CN101865984A - Magnetic relaxation switch based on Fe3O4 at Au and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of magnetic relaxation switch detection, in particular to a magnetic relaxation switch based on Fe3O4 at Au and a preparation method thereof. Probes adopted by the magnetic relaxation switch of the invention are flower-shaped magnetic nanometer particles with identification marks decorated on the surfaces, and the flower-shaped magnetic nanometer particles are in Fe3O4 at Au nuclear shell structures, the particle diameter of the probes is ranged from 50 to 70nm, and the relaxation degree R2 of the probes is 9.35 mM<-1>.s<-1>. Through decorating identification molecular biotin on gold layers of the nanometer particle surfaces, the invention realizes the fast, simple and convenient detection of the model protein Avidin, and the detection limit is as low as the level of 10 nM. The invention solves the problems of inconvenient implementation and unstable detection results of the existing magnetic relaxation switch technology.

Description

A kind of based on Fe 3O 4Magnetic relaxation switch of@Au and preparation method thereof

Technical field

The invention belongs to magnetic relaxation switch detection technique field, be specifically related to a kind of based on Fe ₃O ₄Magnetic relaxation switch of@Au magnetic nanoparticle and preparation method thereof.

Background technology

Biology sensor based on superparamagnetic nano particle is a kind of efficient detection technology that grew up in recent years, and it has broken through the restriction of magnetic resonance in external application, and this technology is called as magnetic relaxation switch technology (Magnetic RelaxationSwitch).In an externally-applied magnetic field, the magnetic dipole of paramagnetic particle itself can produce a small disturbance of magnetic field around it, cause in the hydrone in this coverage ¹The H nuclear spin is degenerated, like this by the radio wave pulses Sequence Detection ¹The H signal can weaken, and the determined time constant of relaxation indices degenerated curve that obtains is T ₂, when target molecule appeared in the solution, the group on magnetic-particle surface was combined with target molecule, caused that magnetic-particle changes aggregating state into from dispersity, and the original little magnetic field " reunion " that disperses is later on in the hydrone ¹The spin degeneration of H nuclear changes, and causes T ₂Variation, T ₂Variable quantity relevant with concentration of target molecules, can do quantitative analysis to target molecule accordingly, and in detecting the process of target molecule, the magnetic granule density need not to know.

At present the detection of biomolecule is adopted mostly the way of optical analysis, comprised UV, visible light absorption analysis, fluorescence radiation analysis, nephelometric analysis etc., these technology all need sample that good light transmittance is arranged.The advantage of MRSw technology is to detect the content of target molecule in the turbid solution, blood for example, and cell culture fluid, milk etc. can significantly reduce complicated sample pretreatment step like this.

The MRSw technology is in the starting stage at present, although obvious practical prospect is arranged, the T of MRSw ₂The concentration correlation of signal intensity and analyte is not enough stable, and T ₂Signal is before the concentration of analyte reaches the detection range peak and distinct variation tendency arranged afterwards, other detection means of necessary employing is replenished and is proved the relaxation switching technique, improves the actual application value of MRSw in the mode of Multiple detection.On the other hand, general magnetic nanoparticle finishing group also is not easy, and modify especially difficulty of different groups, and utilize the key bond effect of Au and sulfydryl can make probe particle surface modification recognition group or antibody become easy.

With magnetic relaxation switches and Fe ₃O ₄@Au nano particle has been that keyword is respectively to the global patent retrieval since 1992, wherein do not find relevant patent take magnetic relaxation switches as keyword, international monopoly is retrieved as the keyword combination take Magnetic, Relaxation and Sensor in addition, wherein related to 1 US20060269965 of patent (Water Relaxation-Based Sensor) of magnetic relaxation switch; And relate to Fe ₃O ₄The patent of@Au nano particle has 2, is respectively a kind of preparation method (number of applying for a patent CN200810029399.0) of gold magnetic core-shell nano-particle, and this patent has been described a kind of Fe that synthesizes under ultrasonication ₃O ₄The method of@Au nano particle; Another piece patent is that a kind of gold-coating magnetic granule in-situ initiation high-sensibility chemical luminescence detects colibacillary new method (number of applying for a patent CN200810031350.9), and that this patent is described is a kind of Fe of utilization ₃O ₄The oxidized Au that discharges of@Au nano particle ³⁺, cause chemiluminescence, the technology that then Escherichia coli is detected behind the adding luminol.

Summary of the invention

The objective of the invention is to design a kind of based on Fe ₃O ₄Magnetic relaxation switch of@Au core-shell structure magnetic nano particle and preparation method thereof.

The present invention design based on Fe ₃O ₄The magnetic relaxation switch of@Au, the probe of employing are the flower shape magnetic nanoparticle that finishing has the identification molecule, and this flower shape magnetic nanoparticle is Fe ₃O ₄@Au nucleocapsid structure.

Described finishing has the particle diameter of the flower shape magnetic nanoparticle probe of discerning molecule in the 50-70nm scope, and outward appearance is the flower shape.

Described finishing has the element mass ratio (measuring by ICP) of the flower shape magnetic nanoparticle probe of identification molecule to be Fe: Au=1: 3.4.

Described finishing has the ultraviolet and visible absorption peak of the flower shape magnetic nanoparticle probe of discerning molecule at the 578nm place.

It is R that described finishing has the relaxivity of the flower shape magnetic nanoparticle probe of identification molecule ₂=9.35mM ^-1S ^-1

The present invention propose based on Fe ₃O ₄The preparation process of the magnetic relaxation switch of@Au core-shell structure magnetic nano particle is as follows:

A. in dextran solution, synthesize diameter less than the Fe of 5nm by the method for co-precipitation ₃O ₄Particle;

B. be the Fe of 14--16mg Fe/mL at concentration of iron ₃O ₄Method by the Reduction of Glucose gold chloride in the aqueous solution is synthesized flower shape Fe ₃O ₄@Au core-shell structure magnetic nano particle;

C. at Fe ₃O ₄Cystamine in the self assembly of@Au nano grain surface, and by the upper biotin of EDC/NHS catalysis connection, obtain the magnetic nanoparticle Biotin-Fe of functionalization ₃O ₄@Au.

Toward above-mentioned scattered Biotin-Fe ₃O ₄The avidin Avidin that adds different amounts in the@Au aqueous solution, the spin spin relaxation time T of mensuration solution behind the reaction 10min ₂

Compared with prior art, the present invention has following characteristics:

1, the flower shape Fe that adopts ₃O ₄@Au nanoparticle probes relaxivity size to fit can keep stable in solution for a long time;

2, particle size is even, and particle diameter is distributed between the 50-70nm, be very beneficial for according to disperse and reunite between conversion and the enforcement of the magnetic relaxation switch technology that detects has lower detectability;

3, the magnetic nanoparticle surface is coated with the gold layer, can modify upper specific identification molecule or albumen for type and the character of the analyte that will detect easily;

4, core-shell structure particles has special absorption in ultraviolet-visible absorption spectroscopy, can introduce UV, visible light absorption detecting technology easily testing result is confirmed.

Description of drawings

Fig. 1 (A) is flower shape Fe ₃O ₄The TEM close-up view of@Au magnetic nanoparticle; (B) be flower shape Fe ₃O ₄The TEM enlarged drawing of@Au magnetic nanoparticle.

Fig. 2 (A) is flower shape Fe ₃O ₄X-ray energy spectrum (EDX) figure of@Au magnetic nanoparticle; (B) the flower shape Fe that records for EDX ₃O ₄The element mass percent figure of@Au magnetic nanoparticle.

Fig. 3 (A) is flower shape Fe ₃O ₄The ultraviolet-visible absorption spectroscopy figure of@Au solution of magnetic nanoparticles; (B) be by match 1/T ₂The straight line that concerns to Fe concentration is obtained Fe ₃O ₄The relaxivity of@Au magnetic nanoparticle.

Fig. 4 is Biotin-Fe ₃O ₄The Fe of@Au solution and unmodified Biotin ₃O ₄The T of@Au solution ₂The graph of a relation of time and Avidin concentration; Illustration is the interior solution T of the Avidin concentration range of 0-40nM ₂Match linear relationship chart to Avidin concentration.

Fig. 5 is under the different Avidin concentration, Biotin-Fe ₃O ₄The ultraviolet-visible absorption spectroscopy figure of@Au solution; Illustration is the graph of a relation of each absorption line absorption maximum peak position and Avidin concentration.

Fig. 6 (A) is under the different Avidin concentration, Biotin-Fe ₃O ₄The dynamic light scattering particle diameter distribution map of@Au solution particle; (B) be the graph of a relation of each particle diameter distribution map distribution of peaks position and Avidin concentration.

Fig. 7 is a structural diagrams of the present invention.

Embodiment

Embodiment 1: the nanometer Fe of synthetic glucan parcel ₃O ₄

(15%w/w Mw=20000) adds the dense NH of 4mL in the solution at the 15mL glucan aqueous solution under the room temperature ₄OH (＞25%w/w), make the pH value of solution value be about 11.7, water-bath is heated to 25 ℃ under magnetic agitation then.0.75gFeCl ₃6H ₂O and 0.28g FeSO ₄4H ₂O is dissolved in the 5mL water, and with dropwise joining behind the membrane filtration of 0.22 μ m in the dextran solution, solution becomes the black turbid solution, stirs 0.5h.The centrifugal 20min of turbid solution 10000rpm removes bigger particle afterwards, and the orange red clarified solution in upper strata is with the deionized water 24h (use 25kDa bag filter) that dialyses, to remove ammonium ion excessive in the solution, free glucosan etc.By centrifugal ultrafiltration above-mentioned solution is carried out concentration more afterwards.TEM figure shows the Fe that obtains ₃O ₄Nanoparticle size is between 3-5nm, and ICP result shows that the solution concentration after concentrating is 0.602mgFe/mL.

Embodiment 2: synthetic flower shape Fe ₃O ₄@Au magnetic nanoparticle

Fe after getting that 5mL is above-mentioned and concentrating ₃O ₄Nanoparticles solution adds the 4mL deionized water, adds the NH of 200 μ L under the magnetic agitation ₂OH (w/w=1%) aqueous solution stirs 10min, adds 0.5g glucose as reducing agent, adds then the NH of 50 μ L ₄OH (7%) makes pH be about 9.0, divides afterwards the HAuCl of 4 each 100 μ L 0.25% of adding ₄The aqueous solution adds altogether 400 μ L, is spaced apart 10min between per twice, along with HAuCl ₄Adding, solution changes kermesinus into from rufous gradually.Centrifugal 6min under 7000rpm removes supernatant afterwards, and lower sediment is dissolved in the water again, afterwards through the Fe that Au is not wrapped up in 5 removals is washed in ultrasonic dispersion (80W) and centrifugation repeatedly ₃O ₄, finally being dissolved in 1% dextran solution, solution is navy blue, can keep stable in a week.TEM figure shows the similar flower of particle profile, and size distribution is (Fig. 1) between 50-70nm, EDX show in the particle ratio of Fe and Au be 1: 11 (w/w) (Fig. 2), identical with ICP result, UV-Vis is presented at the 578nm place tangible absorption peak (Fig. 3 A).

Embodiment 3: at Fe ₃O ₄@Au magnetic nanoparticle finishing biotin

Cystamine in the particle surface self assembly at first: get the above-mentioned Fe of 2mL ₃O ₄The centrifugal 6mins of@Au solution 7000rpm removes supernatant, the PBS (pH=8.0) that adds 2mL 1mM, ultrasonic limit, limit adds cystamine solution, making its ultimate density is 4mM, continue ultrasonic 10min, magnetic agitation 8h then is more centrifugal and with deionized water washing 3 times, in last ultrasonic PBS (pH=8.0) solution that is dispersed in 5mL 1mM, add the dextran solution of 200 μ L 5% to improve the stability of solution.

Connect Biotin by cystamine then: 2mg Biotin, 10mg EDC, 1mg NHS are dissolved in PBS (pH=7.4) buffer solution of 2mL 2mM, and jolting 10mins joins above-mentioned Fe then gently ₃O ₄In the@Au solution, the centrifugal 6min of 7000rpm behind the magnetic agitation 12h removes supernatant, and with deionized water centrifuge washing 3 times, is dispersed in then in the 3mL deionized water, obtains Biotin-Fe ₃O ₄@Au solution, ICP record that Fe content is 31.65 μ g/mL in the solution, and Au content is 107.47 μ g/mL, records Biotin-Fe by the Magnetic resonance imaging analyzer ₃O ₄The relaxivity R2 of@Au is 9.35mM ^-1S ^-1(Fig. 3 B)

Embodiment 4: magnetic relaxation switch analyzing and testing avidin

Earlier to above-mentioned Biotin-Fe ₃O ₄The ultrasonic dispersion of@Au solution 5min uses the membrane filtration solution of 0.22 μ m, the filter liquor that obtains namely to can be used as mother liquor again and uses.In the nuclear-magnetism pipe, add 150 μ L mother liquors, add the 0.1mg/mLAvidin solution of different amounts, add a certain amount of 2mM PBS (pH=7.4) damping fluid again, making cumulative volume is 200 μ L, and the concentration of Avidin is respectively in each nuclear-magnetism pipe at this moment: 0.00,0.15,3.79,6.06,7.58,22.73,37.88,53.03,75.76,151.52 and 227.27nM.Be placed on after shaking up in 37 ℃ of water-baths and react 5min, nuclear magnetic tube is successively placed on measures spin spin relaxation time T in the Magnetic resonance imaging analyzer afterwards ₂, obtain the T of solution under the different Avidin concentration ₂Time, each concentration determination three times is at last with T ₂To Avidin concentration mapping (Fig. 4), for the reunion that proves particle is to be interacted with the Avidin specificity rather than the non-specific adsorption of Avidin is caused, the past Fe that does not contain Biotin by Biotin ₃O ₄The Avidin that adds 0-150nM in the@Au solution measures the T of solution again ₂Time is with T ₂To Avidin concentration mapping (Fig. 4).

Embodiment 5: UV, visible light (UV-Vis) absorption spectrum and dynamic light scattering (DLS) granularmetric analysis confirmation testing result

Above-mentioned survey finishes T ₂After solution inject micro-cuvette respectively and measure ultraviolet-visible absorption spectroscopy, with deionized water as blank, analyze the peak-seeking mode that adopts, at last the absorption spectrum of each concentration correspondence is plotted among same the figure (Fig. 5), and with the absorption peak position of each absorption spectrum to Avidin concentration mapping (Fig. 5 illustration).

Measure the intensity distributions (Fig. 6 A) of grain diameter in the solution behind 10 times of above-mentioned each solution dilutions by the dynamic light scattering particle size instrument, again with the peak position of grain diameter intensity distributions in each solution to Avidin concentration mapping (Fig. 6 B) in the original solution.

Adopt detection technique of the present invention can detect the protein concentration of 10nM as model protein, detect the range of linearity between 5-35nM with Avidin.At Fe ₃O ₄@Au particle surface is modified dissimilar identification molecules and is made things convenient for manyly than modifying at common magnetic particle surface.And Fe ₃O ₄The compound probe of@Au has peculiar advantage, can introduce easily UV-Vis analysis and DLS granularmetric analysis technology testing result is confirmed mutually, can guarantee to detect higher confidence level.

Claims (6)

1. one kind based on Fe ₃O ₄The magnetic relaxation switch of@Au is characterized in that the probe that adopts is the flower shape magnetic nanoparticle that finishing has the identification molecule, and this flower shape magnetic nanoparticle is Fe ₃O ₄@Au nucleocapsid structure.

2. as claimed in claim 1 based on Fe ₃O ₄The magnetic relaxation switch of@Au is characterized in that finishing has the particle diameter of the flower shape magnetic nanoparticle probe of identifying molecule in the 50-70nm scope.

3. as claimed in claim 2 based on Fe ₃O ₄The magnetic relaxation switch of@Au is characterized in that it is Fe: Au=1 that finishing has the element mass ratio of the flower shape magnetic nanoparticle probe of identification molecule: 3.4.

4. as claimed in claim 2 based on Fe ₃O ₄The magnetic relaxation switch of@Au is characterized in that finishing has the ultraviolet and visible absorption peak of the flower shape magnetic nanoparticle probe of identifying molecule at the 578nm place.

5. as claimed in claim 2 based on Fe ₃O ₄The magnetic relaxation switch of @Au is characterized in that it is R that finishing has the relaxivity of the flower shape magnetic nanoparticle probe of identification molecule ₂=9.35mM ^-1S ^-1

As claim 1 adopt based on Fe ₃O ₄The preparation method of the magnetic relaxation switch of@Au is characterized in that concrete steps are as follows:

A. in dextran solution, synthesize diameter less than the Fe of 5nm by the method for co-precipitation ₃O ₄Particle;

B. be the Fe of 14--16mg Fe/mL at concentration of iron ₃O ₄Method by the Reduction of Glucose gold chloride in the aqueous solution is synthesized flower shape Fe ₃O ₄@Au core-shell structure magnetic nano particle;

C. at Fe ₃O ₄Cystamine in the self assembly of@Au nano grain surface, and by the upper biotin Biotin of EDC/NHS catalysis connection, obtain the magnetic nanoparticle Biotin-Fe of functionalization ₃O ₄@Au.

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