CN106093391A - A kind of based on singlet oxygen passage luminescent quantum point sensor - Google Patents

Abstract

The invention discloses a kind of based on singlet oxygen passage luminescent quantum point sensor, including the quantum dot nano hydrosol and the photosensitive microsphere of functionalization；The quantum dot nano hydrosol is will to launch the wavelength fat-soluble quantum-dot modified one-tenth water-soluble quantum dot between 520～620 nm；Water-soluble quantum dot and thiazine compounds are coated in the nano-emulsion glueballs that carboxyl, amino, hydroxyl, aldehyde radical or sulfonic group are modified and prepare quantum dot nano latex balloon；Quantum dot nano latex balloon surface modification polydextran gel is obtained the quantum dot nano hydrosol；The photosensitive microsphere of functionalization is to be coated by sensitive material phthalocyanine compound in the nano-emulsion glueballs that carboxyl, amino, hydroxyl, aldehyde radical or sulfonic group are modified, and obtains the photosensitive microsphere of functionalization.Sensor of the invention is novel homogeneous fluorescence immunoassay sensor based on singlet oxygen transmission, changes the man-to-man mode of conventional energy part, and breaks through the space detecting distance of 20nm.

Description

A kind of based on singlet oxygen passage luminescent quantum point sensor

Technical field

The present invention relates to technical field of immunoassay, in particular it relates to one is based on singlet oxygen passage luminescent quantum dot Sensor.

Background technology

Carcinoembryonic antigen (Carcinoembryonic antigen, CEA) is a kind of sugar-protein compound, and molecular weight is about About 220KDa, it be by Canada scientist Samuel O.Freedman and Phil Gold in nineteen sixty-five at colon cancer tissue Find first in the middle of extract.Typically, carcinoembryonic antigen is at fetal period, stomach intestinal tissue, pancreas and liver cell close Become, and the most substantially stop synthesis.Therefore in the middle of the serum of normal health subjects, the content of carcinoembryonic antigen maintains the lowest Level, about 2.5 μ g/mL.Generally, it is considered that the rising of CEA level is in gastric cancer, cancer of pancreas, pulmonary carcinoma, breast carcinoma, first in serum In the middle of shape gland medullary carcinoma patient relatively conventional, be also common in the middle of some non-neoplastic diseases, such as ulcerative colitis, pancreas Inflammation, liver cirrhosis, chronic obstructive pulmonary disease, Crohn disease etc..Therefore, in the middle of serum, above-mentioned disease is examined by the detection of CEA content Disconnected have important value with treatment.

The most domestic detect carcinoembryonic antigen mainly uses traditional heterogeneous analysis (such as ELISA, DIPSTICK etc.) And novel high-end quantitative immunological detection technique (such as time resolved fluoro-immunoassay TRFIA, microarray etc.).Many phases Pass technology all uses microsphere, also can accomplish the most highly sensitive qualitative and quantitative analysis, but most of microsphere has used engine dyeing Material, and mostly be solid phase or heterogeneous analysis, whole operating process step is many, needs washing to carry out separating and combining labelling with free Labelling, expends the time long, is difficult to the shortcomings such as automatization.And homogeneous fluorescent Resonance energy transfer analysis just can overcome these to lack Point, analyzes simple to operate, it is not necessary to washing separates free label, analyzes speed fast, easily realizes the advantages such as automatization, at biology Its application of analysis field is more and more extensive.

Existing homogeneous phase time discrimination fluorescence resonance energy transfer is analyzed in (HTRFA) technical system, energy donor be subject to Body is that man-to-man interaction of molecules forms energy transfer, thus realizes qualitative and quantitative analysis.Its technical bottleneck is: a pair Molecular energy Resonance energy transfer (FRET) efficiency of one is the lowest, and the energy of donor can not make full use of, and its sensitivity is by one Fixed restriction；Secondly, the energy acceptor that these analysis methods are used at present is fluorescein (fluorescein), cyanine (cyanine, cy), Ya Likesa dyestuff (Alexa), phycoerythrin (phycoerythrin, PE) or other phycoerythrin Organic dyestuff such as (allophycocyanin, APC).Tens of load has been used, it is well known, however, that there is engine dyeing despite organic dye Expecting that fatal shortcoming is exactly that the Stock displacement of spectrum is comparatively short, fluorescence emission peak is asymmetric and has substantially hangover, anti-light bleaching Property extreme difference so that bioanalysis based on fluorescent value analysis test method, especially HTRFA are the most unstable, inefficiency, in Bigger with an outer analysis deviation.Therefore, the optimum selection of organic dyestuff not HTRFA energy acceptor.And, FRET transmits distance Limit relatively big, the most not can exceed that the space length of 10nm.On the other hand, due to most of organic dyestuff fluorescence emission spectrum widths And having substantially hangover, existing HTRFA technology is suitable only for being analyzed long wavelength or near infrared band, to expensive near-infrared Sensitive detectors requires still to exist.

Summary of the invention

The present invention is in order to overcome the above-mentioned deficiency of prior art, it is provided that a kind of based on singlet oxygen passage luminescent quantum dot Sensor.

To achieve these goals, the present invention is achieved by below scheme:

A kind of based on singlet oxygen passage luminescent quantum point sensor, photosensitive with functionalization including the quantum dot nano hydrosol Microsphere；The preparation process of the described quantum dot nano hydrosol is as follows: will launch the wavelength fat-soluble amount between 520～620nm Son point is modified to water-soluble quantum dot；The water-soluble quantum dot of preparation, thiazine compounds are coated into carboxyl, amino, hydroxyl, aldehyde The nano-emulsion glueballs that base or sulfonic group are modified prepares quantum dot nano latex balloon；Quantum dot nano latex balloon is carried out water-soluble The non-specific process of glue, obtains the quantum dot nano hydrosol by quantum dot nano latex balloon surface modification polydextran gel；Described The preparation method of the photosensitive microsphere of functionalization is: be coated sensitive material phthalocyanine compound into carboxyl, amino, hydroxyl, aldehyde radical or sulphur In the nano-emulsion glueballs that acidic group is modified, obtain the photosensitive microsphere of functionalization.

Sensor of the invention is different from traditional FRET (fluorescence resonance energy transfer) and luminous, is mainly based upon singletstate activity Oxygen and luminous.Sensor of the invention is based on singlet oxygen channel energy Transfer Technology and the quantum dot nano hydrosol (TQPS) The use in conjunction of internal FRET (fluorescence resonance energy transfer).

Preferably, nano-emulsion glueballs when preparing quantum dot nano-sized hydrosol is polystyrene-divinylbenzene carboxylic acid Modified latex, the polystyrene microsphere that nano-emulsion glueballs is surface carboxyl groups during the preparation photosensitive microsphere of functionalization.Nano-emulsion Glueballs has characteristics that surface has porous, carboxyl compound is contained on surface, optics permeability, even particle size distribution.

Fat-soluble quantum dot refers to that quantum dot can be dissolved in the organic solvents such as decane, hexane, chloroform.Preferably, Described fat-soluble quantum dot be cadmium selenide, cadmium sulfide or cadmium telluride be the various semiconductor nanocrystals of core.As the case may be Select the quantum dot of required wavelength, as long as its transmitting wavelength is between 520～620nm, be suitable for the present invention.The biology of quantum dot Application emerges in an endless stream, and because it is anti-light Bleachability extremely strong, brightness is high, and beam of laser light source can excite the quantum of different colours simultaneously Point, and it is amenable to the repeatedly exciting irradiation of laser and fluorescence intensity is without significant change；Additionally, the spectrum Stock position of quantum dot Moving relatively wide, fluorescence emission peak is narrow and symmetrical, and without hangover, this characteristic makes quantum dot nano latex balloon can meet this immunoassay Method sensitivity and precision requirement, it is not necessary to expensive red responsive detectors part.Maximum half peak width of quantum dot fluorescence emission spectrum Narrow, the quantum dot of different wave length can use the characteristics such as same excitation, therefore, have emission peak 525,565 or Its fluorescence emission spectrum of 605nm is overlapping with thiazine compounds fluorescent emission less, and mole excitation spectrum of these quantum dots Overlapping with thiazine compounds fluorescent emission more, it is best suitable for realizing homogeneous fluorescent Resonance energy transfer between thiadiazide and divides Analysis.

Maximum half peak width of quantum dot owing to directly preparing from aqueous solution is generally large, close to 60nm, is not suitable for this Bright CEA analyzes.Therefore, the present invention uses fat-soluble quantum dot, and then modification becomes water-soluble quantum dot.Due to the only amount of being by , there is not the impact on luminescent core material in son point surface modification, the modified fluorescent emission peak position obtained and particle diameter etc. with Oil-soluble compares no significant difference；Method of modifying is simple, workable；

Preferably, the method for described fat-soluble quantum-dot modified one-tenth water-soluble quantum dot is: stabilizer is joined dissolving In the fat-soluble quantum dot of chloroform, mix homogeneously；Add the sodium hydroxide solution oscillation incubation of 0.1M, preserve under room temperature 1～3 hour；Add deionized water and acetone, precipitate, be centrifuged, dissolve and i.e. can get water-soluble quantum dot.

Preferably, described stabilizer is dimercaptosuccinic acid, glutathion or TGA.

It is highly preferred that the method for described fat-soluble quantum-dot modified one-tenth water-soluble quantum dot is: by 25mg dimercaptosuccinic acid Stabilizer joins and is dissolved in CdSe quantum dots in chloroform (1～5 μM) 500 μ L, mix homogeneously, adds the hydrogen-oxygen of 0.1M Change sodium solution 10～100 μ L, oscillation incubation, preserve 1～3 hour under room temperature.Add 400 μ L deionized waters, and 1.8mL acetone, Precipitation, centrifugal, it is dissolved in the deionized water of 0.5mL, i.e. can get water-soluble quantum dot.

Preferably, use before water-soluble quantum dot prepares quantum dot nano-emulsion glueballs, it is possible to use trioctylphosphine oxide (TOPO), just Cetylamine or other carbochains normal alkane modified water-soluble quantum dot surface more than five so that it is surface has hydrophobic and hydrophilic pair Can property group.The method of pretreatment is: joined in water-soluble quantum dot by trioctylphosphine oxide (TOPO)；It is heated to 60 DEG C, is stirred vigorously, It is allowed to be completely dissolved, is cooled to room temperature；Successively add benzyl alcohol, ethylene glycol and the sodium hydroxide solution stirring that concentration is 0.1M is mixed Close uniformly.

In sensor, the quantum dot nano hydrosol is energy acceptor, and the photosensitive microsphere of functionalization is energy donor.Donor be subject to The remote energy up to 200nm can be realized between body shift.

Comprise in quantum dot nano latex balloon and there is chemical reagent, the quantum dot reacted with singlet oxygen.Chemical reagent refers to Be energy acceptor, it comprises thiazine compounds or other have when can react with singlet oxygen and discharge 200～400nm fluorescence Chemical reagent, after the chemical reagent such as thiazine compounds and singlet oxygen generation chemical reaction, the fluorescence energy discharged and quantum dot FRET (fluorescence resonance energy transfer) can occur.

Preferably, the preparation method of described quantum dot nano latex balloon is particularly as follows: add three pungent in water-soluble quantum dot Base phosphine, heating, stir, then modify with sweller, thiazine compounds and carboxyl, amino, hydroxyl, aldehyde radical or sulfonic group Nano-emulsion glueballs mix homogeneously, in 110～120 DEG C of high-temperature hot swelling 5～30min, is cooled to room temperature, and high speed centrifugation precipitation is washed Wash, obtain quantum dot nano latex balloon.

It is highly preferred that described sweller is benzyl alcohol (or other solvents such as chloroform, acetone, ethoxy ethanol), goes Ionized water and the mixture of ethylene glycol, the volume ratio of three is 1:(0.8～1): (8～8.2).Under hot sweller is protected, carboxylic The nano-emulsion glueballs surface that base, amino, hydroxyl, aldehyde radical or sulfonic group are modified at high temperature expands, and will not be made by excessive tearing Become surface functional group skewness, the problem overcoming quantum dot fluorescence cancellation, keep it to be coated front due quantum yield, and have Effect prevents precipitation from assembling, and is greatly improved the stability of quantum dot nano latex balloon.

Preferably, the concentration of described water-soluble quantum dot is generally 0.1～10 μM/mL.Carboxyl, amino, hydroxyl, aldehyde radical or The nano-emulsion glueballs that sulfonic group is modified is generally 1～200mg/mL, and particle diameter is generally 20nm～3 μm, it is further preferred that particle diameter is 20nm ～300nm.It addition, preferably, the particle diameter≤preparation of nano-emulsion glueballs during preparation photosensitive microsphere of functionalization The particle diameter of the nano-emulsion glueballs during quantum dot nano hydrosol.A large amount of carboxyl compound is contained on latex balloon surface, and its carboxyl is close Degree contains 5～12 for every square nanometers, and the latex balloon of this specification is to have passed through special handling, is different from general carboxylated gather Phenylethylene micro ball, surface has micropore can be had the nano-particle of double-functional group by the swollen containing of thermosol, and this latex balloon hydrophilic is relatively By force, there is the strongest optics permeability, be used directly for conjugated biological molecules or surface processes, be suitable for doing clinical diagnosis and divide Analysis.The method of the invention, it is recommended that directly use CPS latex balloon, it is not necessary to use surface micro-with the polystyrene of one layer of carboxyl Ball, also need not carry out the carboxylated or grafting of other groups by water-insoluble polystyrene microsphere.The present invention uses the double of gentleness Function organic reagent, at a temperature of required by most of bioanalysiss, i.e. embeds at 110～120 DEG C of swelling latex balloons Water-soluble quantum dot.Temperature required by most of bioanalysiss is below hot swelling temperature, produces in can avoiding using Leakage problem.Although the distribution of sizes slight variation of nano-emulsion glueballs after being coated quantum dot, but do not interfere with quantum dot nano breast The biologic applications of glueballs.

Preferably, the concrete preparation method of the described quantum dot nano hydrosol is: glycosaminoglycan is dissolved in MES buffering In liquid, regulation pH is to be added dropwise in quantum dot nano latex balloon after 6.0；Stirring adds EDAC solution and MES buffer, room temperature Under continuously stirred 2 hours；Add ethanolamine and hatch that 30min, 17000rmp are centrifugal discards supernatant；Precipitate is dissolved in deionization In water, supersound process, repeats this step 3 time, to obtain final product.

Glucosan is that biological field is widely used a kind of hydrosol.The amination dextran molecule amount purchased is 100KDa, every 7 glucan unit contain 1～2 amino；Coupling agent is that 1-(3-dimethylamino-propyl)-3-ethyl carbon two is sub- Amine hydrochlorate (EDAC) and N-hydroxy thiosuccinimide (Sulfo-NHS), it is possible to use other can be by carboxyl and amino The coupling agent of coupling, the reaction condition also respective change such as corresponding buffer solution；After coupling, quantum dot nano latex balloon institute Surplus carboxyl or amino can continue conjugated biological molecules；Additionally, the glucosan on TQPS top layer greatly prevents the leakage of quantum dot, increase Strong himself stability and bio-compatibility.The TQPS prepared by the present invention is as the important set of detection CEA antigen sensor Becoming part, substantial amounts of hydroxyl, carboxyl and amido functional group are contained in its surface, and energy single dispersing, in biology buffer, overcomes Self, because the high sedimentation problem caused of the negative charge density of carboxyl, also solves the non-specific adsorption to biomolecule and asks Topic.

The TQPS of described sensor and coupled antibody, be by covalent bond effect, coupling agent EDAC's and Sulfo-NHS Under effect, by monoclonal antibody S001 of CEA and TQPS surface carboxyl groups phase coupling, prepare for homogeneous immunity point The functional TQPS of analysis.According to the difference of analysans, other albumen, or end can also be used with the antibody of TQPS coupling With amino or the nucleic acid of carboxyl or peptide chain.

At described 605nm, fluorescence signal comes from TQPS.When near-infrared laser excitation-detection sample, in SA-PS BTHS discharges singletstate active oxygen because absorbing photosensitive energy.This active oxygen is the most diffusible and in the range of penetrating into 200nm TQPS, then with TQPS contained by thiazine compounds react release short wavelength's fluorescence (320～400nm), and with the quantum dot of surrounding There is FRET (fluorescence resonance energy transfer), so that TQPS discharges the fluorescent assay signal of 605nm.

Sensor of the present invention also needs to preparation and the functionalization producing singlet oxygen can be excited by near infrared light photosensitive micro- Ball, its preparation method is: join in ethylene glycol by the nano-emulsion glueballs that carboxyl, amino, hydroxyl, aldehyde radical or sulfonic group are modified, Take phthalocyanine compound to be dissolved in benzyl alcohol, after the two mixing, add deionized water, mix latter 110 DEG C and react 10 minutes, be cooled to Adding ethanol after room temperature, size selectivity is centrifuged.

Described sensitive material phthalocyanine compound is phthalocyanine compound commonly used in the art, such as two silicon hydroxides 2,9,16,23- Tetra-tert-29H, 31H-phthalocyanine, No. CAS: 85214-70-6.

Sensitive material phthalocyanine compound (BTHS) is irradiated by near-infrared laser, discharges substantial amounts of singletstate active oxygen. Having document to report (Macromolecules 25,3399-3405), these singletstate active oxygens can be at general water-soluble medium Middle diffusion, its distance about 200nm, and can penetrate into polystyrene microsphere about, its efficiency reaches 20%.Novel sensing Device, based on this singlet oxygen passage Transfer Technology, the anti-light Bleachability and specific optical character of incorporating quantum point, carries significantly High can transfer efficiency, overcome the spatial obstacle that energy is shifted by hydrosol surface group；Meanwhile, because there is multiple knot on surface Close site can carry out singlet oxygen transmission to light-induced chemiluminescent quantum dot simultaneously, substantially increase the sensitivity of detection.

Described homogeneous fluorescent analysis based on singlet oxygen passage luminescent quantum point sensor application, refers to utilize list In the immunoassay serum that between line state oxygen receiving terminal thiazine compounds and quantum dot, FRET (fluorescence resonance energy transfer) principle is carried out Carcinoembryonic antigen content.In this analysis method, use double-antibody sandwich one-step method, energy acceptor and donor are coupled to antigen In two monoclonal antibodies, by the immunoreation of antibody Yu antigen, energy acceptor TQPS is close with donor SA-PS, work as sense When light microsphere SA-PS and quantum dot nano hydrosol TQPS distance are less than 200nm, oxygen signal can be occurred to transmit, then excite FRET (fluorescence resonance energy transfer) (as shown in Figure 2) between QDS and thiazine compounds in TQPS.

In TQPS, according to FRET (fluorescence resonance energy transfer) principle, need to realize illuminating antenna material thiazine compounds and amount FRET (fluorescence resonance energy transfer) between son point, and Resonance energy transfer to be realized needs emission spectrum and the quantum dot of antenna material Excitation wavelength coupling (as shown in Figure 3).According to select luminescent material thiazine compounds luminescent spectrum and quantum dot mole Extinction spectra, Theoretical Calculation spectra overlapping is:

$J\left(\mathrm{\λ}\right)=\frac{\underset{0}{\overset{\mathrm{\∞}}{\∫}}{\mathrm{\ϵ}}_A\left(\mathrm{\λ}\right)\·I_D\left(\mathrm{\λ}\right)\·{\mathrm{\λ}}^4\·d\mathrm{\λ}}{\underset{0}{\overset{\mathrm{\∞}}{\∫}}{I}_D\left(\mathrm{\λ}\right)\·d\mathrm{\λ}}$

Here A represents quantum dot energy acceptor, and D represents illuminating antenna material.ε_AFor the excitation spectrum function of quantum dot, λ For wavelength, I_DFluorescence emission spectral function for energy donor.Calculate J (λ)=2.86 × 10¹⁶。

Illuminating antenna material and quantum dot are close needsDistance Theory formula:

R₀=(8.79 × 10^-5·κ²·Φ_D·n^-4·J(λ))^1/6

Wherein, Φ_DFor the quantum yield (2.5%) of illuminating antenna material, к²Being generally 2/3 for kinetic parameter, n is molten Liquid refractive index.It is calculated illuminating antenna material and quantum dot generation FRET (fluorescence resonance energy transfer) by this formulaDistance For 2.3nm, this distance is that the average distance of 50% energy transfer efficiency occurs between illuminating antenna material and quantum dot.

And transferring efficiency of fluorescence resonance energy:

$E=\frac{R_0^6}{R_0^6+r^6}$

Wherein r is the space length of energy donor and receptor；In actual application, energy transfer efficiency E is generally 1.5%- 98.5% for effectively to shift scope, and i.e. can get illuminating antenna material with quantum dot distance is 1.6nm < R < 4.6nm.Therefore, exist On space structure, the thiazine compounds used in the present invention and quantum dot are by being simply embedded in polystyrene microsphere, in theory On meet the condition of fluorescence energy transfer.

Because mole excitation spectrum of red quantum point (fluorescence emission peak 605nm, quantum yield is 48%) and thiazine The emission spectrum of compound has greater overlap, Streptavidin and the monoclonal antibody of CEA on photosensitive microsphere and TQPS labelling respectively S001 forms the quantum dot sensor luminous based on singlet oxygen passage, and another strain monoclonal antibody 6F11 of CEA is carried out Biotin labeling.Therefore, when sending out based on singlet oxygen passage luminescent quantum point sensor, biotinylation 6F11 and CEA antigen After raw immunoreation, photosensitive microsphere is furthered with TQPS, and now under the irradiation of excitation source, photosensitive microsphere makes the oxygen of periphery become Becoming the singlet oxygen of upper state, singlet oxygen is delivered to, inside the quantum dot nano hydrosol, then excite thiazine compounds and amount FRET (fluorescence resonance energy transfer) between son point, quantum dot sends signal fluorescence (as shown in Figure 3).When two monoclonal antibodies relatively antigen During CEA excess significantly, the FRET (fluorescence resonance energy transfer) signal detected is by relation (as shown in Figure 4) proportional to the amount of antigen.

Compared with prior art, there is advantages that

The sensor that the present invention provides is novel homogeneous fluorescence immunoassay sensor based on singlet oxygen transmission, and it changes Become the man-to-man mode of prior art energy ligand, and break through the space detecting distance of 20nm.It addition, singlet oxygen passage Luminescent quantum dot is the energy acceptor of a kind of optical characteristics with novelty: the Stock displacement of its spectrum is relatively wide, fluorescent emission Peak is symmetrical and trails without obvious, it is often more important that anti-light Bleachability extremely strong, and this energy acceptor does not relies on the red quick of costliness Detector.This kind of sensor, it is not necessary to rinse excess label, in homogeneous, reaction and detection, be suitable for macromole immunoglobulin Or the detection of the biomolecule such as small haptens, immunologic diagnosis field has potential huge advantage in vitro.

The present invention prepares multifunctional groups originally in singlet oxygen passage luminescent quantum point sensor.This sensor is used TQPS has good water solubility, even size distribution, quantum yield advantages of higher, improves stability and the sensitivity of analysis, also Overcome the problems such as non-specific adsorption, innovatively quantum dot is being combined with oxygen Transfer Technology.Use multifunctional groups in The novel homogeneous oxygen transmission FRET (fluorescence resonance energy transfer) of singlet oxygen passage luminescent quantum point sensor analyzes method, not only can carry High-energy transfer efficiency thus improve sensitivity for analysis, it is also possible to reduce the dependence to expensive red quick photon detector, facing The bed biochemical analysis such as molecular diagnosis and food inspection has very important significance.

Accompanying drawing explanation

Fig. 1 is the grain size distribution of (CPS) (QPS) afterwards before nano-sized hydrosol functionalization.

Fig. 2 is based on singlet oxygen passage luminescent quantum point sensor homogeneous analysis schematic diagram.

Fig. 3 is mole excitation spectrum and the thiazine compounds fluorescence emission spectrum figure of water-soluble quantum dot.

Fig. 4 is the Preliminary Applications analyzing CEA based on singlet oxygen passage luminescent quantum point sensor homogeneous immuno-sandwich method.

Detailed description of the invention

Elaborating the present invention further below in conjunction with Figure of description and specific embodiment, described embodiment is only For explaining the present invention, it is not intended to limit the scope of the present invention.Polystyrene-divinyl base used in following embodiment Benzene carboxylic acid modified latex is purchased from Thermo Fisher Scientific company (article No. 8300-0520100390), fat-soluble amount Sub-point (article No. Q21701MP) is purchased from chemistry such as Invitrogen company, coupling agent EDAC, Sulfo-NHS and trioctylphosphine oxide (TOPO)s It is anti-that reagent is purchased from Sigma-Aldrich, used Cea Monoclonal Antibodies S001 and 6F11 and cancer embryo Proantigen standard substance are purchased from Da An genome company of Zhongshan University, and other chemical reagent are purchased from Chemical Reagent Co., Ltd., Sinopharm Group

Embodiment 1

The core technology of this step is, obtains that quantum yield is high, maximum half peak width of fluorescence emission peak is narrow and symmetry water-soluble Property quantum dot.What the present invention was embodied as using is the core/shell type cadmium selenide/zinc sulfide buied from American I nvitrogen company Quantum dot, its solvent is decane, its wave spectrum Stock shift length from 592nm toward in the relative broad range between short wavelength, therefore ripple Length is shorter than 592nm and all can excite this quantum dot；Fluorescence emission peak is 605 ± 0.5nm, and maximum half peak width is 27 ± 1.5nm, quantum Productivity 76%；The quantum dot of such optical characteristics, receives " antenna " thiazine compounds fluorescence emission spectrum relatively with singlet oxygen Join, the most all overcome the deficiency of organic dyestuff, be suitable for replacement, to reduce measurement error, improve Fluorescence Resonance Energy Transfer (FRET) efficiency, thus improve sensitivity for analysis.

A kind of based on singlet oxygen passage luminescent quantum point sensor, photosensitive with functionalization including the quantum dot nano hydrosol Microsphere；The preparation method of quantum dot nano hydrosol microsphere photosensitive with functionalization is as follows:

One, the preparation of high performance water-soluble quantum dot: be placed in centrifuge tube by the fat-soluble quantum dot that 50 μ L buy, adds The absolute methanol of 200 μ L；Mix homogeneously, seals centrifuge tube, at 3000rpm, 4 DEG C of centrifugal 5min；Discard supernatant, add 50 μ L Chloroform, fully dissolves；Adding 25mg dimercaptosuccinic acid stabilizer and the sodium hydrate aqueous solution 20 μ L of 0.1M, mixing is all Even, oscillation incubation on shaking table, preserve 3 hours under room temperature；Add 100 μ L deionized water and 1.8mL acetone, precipitation, 6000rpm Centrifugal 5min；It is dissolved in the deionized water of 0.2mL, obtains water-soluble quantum dot stand-by.The water-soluble quantum dot obtained uses Its emission spectrum analyzed by spectrofluorophotometer, its fluorescence emission peak 605nm.

Two, preparation based on singlet oxygen passage luminescent quantum dot nano-emulsion glueballs (TQPS):

1, the pretreatment of nano-emulsion glueballs (CPS): by 100 μ L polystyrene-divinylbenzene carboxyl acid modified latex (particle diameters 205 ± 3nm, mass concentration is 100mg/mL, and solid concentration is 10%) in 14000rmp, 4 DEG C are centrifuged；Abandoning supernatant, lays equal stress on It is suspended from 100 μ L deionized waters；

2, the pretreatment of water-soluble quantum dot: the trioctylphosphine oxide (TOPO) of 2mg is joined the water solublity that above-mentioned steps one prepares In quantum dot；It is heated to 60 DEG C, is stirred vigorously, be allowed to be completely dissolved, be cooled to room temperature；Successively add 2mL benzyl alcohol, 2.8mL Ethylene glycol and the sodium hydroxide solution that 100 μ L concentration are 0.1M are uniformly mixed；

3, then the quantum dot aqueous solution in step 2 and thiazine compounds (4mg is dissolved in the benzyl alcohol of 100 μ L) are dripped Add in the nano-emulsion glueballs of step 1, at 105 DEG C, be stirred vigorously 15 minutes；It is cooled to room temperature, sinks with the speed of 12000rpm Form sediment centrifugal 25 minutes；It is dissolved in 2-(N-quinoline) ethanesulfonic acid buffer (MES, 50mM, pH6) of 200 μ L；The high-performance obtained is water-soluble Property quantum dot nano latex balloon (QPS), its distribution of sizes is as it is shown in figure 1, square and triangle solid line represent package amount respectively Nano-emulsion glueballs distribution of sizes situation before and after son point.The preparation method of described thiazine compounds is as follows: by 10～15g to bromobenzene Amine solvent, in 100mL dimethyl sulphoxide solution, is separately added into 1-bromine n-tetradecane and 30mL N, the N '-diisopropyl of 40mL Ethamine.Under nitrogen protection, reactant liquor continues to be heated to 90 DEG C, is cooled to room temperature after reacting 16 hours.Again add toward reactant liquor Enter 20mL1-bromine n-tetradecane and 15mL N, N '-diisopropylethylamine.It is little that reaction mixture is again heated to 90 DEG C of reactions 15 Time.Reactant liquor is cooled to room temperature, is vacuum dried and by debris 200mL dchloromethane. use the NaOH (2 of 1N respectively ×) aqueous solution, water and saline extracting and washing, finally use Na₂SO₄It is dried.Reactant liquor is concentrated to give brownish black oil and is about 55g.Make Preparing liquid phase separation with Agilent 1200HPLC, flowing is normal hexane mutually, obtains yellow oil, and wherein great majority are product 4- Bromo-N, N '-dimethyl-to aniline and a small amount of 1-bromomethane. the latter by decompression distillation (bp 105～110 DEG C, 0.6mmH) can remove, finally give target product brown oil.

Three, the preparation of TQPS: TQPS can be modified by quantum dot nano-sized hydrosol surface and prepare, and its step is as follows: Weigh in the MES buffer that 10mg glycosaminoglycan is dissolved in 100 μ L, with 1M hydrochloric acid, pH is transferred to again 6.0；It is added dropwise to In quantum dot nano latex balloon (QPS) in above-mentioned steps two, stir with 300rmp speed；Add the EDAC solution of 300 μ L (80mg/mL) and the MES buffer of 1.4mL, room temperature with constant stirs 2 hours；Add the ethanolamine 700 μ L of 1mM, hatch 30min； It is centrifuged under 17000rmp rotating speed, discards supernatant, precipitate is dissolved in 20 μ L deionized waters, supersound process；Repeat with Upper step 3 time, finally obtains quantum dot nano hydrosol emulsion ball (TQPS).

Four, the preparation of functional photosensitive microsphere:

The polystyrene microsphere of 0.1mL surface carboxyl groups is joined in 0.7mL ethylene glycol, takes 5mg BTHS phthalocyanine compounds Thing is dissolved in 0.1mL benzyl alcohol, adds 0.1mL deionized water after both mixing, is fully warmed up to 110 DEG C after mixing, reaction 10 minutes, adding ethanol after being cooled to room temperature, size selectivity is centrifuged.By photonasty donor microsphere (0.1g) collected with 10% ethanol preserves stand-by.

Embodiment 2

A kind of based on singlet oxygen passage luminescent quantum point sensor, photosensitive with functionalization including the quantum dot nano hydrosol Microsphere；The preparation method of quantum dot nano hydrosol microsphere photosensitive with functionalization is as follows:

One, the preparation of high performance water-soluble quantum dot: 50 μ L fat-soluble quantum dot amount is placed in centrifuge tube, adds 200 μ The absolute methanol of L；Mix homogeneously, seals centrifuge tube, at 3000rpm, 4 DEG C of centrifugal 5min；Discard supernatant, add 40 μ L trichlorines Methane, fully dissolves；Add 21mg glutathion stabilizer and the sodium hydrate aqueous solution 10 μ L of 0.1M, mix homogeneously, shaking Oscillation incubation on bed, preserves 2 hours under room temperature；Adding 100 μ L deionized water and 1.8mL acetone, precipitation, 6000rpm is centrifuged 5min；It is dissolved in the deionized water of 0.5mL, obtains water-soluble quantum dot.The water-soluble quantum dot fluorescence spectrophotometer light prepared Degree meter analyzes its emission spectrum, its fluorescence emission peak 605nm.

Two, preparation based on singlet oxygen passage luminescent quantum dot nano-emulsion glueballs (TQPS):

Three, the preparation of TQPS:

Four, the preparation of functional photosensitive microsphere:

Application examples

Based on singlet oxygen passage luminescent quantum point sensor in homogeneous oxygen transmission FRET (fluorescence resonance energy transfer) is analyzed Application, singlet oxygen passage luminescent quantum point sensor homogeneous analysis principle is as it is shown on figure 3, the step of application is as follows:

One, TQPS and anti-carcinoembryonic antigen monoclonal antibody coupling, step is as follows: the MES buffer of preparation 0.5M, and pH modulates 6.1, take 100 μ L stand-by；With the Sulfo-NHS solution of deionized water preparation 50mg/mL, take 230 μ L stand-by；Join with deionized water The EDAC solution of 50mg/mL processed, takes 240 μ L stand-by；Above stand-by solution is mixed, is settled to 1mL with deionized water, in addition State in TQPS prepared by step 3, resuspended；Concussion reaction 30 minutes under mixture room temperature；16000rmp rotating speed is centrifuged, and removes not Sulfo-NHS and EDAC of reaction, makes to be washed with deionized, resuspended, centrifugal, in triplicate；After the resuspended activation of deionized water TQPS, final mass concentration is 1%, and volume is 1mL；Take monoclonal antibody S001 (1mg/mL) that 500 μ L add 500 μ L； Concussion is hatched 2 hours gently, adds 1.5 μ L ethamine alcohol, and room temperature concussion hatches 30 minutes；Centrifuge washing removes the albumen of non-coupling Matter and ethamine alcohol；Adding the phosphate buffer (wherein containing 0.05%Proclin-300) of the 50mM of 1mL, obtaining coupling has list The quantum dot nano hydrosol emulsion ball of clonal antibody S001, and its concentration is adjusted to 100 μ g/mL, its mole excites Spectrum is as shown in Monas cuspurpureus Went line on the left of Fig. 2.

Two, photosensitive microsphere and the coupling of Streptavidin, by EDAC and Sulfo-NHS, the donor microsphere being prepared into Streptavidin in the coupling of surface, and its concentration is adjusted to 16 μ g/mL.

Three, the biotinylation labelling of anti-carcinoembryonic antigen monoclonal antibody 6F11: antibody is molten according to sigma company description Liquid and biotin solution volume ratio are that 10:1 fully mixes, and after shaking at room temperature hatches 4 hours, use Millipore company of the U.S. The centrifuge tube with filter membrane remove unnecessary biotin, and antibody concentration is adjusted to 5 μ g/mL.

Four, CEA psma ligand is made 0ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, 100ng/mL, 500ng/mL series Concentration；Capillary strip is sequentially added into 25 μ L testing samples, 25 μ μ L biotinylated antibodies, the 25 μ L quantum dot nano hydrosols, sticks on Mounting；Micropore reaction bar hatches 15 minutes 37 DEG C of constant temperature oscillation instrument internal vibrations；Streptavidin is added under the conditions of lucifuge Donor microsphere 175 μ L；Micropore reaction bar hatches 15 minutes 37 DEG C of constant temperature oscillation instrument internal vibrations；In PerkinElmer company Detected signal value on 2300EnSpireTM detector.The logarithm of reference standard product concentration is abscissa, the logarithm of signal value counting For vertical coordinate, double-log mathematical model Log-Log function process, record CEA test kit dose-response curve linear correlation system Number is r=0.9918, as shown in Figure 3.

Carrying out after above detection two hours in, carried out identical fluoroscopic examination every 10 minutes.All samples Result shows, the TQPS of use is anti-light Bleachability extremely strong, and standard concentration is the most overlapping with fluorescent value double-log graph of a relation.

Using zero reference standard product (A point) as specimen repeated measure 20 times, calculate its fluorescence average χ and standard deviation SD, χ+ The fluorescent value of 2SD gained substitutes into standard curve Equation for Calculating and draws its sensitivity, and the lowest detection lower limit i.e. detecting CEA is 0.7ng/mL。

Do the reaction of CEA sample cross survey with hemoglobin, triglyceride, bilirubin, the equal no cross reaction of result.

Claims (8)

1. one kind based on singlet oxygen passage luminescent quantum point sensor, it is characterised in that include the quantum dot nano hydrosol with The photosensitive microsphere of functionalization；The preparation process of the described quantum dot nano hydrosol is as follows: will launch wavelength between 520～620 nm Fat-soluble quantum-dot modified one-tenth water-soluble quantum dot；The water-soluble quantum dot of preparation, thiazine compounds are coated into carboxyl, ammonia The nano-emulsion glueballs that base, hydroxyl, aldehyde radical or sulfonic group are modified prepares quantum dot nano latex balloon；To quantum dot nano latex Ball carries out the non-specific process of the hydrosol, and quantum dot nano latex balloon surface modification polydextran gel is obtained quantum dot nano water Colloidal sol；The preparation method of the photosensitive microsphere of described functionalization is: sensitive material phthalocyanine compound is coated into carboxyl, amino, hydroxyl, In the nano-emulsion glueballs that aldehyde radical or sulfonic group are modified, obtain the photosensitive microsphere of functionalization.

Sensor the most according to claim 1, it is characterised in that described fat-soluble quantum dot be cadmium selenide, cadmium sulfide or Cadmium telluride is the various semiconductor nanocrystals of core.

Sensor the most according to claim 1, it is characterised in that described fat-soluble quantum-dot modified one-tenth water-soluble quantum dot Method be: stabilizer is joined in the fat-soluble quantum dot being dissolved in chloroform, mix homogeneously；Add the hydrogen-oxygen of 0.1M Change sodium solution oscillation incubation, preserve 1～3 hour under room temperature；Add deionized water and acetone, precipitate, be centrifuged, dissolving the most available Water-soluble quantum dot.

Sensor the most according to claim 3, it is characterised in that described stabilizer be dimercaptosuccinic acid, glutathion or TGA.

Sensor the most according to claim 1, it is characterised in that the preparation method of described quantum dot nano latex balloon is concrete For: in water-soluble quantum dot add tri octyl phosphine, heating, stir, then with sweller, thiazine compounds and carboxyl, The nano-emulsion glueballs mix homogeneously that amino, hydroxyl, aldehyde radical or sulfonic group are modified, in 110～120 DEG C of high-temperature hot swelling 5～ 30min, is cooled to room temperature, high speed centrifugation washing of precipitate, obtains quantum dot nano latex balloon.

Sensor the most according to claim 1 or 5, it is characterised in that the preparation method of described thiazine compounds is: by right Bromaniline is dissolved in dimethyl sulphoxide solution, is separately added into 1-bromine n-tetradecane and N, N '-diisopropylethylamine, protects at nitrogen Protect down, be heated to 90 DEG C, after reacting 16 hours, be cooled to room temperature；1-bromine n-tetradecane and N, N '-two is again added toward reactant liquor Wopropyl ethyl amine, is again heated to 90 DEG C and reacts 15 hours；Reactant liquor is cooled to room temperature, is vacuum dried and by debris with two Chloromethanes dilutes, and uses NaOH (2 ×) aqueous solution, water and saline extracting and washing respectively, finally uses Na₂SO₄It is dried；Reactant liquor Being concentrated to give brownish black oil, use HPLC to prepare liquid phase separation, flowing is normal hexane mutually, obtains yellow oil, and decompression is distilled off Impurity, obtains brown oil product.

Sensor the most according to claim 1, it is characterised in that the concrete preparation method of the described quantum dot nano hydrosol For: glycosaminoglycan being dissolved in MES buffer, regulation pH is to be added dropwise in quantum dot nano latex balloon after 6.0；Stirring Adding EDAC solution and MES buffer, room temperature with constant stirs 2 hours；It is centrifugal that addition ethanolamine hatches 30min, 17000rmp Discard supernatant；Precipitate is dissolved in deionized water, supersound process, repeats this step 3 time, to obtain final product.

Sensor the most according to claim 1, it is characterised in that the preparation method of the photosensitive microsphere of described functionalization is: will The nano-emulsion glueballs that carboxyl, amino, hydroxyl, aldehyde radical or sulfonic group are modified joins in ethylene glycol, takes phthalocyanine compound and is dissolved in benzene In methanol, after the two mixing, add deionized water, mix latter 110 DEG C and react 10 minutes, after being cooled to room temperature, add ethanol, size Selectivity is centrifuged.