CN103528996A - Preparation method of gold nanorod SPR probe and method for detecting drug loading release kinetic process of the probe - Google Patents
Preparation method of gold nanorod SPR probe and method for detecting drug loading release kinetic process of the probe Download PDFInfo
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Abstract
The invention discloses a preparation method of a gold nanorod single particle SPR probe and a method for detecting drug loading release kinetic process of the probe. The preparation method is as below: adding a reducing agent ascorbic acid and gold seeds obtained by NaBH4 reduction into HAuCl4 containing a surfactant CTAB to obtain a gold nanorod solution; centrifuging and purifying; regulating the pH value and adding a TEOS ethanol solution as a silicon source under alkaline conditions; stirring at room temperature for reaction; further centrifuging and purifying the obtained solution; and dissolving in water to obtain a solution containing mesoporous silicon coated gold nanorod. The composite plasma probe formed by nuclear shell structure can simulate and monitor macro release process of drug in solution state, and also can be fixed on a pretreated glass surface to form a single particle SPR probe chip. Through the addition of anticancer drugs and removal of anticancer drugs, effective monitoring on release kinetics of nano drug loading system is realized by single particles by means of an SPR dark field spectromicroscope.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to the preparation method of the coated gold nanorods individual particle SPR probe of mesoporous silicon, and utilize this probe in the method for monitoring gold nanorods medicine carrying release dynamics.
Background technology
Nano medicament carrying system has just got more and more people's extensive concerning since being born, and in tradition research, people usually simulate to understand nano-carrier drug release kinetics process in vitro.At this research process, the aids drug that people choose conventionally need have certain fluorescent characteristic, and utilizes this characteristic to understand release dynamics process.But, under reality, be not that all cancer therapy drugs all have fluorescent characteristic, this has limited the research process of people for various drug release kinetics to a certain extent greatly, and the simulation of external solution state and cell self environment still exist larger gap.In addition, researcher finds the introducing of nano-carrier recently, because the biocompatibility of himself is different, can cause it to there is certain cytotoxicity, may bring new harm to human body, therefore, seek a kind of new study hotspot that can be used for the monitoring method of various medicines dispose procedure in born of the same parents just to become current people.
Under this situation, surface plasma resonance (SPR) effect starts to enter in people's the visual field with its unique advantage.Surface plasma resonance (SPR) is that metal surface free electron is subject to the collective oscillation in metal-dielectric interface after electromagnetic radiation, and under resonance state, the energy of electromagnetic field is changed into the collective vibration energy of metal surface free electron effectively.The plasma resonance photonics (Plasmonics) of development, as the important branch in nanocomposite optical field, can realize the observation at nanoscale thus.Because the Rayleigh scattering signal of nano particle is very sensitive to the variation response of its size, composition, pattern, surface charge distribution and surrounding environment thereof, by SPR details in a play not acted out on stage, but told through dialogues spectromicroscope, can design the SPR optical probe for monitoring at single molecules level.Dispose procedure, the bio-toxicity of nano-carrier and the burst size of medicine that SPR optical probe is incorporated in in-situ monitoring nano-carrier born of the same parents have broad application prospects on the correlative studys such as impact of cell.
The present invention adopts SPR effect principle to design the Small-molecule probe of novel nanoscale, adopts modifying interface technology to be modified glass surface, and the highly sensitive and high selectivity that realizes nanoscale on the individual particle SPR probe based on gold nanorods detects.Thereby by medicine, discharge dispose procedure and can understand the release dynamics of nano medicament carrying system to the impact of SPR probe, the method is for the release dynamics performance of evaluating the multiple pharmaceutical carrier based on nano particle, for the application of the nano biological association areas such as pharmaceutical carrier research, formulation development, oncotherapy provides new thinking.It is simple that the method has mechanism, easy to prepare, and the many merits such as sensitive monitoring, for monitoring drug release kinetics process provides a kind of new method.
Summary of the invention
technical matters:the object of the present invention is to provide a kind of preparation method of gold nanorods individual particle SPR probe and the method for using its medicine carrying release dynamics process of this probe in detecting.
technical scheme:technical scheme of the present invention comprises a kind of preparation method of gold nanorods individual particle SPR probe and the method for using its medicine carrying release dynamics process of this probe in detecting.
A kind of gold nanorods SPR probe preparation method of the present invention comprises the following steps:
(a) preparation of 3 ~ 5nm gold seeds solution: in the sample bottle of 20mL, add 10mL, 0.001 ~ 1mol/L cetyl trimethyl ammonium bromide surfactant; Add subsequently 0.25mL, the HAuCl of 0.001 ~ 1mol/L
4solution, is uniformly mixed; And then add rapidly 0.60mL, the NaBH that 0.001 ~ 1mol/L is ice-cold
4solution, vigorous stirring is after 2 seconds, under room temperature standing 2 ~ 8 hours.
(b) growth of gold nanorods: add 40mL in beaker, 1 0.001 ~ 1mol/L cetyl trimethyl ammonium bromide solution; 2.0mL, 0.001 ~ 1mol/L HAuCl
4solution and 10 μ L ~ 1000 μ L, 0.001 ~ 1mol/L AgNO
3solution, is slowly uniformly mixed; Add 10 μ L ~ 1000 μ L thereupon, 1mol/L hydrochloric acid, it is acid regulating pH; Add afterwards 10 μ L ~ 1000 μ L, 0.001 ~ 1mol/L ascorbic acid weak reductant, after stirring, adds 3 ~ 5nm gold seeds solution of 1 ~ 20 times of the dilution prepared in 0.001 ~ 1mL above-mentioned steps, stirs after 1 ~ 10 minute under room temperature standing 6 hours;
(c) purifying of gold nanorods: gold nanorods obtained above is carried out to centrifugal purification, first, carry out centrifugal with low speed, discard the bulky grain of lower floor, then supernatant is carried out to high speed centrifugation, centrifugal twice, stay lower sediment, last, add ultrapure water to original volume;
The preparation of the gold nanorods that step 2, mesoporous silicon are coated:
Get the sample bottle that gold nanorods solution 10mL that above-mentioned centrifugal purification crosses puts into 20mL, put into magneton and carry out rapid stirring, add the sodium hydroxide solution of 0.001 ~ 1mol/L to regulate pH for alkalescence; Subsequently, add 1 ~ 100 μ L, the ethanolic solution containing tetraethyl orthosilicate 1% ~ 80%, added once every 10 ~ 40 minutes, and totally 3 times, under room temperature, rapid stirring 24 hours; Finally the nanoparticles solution obtaining is carried out to centrifugal purification.Obtain the coated gold nanorods solution of mesoporous silicon, i.e. gold nanorods SPR probe.
The method of detection gold nanorods SPR probe medicine carrying release dynamics process of the present invention comprises the following steps:
Ito glass sheet is immersed in order in liquid detergent, acetone, ethanol, ultrapure water solution and carries out, after ultrasonic a period of time 1 ~ 5h, using N after taking-up
2dry up and store for future use; Get gold nanorods solution 1 ~ 100 μ L of claim 1 preparation, after the dilution of 1:1000 ~ 5000, put into ito glass sheet in proportion, soak after regular hour 1 ~ 30min, use N
2dry up, complete on ito glass sheet the fixedly process of the coated gold nano-rod particles of mesoporous silicon;
Step 3, ito glass sheet is placed on objective table, drip upper with isocyatic cancer therapy drug solution, the scattering spectrum of individual particle while taking medicine carrying with dark field microscope; Subsequently, siphon away drug solution, drip the aqueous solution of same volume, with dark field microscope, take medicine time dependent desorption process on gold nano-rod particles;
The gold nanorods individual particle SPR probe that mesoporous silicon of the present invention is coated, it has good Monitoring Performance, can aspect monitoring nano-carrier release dynamics, have good effectiveness in conjunction with SPR technology.This SPR probe is the gold nanorods SPR probe that mesoporous silicon is modified, and its structure is: mesoporous silicon is wrapped in gold nanorods, adsorbs certain cancer therapy drug, SPR spectrum blue shift when cancer therapy drug desorption.
Accompanying drawing explanation
The schematic diagram of Fig. 1 key link of the present invention.
Gold nanorods AuNRs and the coated gold nanorods AuNRs@SiO of mesoporous silicon that Fig. 2 adopts the present invention to prepare
2ultraviolet spectrogram.
Gold nanorods AuNRs and the coated gold nanorods AuNRs@SiO of mesoporous silicon that Fig. 3 adopts the present invention to prepare
2transmission electron microscope photo.
Fig. 4 adopts the Rayleigh Scattering Spectra based on gold nanorods individual particle SPR probe monitoring drug release process prepared by the present invention to scheme over time.
Fig. 5 adopt prepared by the present invention based on gold nanorods individual particle SPR probe monitoring nano medicament carrying system release dynamics process.
Embodiment
In order to understand better content of the present invention, below in conjunction with embodiment and accompanying drawing, further set forth the present invention.The present embodiment be take technology of the present invention as basis enforcement, provided detailed embodiment and operation steps, but protection scope of the present invention is not limited to following embodiment.
This SPR probe is the gold nanorods SPR probe that mesoporous silicon is modified, and its structure is: mesoporous silicon is wrapped in gold nanorods, adsorbs certain cancer therapy drug, SPR spectrum blue shift when cancer therapy drug desorption.Its concrete structure is as shown in Figure 1: 1 is gold nanorods; 2 is the coated gold nanorods of mesoporous silicon; 3 for the mesoporous coated gold nanorods of the complete medicine of load; 4 is the mesoporous silicon coated gold nanorods of medicine desorption after completing.
Embodiment
1, gold nanorods is synthetic
(a) preparation of gold seeds: add 10mL in the sample bottle of 20mL, 0.1mol/L cetyl trimethyl ammonium bromide solution, adds 0.25mL subsequently, the chlorauric acid solution of 0.01mol/L, after stirring, add fast the sodium borohydride that 0.6mL 0.01mol/L is ice-cold.Under room temperature, front standing at least 2h to be used.
(b) growth of gold nanorods: add 40mL in beaker, 0.1mol/L cetyl trimethyl ammonium bromide solution, 2.0mL, 0.01mol/LHAuCl
4with 150 μ L, 0.01mol/L AgNO
3solution, is slowly uniformly mixed.Add hydrochloric acid, regulating pH is 1 ~ 2 thereupon.Add afterwards weak reductant-ascorbic acid (0.32mL, 0.01mol/L), after stirring, add 0.15mL(a) in 3 ~ 5nm gold seeds solution of 5 times of dilutions of preparation.Stir after the some time under room temperature standing 6 hours.Gold nanorods carries out, after centrifugal purification, adding ultrapure water, obtains certain density gold nanorods solution.
2, the preparation of the coated gold nanorods of mesoporous silicon: get the sample bottle that gold nanorods solution 10mL that above-mentioned centrifugal purification crosses puts into 20mL, put into magneton and carry out rapid stirring, adding the sodium hydroxide solution of 0.1mol/L to regulate pH is 10 ~ 11.Subsequently, add the tetraethyl orthosilicate of 5 μ L ethanolic solution (tetraethyl orthosilicate wherein: ethanol=0.2), add at set intervals once, totally 3 times, under room temperature, rapid stirring 24 hours.Finally, the nanoparticles solution obtaining is carried out, centrifugal purification obtains the coated gold nanorods solution of certain density mesoporous silicon.
Be illustrated in figure 2 gold nanorods AuNRs and the coated gold nanorods AuNRs@SiO of mesoporous silicon prepared by the present invention
2ultraviolet spectrogram.As can be seen from the figure gold nanorods longitudinal plasma after being coated mesoporous silicon absorbs certain red shift has occurred.
Be illustrated in figure 3 gold nanorods AuNRs and the coated gold nanorods AuNRs@SiO of mesoporous silicon prepared by the present invention
2transmission electron microscope photo.Left figure can find out the gold nanorods pattern homogeneous of preparation, and it is well coated that right figure shows that mesoporous silicon has on gold nanorods, and thickness is about 15nm.
3, the coated gold nano-rod particles of mesoporous silicon is in fixing and subsequent treatment on glass
(a) processing procedure of the glass sheet of loaded with nano particle: the ito glass with certain length and width of having cut apart is immersed in sequence in acetone, ethanol, ultrapure water solution and carries out, after ultrasonic a period of time, drying up and storing for future use after taking-up with N2.
(b) fixing on the glass sheet of the coated gold nano-rod particles of mesoporous silicon after processing: the nano particle certain volume of getting above-mentioned preparation, after 1:5000 dilution by a certain percentage, put into the glass sheet of processing, soak after the regular hour, with N2, dry up, complete fixation procedure.
(c) the fixing subsequent treatment of the glass sheet of nano particle: the glass sheet that previous step is made is put into the cancer therapy drug solution that concentration is 20 μ g/mL, lucifuge is shaken about 24h with certain speed on shaking table, until glass sheet is taken out while observing dispose procedure again.
4, composite plasma probe is monitored for conventional dispose procedure:
By the coated gold nano-rod particles of certain density mesoporous silicon and cancer therapy drug according to a certain percentage (10:1 ~ 2:1) mix, under dark surrounds, stir centrifugal treating after 24 h, remove supernatant, add the clear water of 2 mL left and right to be placed in bag filter, in 10 ~ 100 mL clear water, dialyse, keep not stopping to stir, take out at set intervals sample test ultra-violet absorption spectrum with monitoring release dynamics process, in this process, shift out and supplement rapidly isopyknic clear water after solution, to keep in observation process liquor capacity constant.
5, nanometer plasma chip monitoring drug release kinetics process:
By on the above-mentioned glass sheet objective table making, then on glass sheet, drip the cancer therapy drug solution with concentration, dark field microscope is carried out to parameters, focusing etc., the scattering spectrum of individual particle while taking medicine carrying; Subsequently, siphon away drug solution, drip the aqueous solution of same volume, take the desorption process of medicine on nano particle.Observation process is 2 h, and when 0.5 h and 1.5 h, supplementing water solution, to supplement the water of volatilization, siphons away solution on glass sheet the fresh water solution that replaces with same volume when 1 h.
6, composite plasma probe is directly monitored dispose procedure in cell:
By certain density containing the coated gold nanorods fresh medium of load cancer therapy drug mesoporous silicon containing adding in the nutrient culture media of tumour cell, through after a period of time, with PBS solution, rinse cell, remove nutrient culture media and be also placed in PBS solution, under dark field microscope, observe gold nanorods spectrum change in time in desorption process.Wherein, the condition of culture of cell is: 37 ℃ containing 5% CO
2environment under be placed in modified nutrient culture media and cultivate, wherein add certain density heat-inactivated hyclone and microbiotic, grow overnight.
Being illustrated in figure 4 the Rayleigh Scattering Spectra based on gold nanorods individual particle SPR probe monitoring drug release process prepared by the present invention schemes over time.As can be seen from the figure in dispose procedure, there is certain blue shift in the Rayleigh Scattering Spectra of gold nanorods individual particle SPR probe, meets the mechanism of dispose procedure in time.
Be illustrated in figure 5 prepared by the present invention based on gold nanorods individual particle SPR probe monitoring nano medicament carrying system release dynamics process.As can be seen from the figure this probe has good monitoring capability to release dynamics process, is discharged into fast in environment just starting medicine, slows down gradually subsequently convergence certain value.
Table 1 is each performance parameter in the 2h time period:
| Time/min | Scattering peak value/nm | Blue shift amount/ |
| 0 | 700.295 | 0 |
| 5 | 698.030 | 2.265 |
| 8 | 697.412 | 2.883 |
| 12 | 697.001 | 3.294 |
| 15 | 697.001 | 3.294 |
| 25 | 697.001 | 3.294 |
| 30 | 696.795 | 3.5 |
| 35 | 697.207 | 3.088 |
| 40 | 697.001 | 3.294 |
| 45 | 696.795 | 3.5 |
| 50 | 697.001 | 3.294 |
| 55 | 696.795 | 3.5 |
| 60 | 696.589 | 3.706 |
| 65 | 694.735 | 5.56 |
| 80 | 694.323 | 5.972 |
| 95 | 694.323 | 5.972 |
| 100 | 694.323 | 5.972 |
| 105 | 694.529 | 5.766 |
| 110 | 694.323 | 5.972 |
| 115 | 694.529 | 5.766 |
| 120 | 694.529 | 5.766 |
As shown in Table 1, based on mesoporous coated individual particle gold nanorods SPR probe, can effectively monitor the dispose procedure of medicine, there is the feature of simple and effective.
The formed probe of this nucleocapsid structure both can be for the monitoring of conventional dispose procedure, also can be used as plasma chip and realize the monitoring that on individual particle, medicine discharges, meanwhile, it can also effectively be monitored drug release process in cell, novel and high-efficiency.The composite plasma probe that this nucleocapsid structure forms can be at macroscopical dispose procedure of solution state Imitating monitoring medicine.In addition, this nucleocapsid structure can also form individual particle SPR probe chip by being fixed on pretreated glass surface, surface plasma resonance (SPR) effect due to gold nanorods, and the Rayleigh scattering signal of nano particle distributes to its size, composition, pattern, surface charge and the sensitive response of the variation of surrounding environment, by adding cancer therapy drug and removal cancer therapy drug to realize individual particle effective monitoring to Nano medication drug-loading system release dynamics by SPR details in a play not acted out on stage, but told through dialogues spectromicroscope.This SPR probe scattering spectrum in monitoring the desorption process of medicine in mesoporous silicon can produce certain blue shift due to the variation of external environment condition, the scattering peak of this probe is positioned at 600 ~ 700nm.The method has the advantages such as simple and effective, on this basis, utilizes this probe also can realize the observation process that medicine in cell is discharged.
Claims (2)
1. a gold nanorods SPR probe preparation method, its feature comprises step:
Step 1, the synthetic gold nanorods solution of use seeded growth method:
(a) preparation of 3 ~ 5nm gold seeds solution: in the sample bottle of 20mL, add 10mL, 0.001 ~ 1mol/L cetyl trimethyl ammonium bromide surfactant; Add subsequently 0.25mL, the HAuCl of 0.001 ~ 1mol/L
4solution, is uniformly mixed; And then add rapidly 0.60mL, the NaBH that 0.001 ~ 1mol/L is ice-cold
4solution, vigorous stirring is after 2 seconds, under room temperature standing 2 ~ 8 hours;
(b) growth of gold nanorods: add 40mL in beaker, 0.001 ~ 1mol/L cetyl trimethyl ammonium bromide solution; 2.0mL, 0.001 ~ 1mol/L HAuCl
4solution and 10 μ L ~ 1000 μ L, 0.001 ~ 1mol/L AgNO
3solution, is slowly uniformly mixed; Add 10 μ L ~ 1000 μ L thereupon, 1mol/L hydrochloric acid, it is acid regulating pH; Add afterwards 10 μ L ~ 1000 μ L, 0.001 ~ 1mol/L ascorbic acid weak reductant, after stirring, adds 3 ~ 5nm gold seeds solution of 1 ~ 20 times of the dilution prepared in 0.001 ~ 1mL above-mentioned steps, stirs after 1 ~ 10 minute under room temperature standing 6 hours;
(c) purifying of gold nanorods: gold nanorods obtained above is carried out to centrifugal purification, first, carry out centrifugal with low speed, discard the bulky grain of lower floor, then supernatant is carried out to high speed centrifugation, centrifugal twice, stay lower sediment, last, add ultrapure water to original volume;
The preparation of the gold nanorods that step 2, mesoporous silicon are coated:
Get the sample bottle that gold nanorods solution 10mL that above-mentioned centrifugal purification crosses puts into 20mL, put into magneton and carry out rapid stirring, add the sodium hydroxide solution of 0.001 ~ 1mol/L to regulate pH for alkalescence; Subsequently, add 1 ~ 100 μ L, the ethanolic solution containing tetraethyl orthosilicate 1% ~ 80%, added once every 10 ~ 40 minutes, and totally 3 times, under room temperature, rapid stirring 24 hours; Finally the nanoparticles solution obtaining is carried out to centrifugal purification.
2. a method that detects medicine carrying gold nanorods SPR probe release dynamics process, its feature comprises step:
Step 1, the coated gold nano-rod particles of mesoporous silicon fixing on ito glass:
Ito glass sheet is immersed in order in liquid detergent, acetone, ethanol, ultrapure water solution and carries out, after ultrasonic 1 ~ 5h, using N after taking-up
2dry up and store for future use; Get claim 1 preparation gold nanorods solution a little, after the dilution of 1:1000 ~ 5000, put into ito glass sheet in proportion, soak after regular hour 1 ~ 30min, use N
2dry up, complete on ito glass sheet the fixedly process of the coated gold nano-rod particles of mesoporous silicon;
Step 2, on ito glass sheet cancer therapy drug in load: the ito glass sheet that is fixed with the coated gold nano-rod particles of mesoporous silicon is put into the cancer therapy drug solution that concentration is 1 ~ 1000 μ g/mL, and lucifuge is shaken about 24h with 1 ~ 1000r/min on shaking table, uses subsequently N
2dry up;
Step 3, ito glass sheet is placed on objective table, drip upper with isocyatic cancer therapy drug solution, the scattering spectrum of individual particle while taking medicine carrying with dark field microscope; Subsequently, siphon away drug solution, drip the aqueous solution of same volume, with dark field microscope, take medicine time dependent desorption process on gold nano-rod particles;
Step 4, in the monitoring of conventional dispose procedure: the gold nanorods solution of claim 1 preparation of finite concentration 1 ~ 20 μ g/mL is mixed with cancer therapy drug, under details in a play not acted out on stage, but told through dialogues, stirs 24h; Centrifugal treating, places lower sediment dilution in bag filter subsequently, in clear water, dialyses, and continuous stirring, takes out sample every 30 ~ 60min and survey ultraviolet spectrum, and with the clear water of same volume, supplement immediately;
Step 5, in cell Real-Time Monitoring gold nanorods medicine carrying release dynamics process: by the nutrient culture media of cultivating altogether with tumour cell, add finite concentration 1 ~ 20 μ g/mL containing the coated gold nanorods solution of load cancer therapy drug mesoporous silicon, cultivate after a period of time 12 ~ 48h, with PBS solution, rinse cell, remove nutrient culture media and be also placed in PBS solution, under dark field microscope, observe the gold nanorods that enters in cell spectrum change in time in desorption process.
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