CN102565380A - Method for preparing surface plasma chiral probe - Google Patents
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- CN102565380A CN102565380A CN2012100328905A CN201210032890A CN102565380A CN 102565380 A CN102565380 A CN 102565380A CN 2012100328905 A CN2012100328905 A CN 2012100328905A CN 201210032890 A CN201210032890 A CN 201210032890A CN 102565380 A CN102565380 A CN 102565380A
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Abstract
The invention discloses a method for preparing a surface plasma chiral probe and belongs to the field of surface plasma micro/nano-structural biological detectors. The method comprises the following steps of preparing gold nanorod dispersing liquid and a phospholipid molecular template, and hydrating the phospholipid molecular template and a gold nanorod to obtain the probe. The probe can detect the isomeride of cysteine within about 1 minute, is high in detection speed and flexibility, is convenient to operate and is non-toxic and innocent; and the concentration of the gold nanorod used by the probe is at n/mol/L magnitude, and the probe has high flexibility in a low concentration range.
Description
Technical field
The present invention relates to a kind of preparation method of surface plasma chirality probe, belong to surface plasma little/micro-nano structure bioprobe field.
Background technology
In recent years, along with the maturation day by day of synthetic metal nanoparticle investigation of materials, the various plasma sensors that develop based on the unique surface plasma optical property of metal nanoparticle play a significant role at biomedical sector.Because gold nanorods has anisotropic design feature, comprise the spectral characteristic that its tunable surface plasma resonance (SPR) electromagnetic response and surface thereof strengthen, be the focus of present artificial surface plasma sensor research.So far; Biology sensor based on gold nanorods surface plasma body resonant vibration and surperficial enhanced spectrum has been showed great application prospect (X.H.Huang in the conveying of biological sensing, biomedical imaging, gene, medicine, the fields such as detection, diagnosis and treatment of disease; S.Neretina and M.A.El-Sayed; Gold Nanorods:From Synthesis and Properties to Biological and Biomedical Applications; Adv.Mater.2009,21,4880-4910; Z.Nie, A.Petukhova, E.Kumacheva, Properties and emerging applications of self-assembled structures made from inorganic nanoparticles, Nat.Nanotech.2010,5,15-24.).Wherein, Seed mediated growth method is the classical preparation method of gold nanorods, is about to chlorauric acid solution and cetyl trimethyl ammonium bromide (CTAB) solution and mixes, and the adding sodium borohydride solution causes into nuclear reaction and obtains the gold kind; In growth-promoting media, adding gold then plants; Under the effect of CTAB, silver nitrate, reductive agent ascorbic acid, gold ion is by slowly reduction, and is that the core oriented growth is the gold nanorods with certain length-diameter ratio with the gold kind in the solution; The gold nanorods that the control reaction conditions obtains has { 111} crystal face (Anand Gole and Catherine J.Murphy; Seed-Mediated Synthesis of Gold Nanorods:Role of the Size and Nature of the Seed Chem.Mater.2004,16,3633-3640; Babak Nikoobakht and Mostafa A.El-Sayed, Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth Method, Chem.Mater.2003,15,1957-1962; The surface modification of gold nanorods and in the application of biomedical sector, the chemistry circular, 2010 (3), 195-104).
As everyone knows, halfcystine molecule (cysteine, Cys) isomers of two kinds of chiralitys of existence: left-handed halfcystine molecule (L-cys) and D-Cysteine molecule (D-cys).Wherein, L-cys has important effect in life system.Therefore, realize that efficient, the highly sensitive chirality selection of L-cys detection has very high practical value in pharmaceuticals industry.Cys is difunctional micromolecule, and one of which end sulfhydrylation can form strong interaction through the Au-S key with the gold nanorods end, and the other end can form hydrogen bond or zwitter-ion in solution, so Cys can be used for assembling gold nanorods formation linear chain structure.In the linear package assembly of such gold nanorods; Surfaces etc. cause the marked change of surface plasma body resonant vibration (SPR) absorption spectra from the coupling of exciton body; Plasma biology sensor be can be used for making up, organic molecule such as halfcystine molecule are applied to survey with high sensitivity.Though the gold nanorods sensor based on the surface plasma coupling can provide a kind of means (H.Huang, X.Liu, T.Hu that surveys the halfcystine molecule efficiently, in high sensitivity at present; P.K.Chu; Ultra-sensitive detection of cysteine by gold nanorod assembly, Biosensors and Bioelectronics, 2010; 25,2078-2083; P.K.Sudeep, S.T.S.Joseph, K.G.Thomas; Selective Detection of Cysteine and Glutathione Using Gold Nanorods, J.Am.Chem.Soc.2005,127; 6516-6517); Yet weak point can not be distinguished the nuance of chiral molecules isomers at this spr sensor, lacks the chiral Recognition function.Need a kind of surface plasma chirality probe for this reason, be used for the detection of halfcystine molecule and the chiral Recognition of isomers thereof, on pharmacy and biomedicine, have significant application value.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of surface plasma chirality probe, obtained surface plasma chirality probe, be used for the detection of halfcystine molecule and the chiral Recognition of isomers thereof through said preparation method.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
A kind of preparation method of surface plasma chirality probe, said method concrete steps are following:
Wherein, the preparation process of gold nanorods dispersion liquid is following: after preparing gold nanorods dispersion liquid stoste through seed mediated growth method, centrifugal deposition and the supernatant liquid of obtaining wherein is precipitated as the gold nanorods that the surface is wrapped in cetyl trimethyl ammonium bromide (CTAB); After taking out supernatant liquid, in deposition, add water, obtain the gold nanorods dispersion liquid; Free CTAB is arranged in the gold nanorods dispersion liquid.
It is following to prepare the process of gold nanorods dispersion liquid stoste through seed mediated growth method in the preferred steps one:
1. gold is planted and is prepared
Get CTAB solution, stirring condition adds gold chloride (HAuCl down
4) the WS after, add sodium borohydride (NaBH
4) solution, stir 3min, obtain gold and plant subsequent use;
Wherein, the amount of substance of CTAB: the amount of substance of gold chloride: the amount of substance of sodium borohydride=7500: 24.8: 6;
2. growth-promoting media preparation
Get CTAB solution, add gold chloride (HAuCl
4) the WS, silver nitrate aqueous solution, sulfuric acid and aqueous ascorbic acid;
Wherein, the amount of substance of CTAB: the amount of substance of gold chloride: the amount of substance of silver nitrate=10
4: 50.6: 4～7;
The amount of substance of CTAB: the amount of substance of sulfuric acid: the amount of substance of ascorbic acid=10
3: 10
2: 8;
3. the preparation of gold nanorods dispersion liquid
Get the gold kind of step 1 preparation, join in the growth-promoting media of step 2 preparation,, obtain gold nanorods dispersion liquid stoste at 30 ℃ of 12～15h that grow down;
Wherein, the amount of substance of CTAB in the gold kind of the step 1 of getting preparation: amount of substance=1.8 * 10 of CTAB in the growth-promoting media of step 2 preparation
-5: 1;
The phospholipid molecule template is through dimyristoyl phosphatidyl choline (DMPC) phospholipid molecule is dissolved in chloroform, and behind the chloroform rotary evaporation, freeze drying obtains;
Wherein, the quality (mg) of preferred DMPC phospholipid molecule: the volume of chloroform (ml)≤0.5.
Wherein in phospholipid molecule, can add luminescent dye molecule, said luminescent dye molecule is the conventional reagent of biological and chemical field, like rhodamine-DHPE, NBD-PE.
After in the phospholipid molecule template, adding the gold nanorods dispersion liquid and phosphate (PBS) damping fluid that obtains in the step 1,, obtain reactant liquor 60～70 ℃ of following hydrations 2～4 hours;
Wherein, the long 30～60nm of gold nanorods in the said gold nanorods dispersion liquid, wide 12～20nm, two ends contain that { 111} face, length-diameter ratio are 1.5～5;
Be free in the amount of substance of the CTAB in the gold nanorods dispersion liquid: the amount of substance of phospholipid molecule template=1: 3～4;
The volume of gold nanorods dispersion liquid: the volume of PBS damping fluid=1: 1～4;
The PBS damping fluid is 1 * PBS, and concentration is 10mmol/L, and the pH value is 7.4;
Put into refrigerator after step 3, the reactant liquor cooling that step 2 is obtained, left standstill 24 hours under 4～5 ℃, obtain a kind of surface plasma chirality probe, gold nanorods concentration is 0.7～1.5nmol/L in the preferred probe.
The kind of surveying cysteine with said probe is during with concentration, adding L-cysteine or D-halfcystine solution in probe, and probe combines with halfcystine, and (CD) surveys with circular dichroism spectrum.
Beneficial effect
1. probe according to the invention can detect the isomers L-cys and the D-cys of halfcystine about 1 minute, speed of detection is fast, highly sensitive, and is simultaneously easy and simple to handle, nontoxic; The concentration linear measurement range of the detectable halfcystine molecule of said probe is 22.4-111.6 μ mol/L; With use 50 μ mol/L silver nano-grains such as X.P.Yan and concentration is that the L-halfcystine of 50 μ mol/L mixes the back and surveys and compare (N.Jing and X.P.Yan; A Circular Dichroism Probe for l-Cysteine Based on the Self-Assembly of Chiral Complex Nanoparticles; Chem.Eur.J.2010,16,423-427); The used gold nanorods concentration of probe of the present invention is in the nmol/L magnitude, and in lower concentration range, has higher sensitivity;
2. this method is all having characteristic peak in the ultraviolet region with visible/near infrared region; Than the classic method that absorbs the circular dichroism spectrographic detection at the electron resonance of ultraviolet region, realize that at the surface plasma resonance absorption circular dichroism spectrum of visible/near infrared region the method that chirality is surveyed has efficient and advantages of being cheap.
Description of drawings
Fig. 1 is the environmental scanning electronic microscope figure of embodiment 1 said probe;
Fig. 2 is the circular dichroism spectrogram of the isomers of embodiment 1 said probe identification halfcystine;
Fig. 3 is the environmental scanning electronic microscope figure of embodiment 2 said probes;
Fig. 4 is the circular dichroism spectrogram of embodiment 2 said probe identification halfcystine isomerss;
Fig. 5 is the linear change figure of the PCD peak value of embodiment 3 said probe identification L-halfcystines and D-halfcystine with the halfcystine molecular conecentration;
Fig. 6 is the circular dichroism spectrogram of the isomers of embodiment 4 said probe identification halfcystines.
Embodiment
Describe the present invention in detail through specific embodiment below:
In embodiment 1～4, through ultraviolet-visible coefficient spectrometer, detect gold nanorods concentration and free CTAB concentration in the gold nanorods dispersion liquid, and the gold nanorods concentration in the surface plasma chirality probe; Through environmental scanning electron microscope, obtain the pattern and the structure of said probe; Through the circular dichroism spectrometer, detect the spectral signature of said probe identification L-halfcystine and D-halfcystine.
Wherein, the preparation process of gold nanorods is following:
1. gold is planted and is prepared
Get the CTAB solution 7.5ml of 0.1mol/L, add 1.8ml water, it is the gold chloride (HAuCl of 24.7mmol/L that stirring condition adds concentration down
4) the WS 100.4 μ l after, add the sodium borohydride (NaBH of 0.01mmol/L
4) solution 600 μ l, stir 3min, obtain gold and plant subsequent use;
2. growth-promoting media preparation
Get the CTAB solution 100ml of 0.1mol/L, adding concentration is the gold chloride (HAuCl of 24.7mmol/L
4) WS 2.05ml, concentration is the silver nitrate aqueous solution 0.5ml of 0.01mol/L, concentration is the sulfuric acid 2ml of 0.5mol/L, concentration is the aqueous ascorbic acid 0.8ml of 0.1mol/L.
3. the preparation of gold nanorods dispersion liquid
The gold of getting step 1 preparation is planted 240 μ L and is joined in the growth-promoting media of step 2 preparation, at 30 ℃ of growth 12h down, obtains gold nanorods dispersion liquid stoste.Said gold nanorods stoste after centrifuging, is obtained deposition and supernatant liquid, wherein be precipitated as the gold nanorods that the surface is wrapped in CTAB; Behind pipettor taking-up supernatant liquid, in deposition, add 20ml water, obtain the gold nanorods dispersion liquid;
In said gold nanorods dispersion liquid, gold nanorods concentration is 2nmol/L, and free CTAB concentration is 5mmol/L; Record the long 30～60nm of said gold nanorods through transmission electron microscope (TEM), wide 12～20nm, two ends contain that { 111} face, length-diameter ratio are 1.5～5.
The preparation process of phospholipid molecule template is following:
8mg DMPC phospholipid molecule is dissolved in the 250mL round-bottomed flask of packing into behind the 20mL chloroform,, obtains product 1, product 1 freeze drying after 36 hours, is obtained the phospholipid molecule template 40 ℃ of following rotary evaporations 7 hours;
Wherein, freeze drying places vacuum drying chamber for after product 1 is placed the container that fills liquid nitrogen with said container, and vacuum drying chamber is provided with vacuum tightness to maximum range, carries out vacuum drying treatment.
After adding gold nanorods dispersion liquid 1.5ml and PBS damping fluid 2ml in the phospholipid molecule template that in step 1, obtains,, obtain reactant liquor 60 ℃ of following hydrations 4 hours;
Wherein, be free in the amount of substance of the CTAB in the gold nanorods dispersion liquid: the amount of substance of phospholipid molecule template=1: 4;
The volume of gold nanorods dispersion liquid: the volume of PBS solution=1: 1.3;
The PBS damping fluid is 1 * PBS, and concentration is 10mmol/L, pH=7.4;
Put into refrigerator after step 3, the reactant liquor cooling that step 2 is obtained, left standstill 24 hours under 5 ℃, obtain a kind of surface plasma chirality probe; Gold nanorods concentration is 0.7nmol/L in the said probe.
Fig. 1 is the environmental scanning electronic microscope figure of embodiment 1 described probe, from figure, obtains the pattern and the microstructure of probe, and wherein dotted line is irised out is the arrangement architecture shoulder to shoulder of surface plasma probe.
Get probe difference called after a1, a2, a3 that three parts of volumes are 600 μ l; In a1, add L-cys solution, in a2, add D-cys solution, a3 is as blank; Add behind the halfcystine solution that semicystinol concentration is 166.7 μ mol/L among the a1 and a2; It is as shown in Figure 2 to obtain circular dichroism (CD) spectrum, and wherein horizontal ordinate is an excitation wavelength, and unit is a nanometer; Ordinate is the CD signal intensity, and unit is a milliradian, and corresponding probe a1, a2, a3 distinguish called after A1, A2, A3 successively with spectral line;
For A3, probe does not have and halfcystine combines, and is the ultraviolet band of 210～350nm at wavelength, and the CD signal is from CTAB and phospholipid molecule template, and the corresponding wavelength in peak value place is 220nm, and gained CD spectrum is that electron resonance absorbs circular dichroism (ECD) spectrum; And at visible/near-infrared band of wavelength 350～820nm, the CD signal approaches 0, and gained CD spectrum is that surface plasma body resonant vibration absorbs circular dichroism (PCD) spectrum; From A1, A2, can find out, when probe and L-cys or D-cys combine,, explain that probe can discern the not halfcystine of isomorphism type in visible/near infrared and ultraviolet band generation mirror image spectral response.
Wherein, the gold nanorods dispersion liquid is to prepare among the embodiment 1;
The preparation process of phospholipid molecule template is following:
6.5mg DMPC phospholipid molecule is dissolved in the 250mL round-bottomed flask of packing into behind the 20mL chloroform,, obtains product 1 40 ℃ of following rotary evaporations 4.5 hours, product 1 freeze drying is handled 36 hours after, obtain the phospholipid molecule template;
After adding gold nanorods dispersion liquid 1.5ml and PBS damping fluid 4ml in the phospholipid molecule template that in step 1, obtains,, obtain reactant liquor 70 ℃ of following hydrations 2 hours;
Wherein, be free in the amount of substance of the CTAB in the gold nanorods dispersion liquid: the amount of substance of phospholipid molecule template=1: 3.25;
The volume of gold nanorods dispersion liquid: the volume of PBS solution=1: 2.7;
The PBS damping fluid is 1 * PBS, and concentration is 10mmol/L, pH=7.4;
Put into refrigerator after step 3, the reactant liquor cooling that step 2 is obtained, left standstill 24 hours under 4 ℃, obtain a kind of surface plasma chirality probe, gold nanorods concentration is 1.1nmol/L in the said probe.
Fig. 3 is the environmental scanning electronic microscope figure of embodiment 2 described probes, from figure, can obtain the pattern and the microstructure of probe, and wherein dotted line is irised out is the arrangement architecture shoulder to shoulder of surface plasma probe.
Get probe difference called after b1, b2, b3 that three parts of volumes are 600 μ l; In b1, add L-cys solution; In b2, add D-cys solution, b3 is as blank, and semicystinol concentration is 166.7 μ mol/L among b1 and the b2; It is as shown in Figure 4 to obtain circular dichroism (CD) spectrum, and corresponding probe b1, b2, b3 distinguish called after B1, B2, B3 successively with spectral line;
For B3, probe does not have and halfcystine combines, and is the ultraviolet band of 210～350nm at wavelength, and the CD signal is from CTAB and phospholipid molecule template, and the corresponding wavelength in peak value place is 220nm, and gained CD spectrum is that electron resonance absorbs circular dichroism (ECD) spectrum; And at visible/near-infrared band of wavelength 350～820nm, the CD signal approaches 0, and gained CD spectrum is that surface plasma body resonant vibration absorbs circular dichroism (PCD) spectrum; From B1, B2, can find out, when probe and L-cys or D-cys combine,, explain that probe can discern the not halfcystine of isomorphism type in visible/near infrared and ultraviolet band generation mirror image spectral response.
Wherein, the preparation process of gold nanorods is following:
1. gold is planted and is prepared
Get the CTAB solution 7.5ml of 0.1mol/L, add 1.8ml water, it is the gold chloride (HAuCl of 24.7mmol/L that stirring condition adds concentration down
4) the WS 100.4 μ l after, add the sodium borohydride (NaBH of 0.01mmol/L
4) solution 600 μ l, stir 3min, obtain gold and plant subsequent use;
2. growth-promoting media preparation
Get the CTAB solution 100ml of 0.1mol/L, adding concentration is the gold chloride (HAuCl of 24.7mmol/L
4) WS 2.05ml, concentration is the silver nitrate aqueous solution 0.7ml of 0.01mol/L, concentration is the sulfuric acid 2ml of 0.5mol/L, concentration is the aqueous ascorbic acid 0.8ml of 0.1mol/L.
3. the preparation of gold nanorods dispersion liquid
The gold of getting step 1 preparation is planted 240 μ L and is joined in the growth-promoting media of step 2 preparation, at 30 ℃ of growth 14h down, obtains gold nanorods dispersion liquid stoste.Said gold nanorods stoste after centrifuging, is obtained deposition and supernatant liquid, wherein be precipitated as the gold nanorods that the surface is wrapped in CTAB; Behind pipettor taking-up supernatant liquid, in deposition, add 20ml water, obtain the gold nanorods dispersion liquid;
The preparation process of phospholipid molecule template is following:
6.5mg DMPC phospholipid molecule and dyestuff NBD-PE 0.05mg are dissolved in the 250mL round-bottomed flask of packing into behind the 20mL chloroform; 40 ℃ of following rotary evaporations 4.5 hours; Obtain product 1; Product 1 freeze drying processing after 36 hours, is obtained the phospholipid molecule template, and dye molecule is distributed in the phospholipid molecule template;
After adding gold nanorods dispersion liquid 1.5ml and PBS damping fluid 6ml in the phospholipid molecule template that in step 1, obtains,, obtain reactant liquor 65 ℃ of following hydrations 3 hours;
Wherein, be free in the amount of substance of the CTAB in the gold nanorods dispersion liquid: the amount of substance of phospholipid molecule template=1: 3.25;
The volume of gold nanorods dispersion liquid: the volume of PBS solution=1: 4;
The PBS damping fluid is 1 * PBS, and concentration is 10mmol/L, pH=7.4;
Put into refrigerator after step 3, the reactant liquor cooling that step 2 is obtained, left standstill 24 hours under 4 ℃, obtain a kind of surface plasma chirality probe, gold nanorods concentration is 1.5nmol/L in the said probe.
Under fluorescent microscope, probe sends visible green under blue-light excited, and the phospholipid molecule template of the enough fluorescence recognizing probe of ability is described.
Fig. 5 gets the probe that ten parts of volumes are 600 μ l, to the L-cys solution that wherein adds variable concentrations in five parts, in other five parts, add the D-cys solution of variable concentrations after, obtain circular dichroism (CD) spectrum; Get the PCD peak value mapping that the 527nm place occurs, when obtaining probe identification L-cys and D-cys, the PCD peak value that occurs at 527nm is along with the linear change of semicystinol concentration; Probe is discerned the straight line called after C1 that L-cys obtains, the straight line called after C2 that probe identification D-cys obtains.Wherein horizontal ordinate is a semicystinol concentration, the μ mol/L of unit; Ordinate is the PCD signal intensity, and unit is a milliradian.The measurement range of probe measurement semicystinol concentration is 22.4-111.6 μ mol/L; In measurement range, PCD peak value and semicystinol concentration present good linearity, explain that probe can be used for the accurate detection of L-halfcystine and D-semicystinol concentration.
Wherein, the preparation process of gold nanorods is following:
1. gold is planted and is prepared
Get the CTAB solution 7.5ml of 0.1mol/L, add 1.8ml water, it is the gold chloride (HAuCl of 24.7mmol/L that stirring condition adds concentration down
4) the WS 100.4 μ l after, add the sodium borohydride (NaBH of 0.01mmol/L
4) solution 600 μ l, stir 3min, obtain gold and plant subsequent use;
2. growth-promoting media preparation
Get the CTAB solution 100ml of 0.1mol/L, adding concentration is the gold chloride (HAuCl of 24.7mmol/L
4) WS 2.05ml, concentration is the silver nitrate aqueous solution 0.4ml of 0.01mol/L, concentration is the sulfuric acid 2ml of 0.5mol/L, concentration is the aqueous ascorbic acid 0.8ml of 0.1mol/L.
3. the preparation of gold nanorods dispersion liquid
The gold of getting step 1 preparation is planted 240 μ L and is joined in the growth-promoting media of step 2 preparation, at 30 ℃ of growth 15h down, obtains gold nanorods dispersion liquid stoste.Said gold nanorods stoste after centrifuging, is obtained deposition and supernatant liquid, wherein be precipitated as the gold nanorods that the surface is wrapped in CTAB; Behind pipettor taking-up supernatant liquid, in deposition, add 20ml water, obtain the gold nanorods dispersion liquid;
The preparation process of phospholipid molecule template is following:
7mg DMPC phospholipid molecule and dyestuff NBD-PE 0.05mg are dissolved in the 250mL round-bottomed flask of packing into behind the 20mL chloroform; 40 ℃ of following rotary evaporations 4.5 hours; Obtain product 1; Product 1 freeze drying processing after 38 hours, is obtained the phospholipid molecule template, and dye molecule is distributed in the phospholipid molecule template;
After adding gold nanorods dispersion liquid 1.5ml and PBS damping fluid 4.5ml in the phospholipid molecule template that in step 1, obtains,, obtain reactant liquor 3 65 ℃ of following hydrations 2 hours;
Wherein, be free in the amount of substance of the CTAB in the gold nanorods dispersion liquid: the amount of substance of phospholipid molecule template=1: 3.47;
The volume of gold nanorods dispersion liquid: the volume of PBS solution=1: 3;
The PBS damping fluid is 1 * PBS, and concentration is 10mmol/L, pH=7.4;
Put into refrigerator after step 3, the reactant liquor cooling that step 2 is obtained, left standstill 24 hours under 4 ℃, obtain a kind of surface plasma chirality probe, gold nanorods concentration is 1.2nmol/L in the said probe.
Under fluorescent microscope, probe sends visible green under blue-light excited, and the phospholipid molecule template of the enough fluorescence recognizing probe of ability is described.
Get probe called after d1, d2, d3, d4, d5 that five parts of volumes are 600 μ l, in d1, add L-cys solution, in d2, add D-cys solution, semicystinol concentration is 221.3 μ mol/L among d1 and the d2; In d4, add L-cys solution, in d5, add D-cys solution, semicystinol concentration is 56.01 μ mol/L among d4 and the d5; D3 is as blank, and it is as shown in Figure 6 to obtain circular dichroism (CD) spectrum, and corresponding probe d1, d2, d3, d4, d5 distinguish called after D1, D2, D3, D4, D5 successively with spectral line, and wherein horizontal ordinate is an excitation wavelength, and unit is a nanometer; Ordinate is the CD signal intensity, and unit is a milliradian;
For D3, probe does not have and halfcystine combines, and is the ultraviolet band of 210～350nm at wavelength, and the CD signal is from CTAB and phospholipid molecule template, and the corresponding wavelength in peak value place is 220nm, and gained CD spectrum is that electron resonance absorbs circular dichroism (ECD) spectrum; And at visible/near-infrared band of wavelength 350～820nm, the CD signal approaches 0, and gained CD spectrum is that surface plasma body resonant vibration absorbs circular dichroism (PCD) spectrum; Wherein, D1 and D2, D4 and D5 be respectively in visible/near infrared and ultraviolet band generation mirror image spectral response, explains that probe can discern the not halfcystine of isomorphism type.
Wherein, in embodiment 1～4, said phospholipid molecule and luminescent dye molecule are available from Avanti company; The PBS damping fluid is available from Shanghai Sigma reagent company.
The present invention has obtained the support of state natural sciences fund, and said fund number is 10874015,11174033.
In sum, more than being merely preferred embodiment of the present invention, is not to be used to limit protection scope of the present invention.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. the preparation method of a surface plasma chirality probe, it is characterized in that: said method concrete steps are following:
Step 1, preparation gold nanorods dispersion liquid and phospholipid molecule template
Wherein, the preparation process of gold nanorods dispersion liquid is following: after preparing gold nanorods dispersion liquid stoste through seed mediated growth method, centrifugal deposition and the supernatant liquid of obtaining wherein is precipitated as the gold nanorods that the surface is wrapped in cetyl trimethyl ammonium bromide; After taking out supernatant liquid, in deposition, add water, obtain the gold nanorods dispersion liquid; Free cetyl trimethyl ammonium bromide is arranged in the gold nanorods dispersion liquid;
The phospholipid molecule template is through the dimyristoyl phosphatidyl choline phospholipid molecule is dissolved in chloroform, and behind the chloroform rotary evaporation, freeze drying obtains;
Step 2, with phospholipid molecule template and gold nanorods hydration
After in the phospholipid molecule template, adding the gold nanorods dispersion liquid and phosphate buffer that obtains in the step 1,, obtain reactant liquor 60～70 ℃ of following hydrations 2～4 hours;
Wherein, the long 30～60nm of gold nanorods in the said gold nanorods dispersion liquid, wide 12～20nm, two ends contain that { 111} face, length-diameter ratio are 1.5～5;
Be free in the amount of substance of the cetyl trimethyl ammonium bromide in the gold nanorods dispersion liquid: the amount of substance of phospholipid molecule template=1: 3～4;
The volume of gold nanorods dispersion liquid: the volume of phosphate buffer=1: 1～4;
The concentration of phosphate buffer is 10mmol/L, and the pH value is 7.4;
Put into refrigerator after step 3, the reactant liquor cooling that step 2 is obtained, left standstill 24 hours under 4～5 ℃, obtain a kind of surface plasma chirality probe.
2. the preparation method of a kind of surface plasma chirality probe according to claim 1 is characterized in that: it is following to prepare the process of gold nanorods dispersion liquid stoste through seed mediated growth method in the step 1:
(1) gold is planted and is prepared
Get cetyl trimethyl ammonium bromide solution, stirring condition adds sodium borohydride solution after adding the WS of gold chloride down, stirs 3min, obtains gold and plants subsequent use;
Wherein, the amount of substance of cetyl trimethyl ammonium bromide: the amount of substance of gold chloride: the amount of substance of sodium borohydride=7500: 24.8: 6;
(2) growth-promoting media preparation
Get cetyl trimethyl ammonium bromide solution, add the WS, silver nitrate aqueous solution, sulfuric acid and the aqueous ascorbic acid of gold chloride;
Wherein, the amount of substance of cetyl trimethyl ammonium bromide: the amount of substance of gold chloride: the amount of substance of silver nitrate=10
4: 50.6: 4～7;
The amount of substance of cetyl trimethyl ammonium bromide: the amount of substance of sulfuric acid: the amount of substance of ascorbic acid=10
3: 10
2: 8;
(3) preparation of gold nanorods dispersion liquid
Get the gold kind of step 1 preparation, join in the growth-promoting media of step 2 preparation,, obtain gold nanorods dispersion liquid stoste at 30 ℃ of 12～15h that grow down;
Wherein, the amount of substance of cetyl trimethyl ammonium bromide in the gold kind of the step of getting (1) preparation: amount of substance=1.8 * 10 of cetyl trimethyl ammonium bromide in the growth-promoting media of step (2) preparation
-5: 1.
3. the preparation method of a kind of surface plasma chirality probe according to claim 1 is characterized in that: the quality of phospholipid molecule in the step 1: the volume≤0.5mg of chloroform: 1ml.
4. the preparation method of a kind of surface plasma chirality probe according to claim 1 is characterized in that: gold nanorods concentration is 0.7～1.5nmol/L in the said probe.
5. the preparation method of a kind of surface plasma chirality probe according to claim 1 is characterized in that: step 1 adds luminescent dye molecule in phospholipid molecule in the preparation process of phospholipid molecule template.
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| CN201210032890.5A CN102565380B (en) | 2012-02-14 | 2012-02-14 | Method for preparing surface plasma chiral probe |
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| CN201210032890.5A CN102565380B (en) | 2012-02-14 | 2012-02-14 | Method for preparing surface plasma chiral probe |
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| CN102565380B CN102565380B (en) | 2014-02-05 |
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Cited By (3)
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| CN103163076A (en) * | 2013-02-26 | 2013-06-19 | 江南大学 | Method for detecting L-cysteine concentration by applying of circular dichroism |
| CN105621352A (en) * | 2015-12-31 | 2016-06-01 | 江南大学 | Preparing method of gold nanometer particle single-layer film with chirality |
| CN112834379A (en) * | 2021-01-22 | 2021-05-25 | 盐城工学院 | Cysteine chiral recognition sensor |
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| CN103163076A (en) * | 2013-02-26 | 2013-06-19 | 江南大学 | Method for detecting L-cysteine concentration by applying of circular dichroism |
| CN103163076B (en) * | 2013-02-26 | 2014-11-26 | 江南大学 | Method for detecting L-cysteine concentration by applying of circular dichroism |
| CN105621352A (en) * | 2015-12-31 | 2016-06-01 | 江南大学 | Preparing method of gold nanometer particle single-layer film with chirality |
| CN105621352B (en) * | 2015-12-31 | 2019-03-29 | 江南大学 | A kind of preparation method with chiral gold nanoparticle single thin film |
| CN112834379A (en) * | 2021-01-22 | 2021-05-25 | 盐城工学院 | Cysteine chiral recognition sensor |
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| CN102565380B (en) | 2014-02-05 |
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