CN102507551B - Reagents for detecting AChE (acetylcholinesterase) and preparation method thereof - Google Patents
Reagents for detecting AChE (acetylcholinesterase) and preparation method thereof Download PDFInfo
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Abstract
The invention provides reagents for detecting AChE (acetylcholinesterase), comprising deionized water, rhodamine B-modified gold nanoparticles, thioacetylcholine, and material for preventing aggregation of gold nanoparticles. The invention further provides a preparation method of the reagents for detecting AChE.
Description
Technical field
The present invention relates to a kind of method of utilizing the gold nano grain detection acetylcholinesterase (acetylcholinesterase, AChE) of surface modification.
background technology
Alzheimer disease is a kind of lethal nerve degenerative diseases that carries out sexual development, has become the 4th cause of death of the elderly after cardiovascular and cerebrovascular, tumour, wound.The diagnosis of Alzheimer disease mainly relies on medical history, brain iconography and neuropsychological to detect clinically at present, but sensitivity and the specificity of diagnosis are all bad, especially very difficult to the patient diagnosis in commitment.The Clinics and Practices of Alzheimer disease still faces huge challenge.Examination of cerebrospinal fluid is the important auxiliary diagnosis means of central nervous system after impaired.The state of an illness of Alzheimer disease and some protein levels in Cerebrospinal Fluid in Patients are closely bound up, as A β, and Protein tau etc., but be also difficult at present detect efficiently these albumen.Research shows, along with Alzheimer disease, sb.'s illness took a turn for the worse, and the level of Acetylcholine levels in Cerebrospinal esterase (acetylcholinesterase, AChE) significantly reduces, and therefore, AChE is expected to become an auxiliary diagnostic index of Alzheimer disease.
The classic method that detects AChE is Ellman reagent, and the playback mode of this method is simple, but sensitivity is not high.Along with the development of analytical chemistry, increasing high sensor is for detection of AChE, such as Electrochemical Detection and fluoroscopic examination.These method complex operations, and need expensive large-scale instrument.The development of nanosecond science and technology has brought opportunity to the detection of AChE.In past more than 10 year, the sensor based on gold nano grain obtains fast development.There are two kinds of visible sensors based on gold nano grain for detection of AChE, a kind of is to assemble based on gold nano grain the change color causing, utilize gold nano grain that sodium citrate modifies and acetylthiocholine as detecting reagent, wherein the stable gold nano grain of sodium citrate is electronegative, the thiocholine that AChE catalysis acetylthiocholine generates is positively charged, can with the sodium citrate generation electrostatic interaction of gold surface, cause gold nano grain to be assembled, thereby change color occur; Another kind is that the size based on gold nano grain changes the change color causing, the method is also to generate thiocholine based on AChE catalysis acetylthiocholine, this compound can reduce gold chloride and generate simple substance gold, causes gold nano grain particle diameter to become large, thereby makes its solution colour blue by red stain.The playback mode of two kinds of methods is simple, but sensitivity is still not high, can only reach tens of to hundreds of mU/mL, so, develop efficient biology sensor and still face a severe challenge for detection of AChE.
Summary of the invention
Therefore, the object of the present invention is to provide detection reagent and the detection method of simple, the highly sensitive detection AChE of a kind of playback mode.
The detection reagent that the invention provides a kind of AChE of detection, comprising:
Deionized water;
The gold nano grain that rhodamine B is modified;
Acetylthiocholine;
Prevent the material that gold nano grain is assembled.
According to detection reagent provided by the invention, the concentration of the gold nano grain that wherein rhodamine B is modified is 1nM-5nM.
According to detection reagent provided by the invention, wherein the concentration of acetylthiocholine is 5 μ M-40 μ M.
According to detection reagent provided by the invention, wherein prevent that the material that gold nano grain is assembled from being NaHCO
3with NaOH potpourri or sodium tetraborate.
The present invention also provides a kind of method of preparing above-mentioned detection reagent, comprising:
Prepare gold nano grain, and form gold nano grain aqueous solution;
In gold nano grain aqueous solution, add the material that prevents that gold nano grain from assembling;
With rhodamine B, modify the gold nano grain in gold nano grain aqueous solution;
In described aqueous solution, add acetylthiocholine.
According to the method that detects reagent of preparing provided by the invention, wherein prevent that the material that gold nano grain is assembled from being NaHCO
3with NaOH potpourri or sodium tetraborate.
The present invention also provides the method for AChE of detection a kind of, comprising:
Using above-mentioned detection reagent as the reagent that detects AChE;
In described detection reagent, add testing sample.
According to the method for detection AChE provided by the invention, wherein by judging the change color of described detection reagent determines in testing sample whether contain AChE, becomes blueness or purple if detect reagent from redness, determines in testing sample and contains AChE.
According to the method for detection AChE provided by the invention, wherein by judging whether the fluorescence of described detection reagent recovers to determine in testing sample whether contain AChE, recovers if detect the fluorescence of reagent, determines in testing sample and contains AChE.
According to the method for detection AChE provided by the invention, wherein by judging whether the change color of described detection reagent and fluorescence recover to determine whether contain AChE in testing sample, only have when detect reagent when redness becomes blueness or purple and fluorescence and recovers to occur simultaneously, determine and in testing sample, contain AChE.
According to the method for detection AChE provided by the invention, wherein by judging the variation of the Zeta electric potential of described detection reagent determines in testing sample whether contain AChE, raises if detect the Zeta electric potential of reagent, determines in testing sample and contains AChE.
According to the method for detection AChE provided by the invention, wherein by judging the change in size of the gold nano grain that rhodamine B in described detection reagent is modified determines whether contain AChE in testing sample, if it is large that the size of the gold nano grain that the rhodamine B in detection reagent is modified becomes, determine in testing sample and contain AChE.
The present invention also provides the purposes of a kind of above-mentioned detection reagent in the medicine for the preparation of diagnosis of alzheimer's disease.
Detection reagent detection sensitivity provided by the invention is higher, and minimum detectable concentration can reach 0.1mU/mL, can reduce false positive, simple, quick, can be without any need for large-scale instrument, and cost is extremely low, possesses good application potential.
Accompanying drawing explanation
Referring to accompanying drawing, embodiments of the present invention is further illustrated, wherein:
Fig. 1 is the schematic diagram that AChE causes gold nano grain to be assembled;
Fig. 2 is RB-AuNPs (a) and absorbance variation of (b) afterwards before adding AChE and ATC;
Fig. 3 is RB-AuNPs (a) and change in fluorescence of (b) afterwards before adding AChE and ATC;
Fig. 4 is that RB-AuNPs is adding the Zeta electric potential of AChE and ATC front and back to change;
Fig. 5 is that RB-AuNPs is adding the dynamic light scattering of AChE and ATC front and back to change;
Fig. 6 is RB-AuNPs (a) and TEM photo contrast of (b) afterwards before adding AChE and ATC;
Fig. 7 is that different organic sulfur and enzymes changes response to absorbance of the present invention;
Fig. 8 is that different organic sulfur and enzymes responds change in fluorescence of the present invention;
Fig. 9 is the UV, visible light extinction spectrum of different concentration AChE (0.1-5.0mU/ μ L);
Figure 10 is the fluorescence spectrum of different concentration AChE (0.1-5.0mU/ μ L);
Figure 11 is that different transgenic mice cerebrospinal fluid samples change response to absorbance of the present invention, and wherein sample 1: not ill transgenic mice cerebrospinal fluid; Sample 2: ill transgenic mice cerebrospinal fluid; Sample 3: the ill transgenic mice cerebrospinal fluid after lumbar injection high dose medicament; Sample 4: the ill transgenic mice cerebrospinal fluid after lumbar injection low-dose drugs;
Figure 12 is that different transgenic mice cerebrospinal fluid samples respond change in fluorescence of the present invention: sample 1: not ill transgenic mice cerebrospinal fluid; Sample 2: ill transgenic mice cerebrospinal fluid; Sample 3: the ill transgenic mice cerebrospinal fluid after lumbar injection high dose medicament; Sample 4: the ill transgenic mice cerebrospinal fluid after lumbar injection low-dose drugs.
Embodiment
Detection reagent of the present invention contains gold nano grain (RB-AuNPs) and the acetylthiocholine (ATC) that utilizes rhodamine B (RB) to modify, detect principle as shown in Figure 1, because gold has very strong cancellation ability to fluorescence molecule, cause the RB fluorescence on RB-AuNPs surface to be quenched, if contain AChE in testing sample, the AChE in sample can be hydrolyzed by catalysis acetylthiocholine (ATC), generate thiocholine, due to the sulfydryl of thiocholine and golden adhesion stronger, the RB molecule of gold surface can be replaced, thereby the fluorescence of RB is recovered, simultaneously, quaternary ammonium salt group on thiocholine can with the carboxyl generation electrostatic interaction of the remaining RB of gold surface, cause gold nano grain to be assembled, make the aqueous solution color that contains RB-AuNPs become purple or blueness from redness.Therefore, by the fluorescence recovery of RB and the solution colour meanwhile occurring, change, just can accurately judge the level of AChE in solution, thereby reach the object that detects AChE.
Embodiment 1
The present embodiment provides the detection reagent of a kind of AChE of detection, comprising:
Deionized water;
The gold nano grain (RB-AuNPs) that rhodamine B is modified, concentration is 5nM;
Acetylthiocholine (ATC), concentration is 20 μ M;
NaHCO
3-NaOH potpourri, the pH value that makes to detect reagent is 10.
In the present embodiment, NaHCO
3-NaOH potpourri add the gathering that can prevent RB-AuNPs.
Embodiment bis-
The present embodiment provides the preparation method of the detection reagent of embodiment mono-, comprises the following steps:
1) adopt the synthetic gold nano grain of method of sodium citrate reduction gold chloride, obtaining concentration is the gold nano grain aqueous solution of 10nM;
2) rhodamine B is dissolved in deionized water, making its concentration is 2mM, with masking foil, wraps up lucifuge;
3) NaHCO that compound concentration is 5mM
3-NaOH mixed solution, pH value is 10;
4) get step 1) in freshly prepd gold nano grain solution 5mL and isopyknic NaHCO
3-NaOH solution evenly mixes;
5) get step 2) the rhodamine B solution 6 μ L that obtain, add step 4) in the solution that obtains, make the final concentration of rhodamine B be about 1.2 μ M, this mixed solution at room temperature slowly stirs 2 hours, with masking foil parcel lucifuge, obtain the solution of the gold nano grain (RB-AuNPs) of rhodamine B modification simultaneously;
6) get step 5) the solution 1mL that obtains, in this solution, add acetylthiocholine (ATC) (2 μ L, 10mM), the final concentration of ATC is 20 μ M, now solution colour is red.
According to other embodiments of the invention, wherein the preferred concentration range of RB-AuNPs is 1nM-5nM, and the preferred concentration range of acetylthiocholine (ATC) is 5 μ M-40 μ M.
According to other embodiments of the invention, through NaHCO
3the pH value of the detection reagent that-NaOH adjusts is preferably 9-10, and more preferably 10, in addition can also be with other materials replacements NaHCO
3-NaOH (for example concentration is the sodium tetraborate of 5mM, pH 9.0).
According to other embodiments of the invention, wherein the formation method of RB-AuNPs solution is not limited to the method that above-described embodiment provides, also can form by other methods well known in the art, as long as RB-AuNPs solution is mixed and can prepare detection reagent of the present invention with acetylthiocholine (ATC).
The detection reagent that embodiment 1 provides can profit detect AChE with the following method:
1) the healthy transgenic mice of not suffering from Alzheimer disease of buying from business extracts cerebrospinal fluid with it, to obtain the sample that contains AChE;
2) get the detection reagent 1mL providing in above-described embodiment 1, to the cerebrospinal fluid that adds 1 μ L mouse in this detection reagent, observe solution colour and from redness, become blueness rapidly.
The change color of solution can confirm by ultraviolet-visible extinction spectrum, and as shown in Figure 2, it is red detecting reagent solution, and the unimodal of 520nm place is that (curve a) for its characteristic absorption peak; After adding the cerebrospinal fluid of mouse, follow solution colour blue by red stain, 520nm place absorption peak declines, and meanwhile, at 600nm, to occurring a broad peak (curve b) between 800nm, shows that reunion has occurred RB-AuNPs, has formed larger Micelle-like Nano-structure of Two.
Be accompanied by solution colour and change, the fluorescence of RB is recovered.As shown in Figure 3, in detecting reagent, modify the fluorescence of the RB on AuNPs by Au cancellation, (curve a), adds after the cerebrospinal fluid of mouse in detecting reagent, and the fluorescence of RB is recovered (curve b) to show extremely weak fluorescence.
Variation by change color (becoming blueness from redness) and fluorescence intensity (fluorescence is from cancellation to recovering) can be determined the cerebrospinal fluid of mouse and contains AuNPs.Therefore, from the above results, the detection method that the present embodiment provides can simply, promptly detect in sample, whether to contain AChE.
The playback mode that the present embodiment detects AChE is change color and fluorescence, only have when solution colour is recovered to occur by red stain blue (or purple) and RB fluorescence simultaneously, just determine in testing sample and necessarily have AChE, and the solution colour that other disturbed conditions cause changes (as high level salt solution) or fluorescence recovers (as biological sulfur molecule exists), neither can disturb the detection of the present invention to AChE, so the AChE detection method that the present embodiment provides can reduce false-positive appearance greatly.
Certainly, in the less demanding situation of degree of accuracy, also can use single playback mode, for example, when solution colour is occurred by red stain blue (or purple), can roughly judge and in testing sample, have AChE, or when RB fluorescence recover to occur, can roughly judge and in testing sample, have AChE.Wherein the variation of solution colour can be read by human eye is visual, or by ultraviolet-visible extinction spectrum, determines the variation of solution colour.
Except change color and these two kinds of playback modes of fluorescence, can also utilize other playback modes, for example Zeta electric potential changes.RB modifies AuNPs surface, AuNPs outermost layer is the carboxyl of RB, so RB-AuNPs integral body is electronegative, by Ma Erwen laser particle analyzer (Zeta Sizer Nano ZS), measure and detect actual Zeta electric potential value, for-38mV, as shown in Figure 4, after adding the cerebrospinal fluid of ill mouse not, RB-AuNPs assembles, become-0.27mV of its Zeta electric potential value.Reason is that the thiocholine that AChE hydrolysis ATC produces is combined with Au by its sulfydryl, quaternary ammonium salt group positively charged on choline, by electrostatic interaction, has neutralized surface charge with the carboxyl of the upper residue of AuNPs RB, cause Zeta electric potential to raise, caused AuNPs gathering simultaneously.
In addition, other playback mode also comprises the change in size that detects RB-AuNPs.As shown in Figure 5, the diameter of RB-AuNPs is also recorded by Ma Erwen laser particle analyzer (Zeta Sizer Nano ZS), the mean diameter that detects monodispersed RB-AuNPs in reagent is 21nm (as shown in Figure 6 a), add after the cerebrospinal fluid of ill mouse not, the RB-AuNPs that makes to detect in reagent assembles, and the mean diameter of its aggregation increases to 400nm (as shown in Figure 6 b).The variation of this size and TEM photo coincide (Fig. 6 a and Fig. 6 b).
In order to study the present invention, detect the selectivity of reagent and detection method, whether we have explored biological sulphur organic compound and the conventional enzyme that in other body fluid, may contain can have interference to the present invention.These organosulfur compounds comprise halfcystine (Cys), homocysteine (Hcy), glutathione (GSH) and human serum albumins (HSA); Enzyme used comprises alkaline phosphatase (P.Alka), acid phosphatase (P.Acid), acylase I (Acy.I), beta galactosidase (Glac) and glucose oxidase (Glu.O).With the positive contrast of AChE.By these biological sulphur (1 μ L, 100mM) and enzyme (1 μ L, 1U/ μ L) join respectively in the detection reagent that embodiment 1 provides, only have AChE can make the color of solution change, within several seconds, from redness, become purple, illustrate that nano particle assembles.By ultraviolet-visible spectrophotometer, detecting the variation of its absorption value, there is variation in the absorption value of the gold nano grain solution after only having AChE to add: 520nm place absorption peak declines, and occurs broad peak (Fig. 7) between 600nm-800nm simultaneously.By fluorescence spectrum, find, sulfocompound all can make the fluorescence that detects the RB in reality recover (Fig. 8).Only have AChE can make AuNPs assemble, can make again the fluorescence of RB be recovered, show that the present invention has good selectivity and reliability to the detection of AChE.
In order to study the sensitivity of detection reagent of the present invention and detection method, utilize the AChE that business is buied to be mixed with the aqueous solution that concentration is different.The change color causing in order to study variable concentrations AChE, inventor has prepared concentration and has been respectively 0mU/ μ L, 1.0mU/ μ L, 2.0mU/ μ L, 3.0mU/ μ L, 4.0mU/ μ L, the AChE aqueous solution of 5.0mU/ μ L, then get respectively 1 μ L, they are joined in the detection reagent that embodiment 1 provides, the AChE of high concentration adds can make RB-AuNPs solution colour become rapidly blueness from redness.Along with AChE concentration reduces, RB-AuNPs solution colour becomes purple, can distinguish that the AChE minimal detectable concentration that RB-AuNPs solution colour changes is 1mU/mL by naked eyes.We have tested the variation of the gold nano grain solution absorbance under different AChE concentration with ultraviolet-visible spectrophotometer, the absorbance at 520nm place is along with AChE concentration increases and reduces, between 600nm-800nm, absorbance, along with AChE concentration increases and increases (Fig. 9), illustrates that its color changes consistent to purple variation tendency with absorbance by red simultaneously.
The change in fluorescence and the checking to change color thereof that in order to study variable concentrations AChE, cause, inventor has prepared concentration and has been respectively 0mU/ μ L, 0.1mU/ μ L, 0.3mU/ μ L, 0.6mU/ μ L, 1.0mU/ μ L, the AChE aqueous solution of 3.0mU/ μ L, find that the intensity that fluorescence recovers is directly proportional (as shown in figure 10) to AChE concentration, the AChE minimal detectable concentration that can detect by fluorescence intensity change is 0.1mU/mL.
In order to illustrate that the present invention can be used for auxiliary diagnosis and the state of an illness detection of Alzheimer disease, inventor uses the transgenic mice of suffering from Alzheimer disease as model, the same kind transgenic mice of not suffering from Alzheimer disease of take is contrast, has detected the level of AChE in this two classes transgenic mice cerebrospinal fluid.The 0.5 μ L cerebrospinal fluid sample of getting respectively two mouse joins in the detection reagent that the embodiment 1 of 1mL provides, standing 20 minutes of room temperature.For quantitatively account for color variation, we verify detecting the variation of reagent color with uv-visible absorption spectra, result as shown in figure 11, find not ill transgenic mice cerebrospinal fluid (Figure 11 of control group, sample 1) make solution colour purple by red stain, and transgenic mice cerebrospinal fluid (Figure 11 of trouble Alzheimer disease, sample 2) do not make solution colour generation significant change, show to suffer from the level of AChE in the transgenic mice cerebrospinal fluid of Alzheimer disease lower than the level of the AChE of control group, show that the present invention can be used as the reliable means that in cerebrospinal fluid, AChE measures.
The present invention can also be for monitoring the PD of Alzheimer disease.Inventor give suffer from Alzheimer disease transgenic mice lumbar injection the medicine neostigmine of various dose, after 10 days, with the present invention, survey AChE level in its cerebrospinal fluid.Find that solution colour after medication changes and all has in various degree purple by red stain, wherein, high dose group (Figure 11, sample 3) than low dose group (Figure 11, sample 4) the solution colour purpling causing is more obvious, the treatment that shows high dose group causes AChE level in cerebrospinal fluid higher than low dose group, and transgenic mice also shows more flexiblely in behavior, shows that the result for the treatment of of high dose group is better than low dose group.In order to illustrate that solution colour changes, be that AChE causes in cerebrospinal fluid, inventor has added AChE inhibitor galanthamine in detection system, add again above-mentioned four groups of samples, it is red that all solution colours all keep, show that solution colour variation is that the thiocholine being produced by its substrate A of AChE catalysis TC causes, change color is directly proportional by the trend of red stain purple and the level of AChE.By fluorescence spectrum test, detected the change in fluorescence of reagent, as shown in figure 12, sample 1 to sample 4 all can cause fluorescence in various degree to recover to result, and fluorescence recovery extent is directly proportional to the level of AChE, and with the sample of galanthamine pre-treatment, obvious fluorescence does not all occur and recover.
According to any one embodiment of the present invention, the concentration that wherein detects the RB-AuNPs in reagent is not limited to 5nM, for example can be in (1nM-5nM) scope.But the RB-AuNPs concentration in detection reagent is unsuitable too low, otherwise it is not obvious to cause solution colour to change, and impact detects.
According to any one embodiment of the present invention, wherein the diameter of gold nano grain is preferably 13nm left and right.The preparation of gold nano grain can adopt existing sodium citrate reduction gold chloride preparation method, electrochemical reducing, radiation reduction method etc.
According to the detection method of any one embodiment of the present invention, wherein variable containing the sample of AChE and the addition sequence of acetylthiocholine (ATC),, can be as embodiment 2, in gold nano grain (RB-AuNPs) solution of first modifying to rhodamine B, add ATC to obtain detecting reagent, to detecting in reagent, add the sample containing AChE again, in gold nano grain (RB-AuNPs) solution that also can first modify to rhodamine B, add the sample containing AChE, then add ATC.Change containing the sample of AChE and the addition sequence of acetylthiocholine (ATC) can not affect testing result.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described.Although the present invention is had been described in detail with reference to embodiment, those of ordinary skill in the art is to be understood that, technical scheme of the present invention is modified or is equal to replacement, do not depart from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (9)
1. a detection reagent that detects AChE, comprising:
Deionized water;
The gold nano grain that rhodamine B is modified;
Acetylthiocholine;
Preventing the material that gold nano grain is assembled, is 5mM, the pH value NaHCO that is 10
3with NaOH potpourri or 5mM, the pH value sodium tetraborate that is 9,
The concentration of the gold nano grain that wherein rhodamine B is modified is 1nM-5nM, and the concentration of acetylthiocholine is 5 μ M-40 μ M.
2. a method of preparing the detection reagent of claim 1, comprising:
Prepare gold nano grain, and form gold nano grain aqueous solution;
In gold nano grain aqueous solution, add the material that prevents that gold nano grain from assembling;
With rhodamine B, modify the gold nano grain in gold nano grain aqueous solution;
In described aqueous solution, add acetylthiocholine.
3. a method that detects AChE, comprising:
Using detection reagent claimed in claim 1 as the reagent that detects AChE;
In described detection reagent, add testing sample.
4. detection method according to claim 3, wherein by judging the change color of described detection reagent determines in testing sample whether contain AChE, becomes blueness or purple if detect reagent from redness, determines in testing sample and contains AChE.
5. detection method according to claim 3, wherein by judging whether the fluorescence of described detection reagent recovers to determine in testing sample whether contain AChE, recovers if detect the fluorescence of reagent, determines in testing sample and contains AChE.
6. detection method according to claim 3, wherein by judging whether the change color of described detection reagent and fluorescence recover to determine whether contain AChE in testing sample, only have when detect reagent when redness becomes blueness or purple and fluorescence and recovers to occur simultaneously, determine and in testing sample, contain AChE.
7. detection method according to claim 3, wherein by judging the variation of the Zeta electric potential of described detection reagent determines in testing sample whether contain AChE, raises if detect the Zeta electric potential of reagent, determines in testing sample and contains AChE.
8. detection method according to claim 3, wherein by judging the change in size of the gold nano grain that rhodamine B in described detection reagent is modified determines whether contain AChE in testing sample, if it is large that the size of the gold nano grain that the rhodamine B in detection reagent is modified becomes, determine in testing sample and contain AChE.
9. the purposes of the detection reagent of claim 1 in the medicine for the preparation of diagnosis of alzheimer's disease.
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