggests that amyloid is capable of forming amyloid plaques in the brain of patients, impairing communication between nerve cells. A beta is the main component of neuritic plaques, of which A beta is the main component 1-40 And Abeta 1-42 Two kinds ofThey spontaneously form aggregates of different morphologies, such as oligomers, fibrils and plaques (Spies, p.e.; slips, d.; sjogren, j.m.c.; kremer, b.p.h.; verhey, f.r.j.; rikkert, m.; verbeek, m.m.curr. Alzheimer. Res.2010,7,470-476.Wogulis, m.; wright, s.; cunningham, d.; chilcotle, t.; powell, k.; rydel, r.e.j.neurosci.2005,25, 1071-1080.). In amyloid plaques, A β 1-40 In an amount much higher than Abeta 1-42 Exhibit faster rates of Aggregation, produce more free radical damage, and are considered to be more neurotoxic substances (pelalin, r.; caflisch, a. Inhibiting the Aggregation Kinetics of Amyloid peptides, j. Mol. Biol.2006,360 (4), 882-892.). Studies have shown that in amyloid plaques, the content of some redox-active ions (e.g. copper ions, iron ions) is relatively high, which may be associated with the onset of alzheimer's disease. Wherein the iron ion is mainly present as a counter ion in heme. Heme can be combined with a β to form a complex, and this combination can reduce the biological regulation ability of Heme, resulting in Heme deficiency necessary for normal physiological function (Active Site Environment of blood-Bound Amyloid Peptide Associated with Alzheimer's Disease superb Debase yoti Pramanik, somdata Ghosh Dey, J.Am.chem.Soc.2011,133, 81-87.). The analytical methods commonly used at present for Α β are mainly immunoassays, mass spectrometry and western blotting (j. -h.kang, m.koreka, j.b.toledo, j.q.trojanowski and l.m.shaw, clin.chem.,2013,59,903-916.g.grasso, massspectrum.rev., 2011,30,347-365.n.ida, t.hartmann, j.patel.j.
Figure BDA0002371005180000011
R.Zerfass,H.
Figure BDA0002371005180000012
Sandbrink, C.L.Masters and K.Beyreuther, J.biol.Chem.,1996,271,22908-22914.J.Pannee, E.Portellius, M.Oppermann, A.Atkins, M.Hornshaw, I.Zegers, P.Hojrup, L.Minthon, O.Hansson, H.Zetterg, K.Blennow and J.Gobom, J.Alzheimer's Dis.,2013,33, 1021-1032). Although these methods are well established and are described inAnd the method is used as a reference technology, but miniaturization detection is difficult to realize, so that development of a simple, convenient and miniaturized detection technology has a better application prospect.
Electrochemiluminescence (ECL) is an emerging detection technology, in the sense that ECL is an ideal combination of electrochemical and spectroscopic methods. Therefore, the ECL not only has the sensitivity and wider dynamic range of the traditional chemiluminescence, but also has the advantages of simple, stable and convenient electrochemical method and the like. Terpyridyl ruthenium, luminol, metal nanoclusters, inorganic nitrides and semiconductor quantum dots (Yuan, j., li, t., yin, x.b., guo, l., jiang, x., jin, w., yang, x, wang, e, 2006, anal, chem.78,2934-2938.Li, x, lu, p, wu, b, wang, y, wang, h, du, b, pang, x, wei, q, 2018, biosens, bioelectrron.112, 40-47, zhou, y, chen, m., zhuo, y, chai, y, xu, w, yuan, r, 2017, anal, chem.89,6787-6793, sha, h, zhang, y, wang, y, ke, h, xiong, x, jia, n, 2019, biosens, bioelectron.124-125,59-65, liu, y, lei, j, huang, y, ju, h, 2014, anal.chem.86, 8735-8741) are all commonly used superior luminescent materials. In recent years, carbon nitride nanosheets (g-C) 3 N 4 ) The blue fluorescent label has good biocompatibility and inherent blue fluorescent emission characteristic under ultraviolet irradiation, and can be used as a signal label (Zhang, X.D.; xie, x.; wang, h.; zhang, j.j.; pan, b.c.; xie, y.j.am.chem.soc.2013,135, 18-21.). g-C 3 N 4 The inherent blue fluorescence emission of Cu is also used 2+ Fluorescence Detection of (Tian, J.; liu, Q.; asiri, A.M.; al-Youbi, A.O.; sun, X.Ultrathin graphic Carbon Nitride Nanosheet: AHighly effective fluorescence sensor for Rapid, ultrasensive Detection of Cu 2+ Anal. Chem.2013,85, 5595-5599.). To widen g-C 3 N 4 The application prospect of (a) is that 3 N 4 Compounding with Hemin (Hemin) to successfully prepare the g-C 3 N 4 -Heme composite material having peroxidase activity and capable of catalyzing g-C by utilizing A beta-Heme complex 3 N 4 Electrogenerated chemiluminescence is characterized by the fact that g-C 3 N 4 -the Heme composite for the detection of amyloid peptides.
Disclosure of Invention
Therefore, the present invention is directed to overcoming the drawbacks of the prior art, and the present invention is directed to providing a novel composite material for detecting amyloid peptide, a biosensor for detecting β -amyloid material, and a method for constructing the same.
The term "a β" refers to: beta-amyloid protein (1-40). The term "Hemin" refers to: hemin. The term "Heme" refers to: heme. The term "g-C 3 N 4 "means that: carbon nitride nanosheets. The term "g-C 3 N 4 -Heme "means: a carbon nitride nanosheet-heme complex.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biosensor for detecting beta-amyloid comprises a gold electrode, a DNA fixed chain with sulfydryl at the tail end, beta-amyloid and a carbon nitride nanosheet-heme complex, wherein the DNA fixed chain with sulfydryl at the tail end is loaded on the surface of the gold electrode, the surface of the DNA fixed chain with sulfydryl at the tail end captures the beta-amyloid, and the surface of the beta-amyloid captures the carbon nitride nanosheet-heme complex.
A construction method of a biosensor for detecting beta-amyloid protein specifically comprises the following steps:
(1) Aluminum powder is used for polishing a gold electrode on polishing cloth, the polished electrode is sequentially subjected to ultrasonic treatment in secondary deionized water, ethanol and secondary deionized water for three minutes, then the electrode is dried by nitrogen, and the electrode is placed at 4 ℃ for later use;
(2) Dripping the DNA fixed chain with the sulfydryl at the tail end onto the electrode pretreated in the step (1), and putting the electrode into an oven at 37 ℃ for overnight reaction;
(3) Mixing appropriate amount of carbon nitride nanosheet-heme complex (g-C) 3 N 4 -Heme) suspension was added to different concentrations of beta-amyloid (a β) solution and the electrode treated in step (2) was then placed in this solution.
Further, the g-C 3 N 4 Suspension of Heme, a novel composite for detecting amyloid peptidesThe preparation method specifically comprises the following steps:
(101) Taking a proper amount of g-C 3 N 4 Adding the suspension and the Hemin solution into a round-bottom flask, stirring, mixing the suspension and the Hemin solution for 15 minutes, increasing the rotation speed, adding 400 mu L of ammonia water and 60 mu L of hydrazine hydrate under the condition of high-speed stirring, quickly stirring for 5 minutes, then placing in a water bath kettle at 60 ℃ for keeping for 4 hours, and then placing in a refrigerator at 4 ℃ overnight.
(102) Centrifugally washing the suspension obtained in the step (101) at the rotating speed of 10000 r/min to remove unreacted Hemin, and then dispersing the Hemin in secondary deionized water again to obtain g-C 3 N 4 -a Heme suspension.
Preferably, in said step (101), g-C 3 N 4 The mass ratio to Hemin is (1: 1.
preferably, the solution of step (3) contains amyloid beta and g-C 3 N 4 -the reaction time of Heme is from 2 to 24 hours; most preferably, the reaction time is 12 hours.
Preferably, the pH of the amyloid beta solution in step (3) is: 5.9-8.4; most preferably, the pH is 7.4.
Preferably, g to C in step (3) 3 N 4 -the amount of the Heme suspension added is: 10-60 mu L; most preferably, the material is used in an amount of 50 μ L.
Preferably, said g-C 3 N 4 Has electrochemiluminescence signals under the wavelengths of 425nm, 440nm, 460nm, 490nm, 535nm, 555nm and 575nm, and the optimal electrochemiluminescence signal wavelength is 460nm.
Preferably, the concentration of potassium persulfate in the detection solution of the biosensor is: 0mol/L, 0.5X 10 -1 mol/L、10 -1 mol/L、10 -2 mol/L、10 -3 mol/L; most preferably, the potassium persulfate concentration is10 -1 mol/L。
g-C prepared by the invention 3 N 4 the-Heme can also be applied to the fields of photocatalysis, electrocatalysis and the like.
The invention has the beneficial effects that:
(1) Book (I)The biosensor prepared by the invention can be used for detecting amyloid protein at 1.0X 10 -14 mol/L～1.0×10 -7 Good linearity in the mol/L range.
(2)g-C 3 N 4 the-Heme is applied to an amyloid protein system, is beneficial to the combination of Heme and amyloid protein polypeptide, realizes the detection of amyloid protein behaviors, has better specificity on amyloid protein, and can quickly and sensitively realize the early detection of the amyloid protein.
(3) The biosensor prepared by the invention has the advantages of simple construction method, low cost, good biocompatibility and the like, and enriches new methods for detecting amyloid protein.
Drawings
FIG. 1 is an electrochemical luminescence test chart for verifying the feasibility of the biosensor constructed according to the present invention (a indicates that beta-amyloid is not added, b indicates that beta-amyloid is added, and c indicates that beta-amyloid and methylene blue are added);
FIG. 2 shows Hemin, g-C in the present invention 3 N 4 And g-C 3 N 4 Infrared absorption spectrum of-Heme (a is Hemin, b is g-C) 3 N 4 -Heme, C is g-C 3 N 4 )；
FIG. 3 shows Hemin, g-C in the present invention 3 N 4 And g-C 3 N 4 Ultraviolet absorption spectrum of-Heme (a is g-C) 3 N 4 B is g-C 3 N 4 -Heme, c is Hemin);
FIG. 4 shows g-C prepared in the present invention 3 N 4 -XPS spectrogram of Heme;
FIG. 5 is an electrochemical luminescence detection chart (A) and a corresponding standard curve (B) of a biosensor constructed according to the present invention for different concentrations of amyloid beta.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.
This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known for the purpose of carrying out the invention, the invention is nevertheless described herein in detail as is practicable.
The beta-amyloid used in the following examples was purchased from Shanghai Biopsis GmbH, unless otherwise specified. The beta-amyloid sequence is: DAEFR HDSGY EVHHQ KLVFF AEDVG SNKGA ILGLM VGGVGV.
Unless otherwise specified, the DNA used in the following examples was purchased from Shanghai Biometrics, inc. and had the DNA sequence GCC TGT GGT GTT GGG GCG GGT GCG.
The solvents of the aqueous solutions used in the following examples were twice deionized water unless otherwise specified.
All reagents used in the following examples were analytical reagents unless otherwise specified.
The reagents and instrumentation used in the following examples are as follows:
reagent:
urea, hemin (Hemin), ammonia, hydrazine hydrate, potassium chloride, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium persulfate, and Hexafluoroisopropanol (HFIP) were all purchased from Aladdin reagent, inc.
The instrument comprises the following steps:
infrared absorption Spectroscopy, model FT-IR Nicolet iS10.
An ultraviolet absorption spectrometer was purchased from Hitachi, ltd., model No. UH5300.
X photoelectron spectrometer, available from sehmer fisher science, usa, model Thermo ESCALAB 250Xi.
Electrochemiluminescence analyzer, available from sienna ruimmy, model MPI-E.
Example 1
g-C in the present example 3 N 4 The preparation method of the Heme specifically comprises the following steps:
(1) Adding 10g urea into crucible, placing into oven, heating at 20 deg.C/min, maintaining at 550 deg.C for two hours, and naturally cooling to room temperature to obtain block C 3 N 4 。
(2) Taking 100mg blocksC 3 N 4 Dissolving in 100mL of secondary deionized water, ultrasonically crushing for two hours by using a cell crusher, and ultrasonically treating the well-treated g-C 3 N 4 The suspension was stored at 4 ℃ until use.
(3) 5mL of g-C was taken 3 N 4 Adding the suspension and 5mL of 1mg/mL Hemin solution into a round-bottom flask, stirring, mixing the suspension and the Hemin solution for 15 minutes, increasing the rotation speed, adding 400 mu L of ammonia water and 60 mu L of hydrazine hydrate under the condition of high-speed stirring, quickly stirring for 5 minutes, then placing in a 60 ℃ water bath kettle for keeping for 4 hours, and then placing in a 4 ℃ refrigerator overnight.
(4) Centrifuging the suspension obtained in the step (3) for 10 minutes at the rotating speed of 10000 r/min, centrifuging and washing for 3 times, removing unreacted Hemin, and re-dispersing the washed material in secondary deionized water to obtain g-C 3 N 4 -the Heme suspension, kept in a refrigerator at 4 ℃ for future use.
For raw material Hemin, and g-C prepared 3 N 4 And g-C 3 N 4 And (4) performing infrared spectrum characterization on the-Heme, and characterizing the successful preparation of the material through the change of the position of each absorption peak of the infrared absorption spectrum, as shown in figure 2. For raw material Hemin, and g-C prepared 3 N 4 And g-C 3 N 4 the-Heme is subjected to ultraviolet absorption spectrum characterization, and in the ultraviolet absorption, g-C 3 N 4 The significant red shift of Heme due to the incorporation of Heme at the position of the absorption peak, as shown in fig. 3. For g-C 3 N 4 Heme performs X Photoelectron Spectroscopy (XPS) in which the success of preparing the material is indicated by the detection of elements C, N, fe.
Example 2
Preparation of a β solution:
(1) Dissolving the A beta powder in Hexafluoroisopropanol (HFIP) to prepare a Hexafluoroisopropanol (HFIP) solution of 1.0mg/mL, and freeze-drying the prepared solution by using a freeze dryer to form an A beta layered film at the bottom of the test tube.
(2) The freeze-dried a β layered film was dissolved in PBS buffer (pH = 7.4) and formulated into 1.0 × 10 - 6 mol/L of A beta solution at-20 ℃ for standby.
Detection of A beta:
(1) Before experimental testing, a gold electrode is polished on polishing cloth by using 0.05 mu M aluminum powder, the polished electrode is respectively subjected to ultrasonic treatment for three minutes in secondary deionized water, ethanol and secondary deionized water in sequence, the electrode subjected to ultrasonic treatment is dried by using nitrogen, and the electrode is placed at 4 ℃ for standby;
(2) The DNA reagent was allowed to stand at room temperature for 2 minutes, centrifuged at 10000 rpm for 10 minutes, and added with PBS buffer (pH = 7.4) to prepare a solution of a certain concentration. The prepared DNA solution and TCEP solution are mixed to cut the disulfide bonds in the DNA solution, and a DNA fixed chain with a thiol group at the end is obtained.
(3) The DNA-immobilized chain with a thiol group at the end was dropped onto the pretreated electrode, and placed in an oven at 37 ℃ for reaction overnight.
(4) Preparation g-C 3 N 4 Ultrasonically dispersing the Heme suspension for 5 minutes to uniformly disperse the Heme suspension, and taking a certain amount of g-C 3 N 4 The Heme suspension was added to a tube, then different amounts of a β solution were added to make a β standard solution, the treated electrodes were rinsed with PBS buffer solution (pH = 7.4), then placed in a tube, and placed in a constant temperature oven at 37 ℃ for 12 hours. And after incubation, putting the electrode into a detection pool filled with potassium persulfate detection liquid to carry out electrochemiluminescence signal detection, and obtaining a standard curve of the concentration of Abeta and the electrochemiluminescence intensity. Wherein the concentration of the potassium persulfate detection solution is10 -1 mol/L。
In addition, a certain amount of preparation g-C 3 N 4 Ultrasonically dispersing the Heme suspension for 5 minutes to uniformly disperse the Heme suspension, and taking a certain amount of g-C 3 N 4 The Heme suspension was added to the tube, followed by the addition of the a β solution and methylene blue, respectively, and the treated electrode was rinsed with PBS buffer (pH = 7.4), then placed in the tube, and placed in a thermostatic oven at 37 ℃ for 12 hours. After incubation, the electrode is put into a detection cell filled with potassium persulfate detection liquid for electrochemiluminescence signal detection, and a blank sample without A beta solution is set in the process, as shown in figure 1.

Claims (5)

1. A biosensor for detecting beta-amyloid is characterized by comprising a gold electrode, a DNA fixed chain with sulfydryl at the tail end, beta-amyloid and a carbon nitride nanosheet-heme complex, wherein the DNA fixed chain with sulfydryl at the tail end is loaded on the surface of the gold electrode, the surface of the DNA fixed chain with sulfydryl at the tail end captures the beta-amyloid, and the surface of the beta-amyloid captures the carbon nitride nanosheet-heme complex.
2. The method for constructing a biosensor for detecting amyloid beta according to claim 1, comprising the steps of:
(1) Aluminum powder is used for polishing a gold electrode on polishing cloth, the polished electrode is sequentially subjected to ultrasonic treatment in secondary deionized water, ethanol and secondary deionized water for three minutes, then the electrode is dried by nitrogen, and the electrode is placed at 4 ℃ for later use;
(2) Dripping the DNA fixed chain with the sulfydryl at the tail end onto the electrode pretreated in the step (1), and putting the electrode into an oven at 37 ℃ for overnight reaction;
(3) Adding a proper amount of uniformly mixed carbon nitride nanosheet-heme compound suspension into beta-amyloid solutions with different concentrations, and then placing the electrode treated in the step (2) into the solutions;
the preparation method of the carbon nitride nanosheet-heme compound comprises the following steps:
(101) Taking a proper amount of g-C 3 N 4 Uniformly mixing the suspension and the Hemin solution, adding ammonia water and hydrazine hydrate under the condition of high-speed stirring, placing in a water bath kettle at 60 ℃ for 4 hours, and then placing in a refrigerator at 4 ℃ overnight;
(102) And (4) centrifugally washing the suspension finally obtained in the step (101) at the rotating speed of 10000 r/min to remove unreacted Hemin, and then re-dispersing in secondary deionized water.
3. The method for constructing a beta-amyloid-detecting biosensor in accordance with claim 2, wherein in step (101), g-C is used 3 N 4 Substance of HeminThe mass ratio is (1; beta-amyloid and g-C in the solution of the step (3) 3 N 4 -the reaction time of Heme is from 2 to 24 hours; the pH of the beta-amyloid solution in the step (3) is as follows: 5.9-8.4; g-C in step (3) 3 N 4 The amount of the Heme suspension added was: 10-60 mu L; the g to C 3 N 4 Heme has an electrochemiluminescent signal at wavelengths of 425nm, 440nm, 460nm, 490nm, 535nm, 555nm, 575 nm.
4. The method of claim 2, wherein in the step (101), g-C is used as the reference in the step (1) 3 N 4 The mass ratio of the mixed solution to Hemin is 1:1; beta-amyloid and g-C in the solution of the step (3) 3 N 4 -a reaction time of Heme of 12 hours; the pH of the beta-amyloid solution in the step (3) is as follows: 7.4 of the total weight of the mixture; g-C in step (3) 3 N 4 The amount of the Heme suspension added was 50. Mu.L; the g to C 3 N 4 Heme has the strongest electrochemiluminescence signal at a wavelength of 460nm.
5. The method of claim 2, wherein the concentration of potassium persulfate in the detection solution of the biosensor is: 0mol/L, 0.5X 10 -1 mol/L、10 -1 mol/L、10 -2 mol/L or 10 -3 mol/L。
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