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

Abstract

The invention discloses a luminescent quantum dot sensor based on a singlet oxygen channel, comprising quantum dot nano hydrosol and functionalized photosensitive microspheres; the quantum dot nano hydrosol is a fat-soluble quantum dot with an emission wavelength between 520 and 620 nm Modified into water-soluble quantum dots; water-soluble quantum dots and thiazine compounds are coated into nano-emulsion balls modified by carboxyl, amino, hydroxyl, aldehyde or sulfonic acid groups to prepare quantum dot nano-emulsion balls; the quantum dot nano-emulsion The sphere surface is modified with dextran gel to obtain quantum dot nano-hydrosol; the functionalized photosensitive microsphere is to coat the photosensitive material phthalocyanine compound into the nano-emulsion ball modified by carboxyl, amino, hydroxyl, aldehyde or sulfonic acid group to obtain Functionalized photosensitive microspheres. The sensor of the present invention is a novel homogeneous fluorescent immunoassay sensor based on singlet oxygen transfer, which changes the traditional one-to-one mode of energy ligands, and breaks through the spatial detection distance of 20nm.

Description

A Luminescent Quantum Dot Sensor Based on Singlet Oxygen Channel

technical field

The invention relates to the technical field of immune detection, in particular to a singlet oxygen channel-based luminescent quantum dot sensor.

Background technique

Carcinoembryonic antigen (CEA) is a glycoprotein complex with a molecular weight of about 220KDa. It was first discovered in colon cancer tissue extracts by Canadian scientists Samuel O. Freedman and Phil Gold in 1965. Generally speaking, carcinoembryonic antigen is synthesized by gastrointestinal tract tissue, pancreas and liver cells during fetal period, and basically ceases to be synthesized after birth. Therefore, the content of carcinoembryonic antigen in the serum of normal healthy individuals is maintained at a very low level, about 2.5 μg/mL. It is generally believed that elevated levels of CEA in serum are more common in patients with gastric cancer, pancreatic cancer, lung cancer, breast cancer, and medullary thyroid cancer, and are also common in some non-neoplastic diseases, such as ulcerative colitis, pancreatitis, liver Cirrhosis, chronic obstructive pulmonary disease, Crohn's disease, etc. Therefore, the detection of CEA content in serum has important value for the diagnosis and treatment of the above diseases.

At present, the detection of carcinoembryonic antigen in China mainly adopts traditional heterogeneous analysis (such as ELISA, DIPSTICK, etc.) and new high-end quantitative immunological detection techniques (such as time-resolved fluorescent immunoassay TRFIA, microarray, etc.). Many related technologies use microspheres, which can also achieve high-sensitivity qualitative and quantitative analysis. However, most microspheres use organic dyes, and most of them are solid-phase or heterogeneous phase analysis. The entire operation process has many steps. Washing is required to separate the bound label from the free label, which takes a long time and is not easy to automate. Homogeneous fluorescence resonance energy transfer analysis can overcome these shortcomings. It has the advantages of simple analysis operation, no need for washing to separate free markers, fast analysis speed, and easy automation. It is more and more widely used in the field of biological analysis.

In the existing homogeneous time-resolved fluorescence resonance transfer analysis (HTRFA) technology system, the energy donor and the acceptor are one-to-one molecular interactions to form energy transfer, thereby realizing qualitative and quantitative analysis. The technical bottleneck lies in: the efficiency of one-to-one molecular energy resonance energy transfer (FRET) is very low, the energy of the donor cannot be fully utilized, and its sensitivity is limited to a certain extent; secondly, the energy acceptor used in these analysis methods is fluorescent Organic dyes such as fluorescein, cyanine (cy), Alexa dye (Alexa), phycoerythrin (PE) or allophycoerythrin (APC). Although organic dyes have been used for decades, it is well known that the fatal disadvantages of organic dyes are that the stock shift of the spectrum is relatively short, the fluorescence emission peaks are asymmetrical and have obvious tailing, and the resistance to photobleaching is extremely poor, making analysis based on fluorescence values The bioanalysis of the test method, especially HTRFA, is very unstable and inefficient, and the analysis deviation between the interval and the interval is large. Therefore, organic dyes are not the best choice for HTRFA energy acceptors. Moreover, the FRET transmission distance is limited, and generally cannot exceed the spatial distance of 10nm. On the other hand, due to the broad fluorescence emission spectrum of most organic dyes and obvious tailing, the existing HTRFA technology is only suitable for analysis in the long-wavelength or near-infrared band, and the requirement for expensive near-infrared sensitive detectors still exists.

Contents of the invention

In order to overcome the above-mentioned shortcomings of the prior art, the present invention provides a singlet oxygen channel-based luminescent quantum dot sensor.

In order to achieve the above object, the present invention is achieved through the following schemes:

A luminescent quantum dot sensor based on a singlet oxygen channel, comprising quantum dot nano hydrosol and functionalized photosensitive microspheres; the preparation steps of the quantum dot nano hydrosol are as follows: fat-soluble quantum Points are modified into water-soluble quantum dots; the prepared water-soluble quantum dots and thiazine compounds are coated into nano-latex balls modified by carboxyl, amino, hydroxyl, aldehyde or sulfonic acid groups to prepare quantum dot nano-latex balls; Quantum dot nano-emulsion balls are subjected to non-specific treatment with hydrosol, and the surface of quantum dot nano-emulsion balls is modified with dextran gel to obtain quantum dot nano-hydrosol; the preparation method of the functionalized photosensitive microspheres is: the photosensitive material phthalocyanine compound The functionalized photosensitive microspheres are obtained by encapsulating into nano latex spheres modified by carboxyl groups, amino groups, hydroxyl groups, aldehyde groups or sulfonic acid groups.

The sensor of the present invention emits light differently from traditional fluorescence resonance energy transfer, and mainly emits light based on singlet active oxygen. The sensor of the present invention is based on the joint application of the singlet oxygen channel energy transfer technology and the fluorescence resonance energy transfer inside the quantum dot nano hydrosol (TQPS).

Preferably, the nano-emulsion ball used when preparing quantum dot nano-hydrosol is polystyrene-divinylbenzene carboxylic acid modified latex, and the nano-emulsion ball used when preparing functionalized photosensitive microspheres is surface carboxylated polystyrene Microspheres. Nano-emulsion balls have the following characteristics: the surface is porous, the surface contains carboxyl compounds, optical permeability, and uniform particle size distribution.

Fat-soluble quantum dots mean that quantum dots can be dissolved in organic solvents such as decane, hexane, and chloroform. Preferably, the fat-soluble quantum dots are various semiconductor nanocrystals with cadmium selenide, cadmium sulfide or cadmium telluride as cores. Select the quantum dots with the required wavelength according to the actual situation, as long as the emission wavelength is between 520nm and 620nm, it is suitable for the present invention. The biological applications of quantum dots emerge in endlessly, because of their strong resistance to photobleaching and high brightness, a laser light source can simultaneously excite quantum dots of different colors, and can withstand multiple laser excitations without significant changes in fluorescence intensity; in addition, The spectral Stock shift of quantum dots is relatively wide, the fluorescence emission peak is narrow and symmetrical, and there is no tailing. This characteristic enables quantum dot nano-emulsion balls to meet the sensitivity and precision requirements of the immunoassay method without the need for expensive red-sensitive detection devices. The maximum half-maximum width of the fluorescence emission spectrum of quantum dots is narrow, and quantum dots of different wavelengths can be excited by the same excitation light. Therefore, the fluorescence emission spectra with emission peaks at 525, 565 or 605nm overlap less with the fluorescence emission of thiazide compounds. The molar excitation spectrum of these quantum dots overlaps more with the fluorescence emission of thiazide compounds, and is most suitable for the realization of homogeneous fluorescence resonance energy transfer analysis with thiazide compounds.

Since the maximum half-peak width of quantum dots prepared directly from aqueous solution is generally large, close to 60nm, it is not suitable for the CEA analysis of the present invention. Therefore, the present invention uses fat-soluble quantum dots, which are then modified into water-soluble quantum dots. Since only the surface modification of quantum dots is carried out, there is no influence on the luminescent core material, and the fluorescence emission peak position and particle size obtained after modification are not significantly different from those of oil solubility; the modification method is simple and easy to operate. powerful;

Preferably, the method for modifying the fat-soluble quantum dots into water-soluble quantum dots is: adding a stabilizer to the fat-soluble quantum dots dissolved in chloroform, mixing uniformly; adding 0.1M sodium hydroxide solution for shaking incubation , stored at room temperature for 1 to 3 hours; adding deionized water and acetone, precipitation, centrifugation, and dissolution to obtain water-soluble quantum dots.

Preferably, the stabilizer is mercaptosuccinic acid, glutathione or thioglycolic acid.

More preferably, the method for modifying the fat-soluble quantum dots into water-soluble quantum dots is: adding 25 mg of mercaptosuccinic acid stabilizer to 500 μL of cadmium selenide quantum dots (1-5 μM) dissolved in chloroform, and mixing Evenly, add 10-100 μL of 0.1M sodium hydroxide solution, incubate with shaking, and store at room temperature for 1-3 hours. Add 400 μL of deionized water and 1.8 mL of acetone, precipitate, centrifuge, and dissolve in 0.5 mL of deionized water to obtain water-soluble quantum dots.

Preferably, before using water-soluble quantum dots to prepare quantum dot nano latex balls, trioctylphosphine oxide, n-hexadecylamine or other normal alkanes with a carbon chain greater than five can be used to modify the surface of water-soluble quantum dots to make the surface hydrophobic and Hydrophilic bifunctional groups. The pretreatment method is: adding trioctylphosphine oxide to the water-soluble quantum dots; heating to 60°C, vigorously stirring to dissolve it completely, and cooling to room temperature; successively adding benzyl alcohol, ethylene glycol and 0.1M The sodium hydroxide solution was stirred and mixed evenly.

The quantum dot nano hydrosol in the sensor is the energy acceptor, and the functionalized photosensitive microsphere is the energy donor. Long-distance energy transfer up to 200nm can be achieved between the donor and the acceptor.

The quantum dot nano latex ball contains chemical reagents and quantum dots that can react with singlet oxygen. Chemical reagents refer to energy acceptors, which include thiazine compounds or other chemical reagents that can release 200-400nm fluorescence when reacting with singlet oxygen. After chemical reagents such as thiazine compounds react with singlet oxygen, they release The fluorescence energy of the quantum dot can undergo fluorescence resonance energy transfer.

Preferably, the preparation method of the quantum dot nano-emulsion ball is as follows: adding trioctylphosphine to the water-soluble quantum dot, heating, stirring evenly, and then mixing with swelling agent, thiazine compound and carboxyl group, amino group, hydroxyl group, aldehyde group or sulfonic acid group-modified nano-emulsion balls are evenly mixed, thermally swelled at a high temperature of 110-120°C for 5-30 minutes, cooled to room temperature, washed by high-speed centrifugation, and the quantum dot nano-emulsion balls are obtained.

More preferably, the swelling agent is a mixture of benzyl alcohol (or other solvents such as chloroform, acetone, ethoxyethanol), deionized water and ethylene glycol, and the volume ratio of the three is 1:(0.8～1 ): (8 ~ 8.2). Under the protection of thermal swelling agent, the surface of nano latex balls modified by carboxyl group, amino group, hydroxyl group, aldehyde group or sulfonic acid group will expand at high temperature, and will not be excessively torn to cause uneven distribution of surface functional groups, and overcome quantum dot fluorescence quenching The problem is to maintain the quantum yield before coating, and effectively prevent precipitation and aggregation, and greatly improve the stability of quantum dot nano latex balls.

Preferably, the concentration of the water-soluble quantum dots is generally 0.1-10 μM/mL. The nanoemulsion balls modified by carboxyl, amino, hydroxyl, aldehyde or sulfonic acid groups are generally 1-200 mg/mL, and the particle size is generally 20 nm-3 μm, more preferably, the particle size is 20 nm-300 nm. In addition, as a preferred embodiment, the particle size of the nano-emulsion spheres used in the preparation of the functionalized photosensitive microspheres is ≤ the particle size of the nano-emulsion spheres used in the preparation of the quantum dot nano-hydrosol. The surface of the latex ball contains a large number of carboxyl compounds, and its carboxyl density is 5 to 12 per square nanometer. The latex ball of this specification has undergone special treatment, which is different from the general carboxylated polystyrene microspheres. There are micropores on the surface that can pass through Thermal swelling contains nanoparticles with dual functional groups. The latex spheres are highly hydrophilic and have strong optical permeability. They can be directly used for coupling biomolecules or surface treatment, and are suitable for clinical diagnostic analysis. In the method of the present invention, it is recommended to directly use CPS latex balls, without using polystyrene microspheres with a layer of carboxyl groups on the surface, and without carrying out carboxylation or grafting of other groups to water-insoluble polystyrene microspheres. The invention adopts a mild bifunctional organic reagent to swell latex balls to embed water-soluble quantum dots at a temperature higher than that required by most biological analysis, that is, at 110-120°C. The temperature required for most biological analysis is lower than the thermal swelling temperature, which can avoid the leakage problem in use. Although the size distribution of nanolatex spheres changed slightly after being coated with quantum dots, it did not affect the biological application of quantum dot nanolatex spheres.

Preferably, the specific preparation method of the quantum dot nano-hydrosol is: dissolving aminodextran in MES buffer solution, adjusting the pH to 6.0 and adding it dropwise to quantum dot nano-emulsion balls; stirring and adding EDAC solution and MES buffer solution, and kept stirring at room temperature for 2 hours; adding ethanolamine to incubate for 30 minutes, centrifuging at 17000rmp and discarding the supernatant; dissolving the precipitate in deionized water, ultrasonic treatment, and repeating this step three times to obtain.

Dextran is a hydrosol widely used in the biological field. The purchased aminated dextran has a molecular weight of 100KDa, and every 7 dextran units contain 1 to 2 amino groups; the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt Hydroxysulfosuccinimide (EDAC) and N-hydroxysulfosuccinimide (Sulfo-NHS), other coupling agents that can couple the carboxyl group to the amino group can also be used, and the reaction conditions such as the corresponding buffer solution also change accordingly; through coupling After coupling, the remaining carboxyl groups or amino groups of quantum dot nano latex balls can continue to couple with biomolecules; in addition, the dextran on the surface of TQPS greatly prevents the leakage of quantum dots and enhances its own stability and biocompatibility. The TQPS prepared by the present invention is used as an important component of the CEA antigen sensor for detection. Its surface contains a large amount of hydroxyl, carboxyl and amino functional groups, which can be monodispersed in the buffer for biology, and overcomes its own disadvantages due to the high negative charge density of the carboxyl group. The resulting precipitation problem also solves the problem of non-specific adsorption of biomolecules.

The TQPS of the sensor and the conjugated antibody are prepared by coupling the monoclonal antibody S001 of the carcinoembryonic antigen CEA with the carboxyl group on the surface of the TQPS through covalent bonding under the action of coupling agents EDAC and Sulfo-NHS. Functional TQPS for homogeneous immunoassays. Depending on the analyte, the antibody coupled to TQPS can also use other proteins, or nucleic acid or peptide chains with amino or carboxyl groups at the end.

The fluorescence signal at 605nm is from TQPS. When the near-infrared laser excites the detection sample, the BTHS in the SA-PS releases singlet reactive oxygen species due to the absorption of photosensitive energy. The active oxygen can diffuse and penetrate into the TQPS in the range of 200nm, then react with the thiazide compound contained in the TQPS to release short-wavelength fluorescence (320-400nm), and undergo fluorescence resonance energy transfer with the surrounding quantum dots, thereby making the TQPS release A fluorescence detection signal at 605nm was produced.

The sensor of the present invention also needs to prepare functionalized photosensitive microspheres that can be excited by near-infrared light to generate singlet oxygen. The preparation method is: adding nano latex balls modified by carboxyl, amino, hydroxyl, aldehyde or sulfonic acid groups In ethylene glycol, dissolve the phthalocyanine compound in benzyl alcohol, mix the two, add deionized water, react at 110°C for 10 minutes after mixing, add ethanol after cooling to room temperature, and size-selectively centrifuge.

The photosensitive material phthalocyanine compound is a commonly used phthalocyanine compound in the field, such as silicon dihydroxide 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine, CAS number: 85214-70-6.

When the photosensitive material phthalocyanine compound (BTHS) is irradiated by near-infrared laser, a large number of singlet reactive oxygen species are released. It has been reported in the literature (Macromolecules 25, 3399-3405) that these singlet reactive oxygen species can diffuse in general water-soluble media with a distance of about 200nm, and can penetrate into the surrounding polystyrene microspheres with an efficiency of 20%. The new sensor is based on the singlet oxygen channel transfer technology, combined with the anti-photobleaching and specific optical properties of quantum dots, which greatly improves the energy transfer efficiency and overcomes the steric barrier of energy transfer by the surface groups of the hydrosol; at the same time, because There are multiple binding sites on the surface that can transfer singlet oxygen to photo-excited chemiluminescence quantum dots at the same time, which greatly improves the detection sensitivity.

The application of homogeneous fluorescence analysis based on the singlet oxygen channel luminescent quantum dot sensor refers to the immunoassay of carcinoembryonic cells in serum using the principle of fluorescence resonance energy transfer between the thiazine compound at the receiving end of singlet oxygen and the quantum dot. Antigen content. In this analysis method, the double-antibody sandwich one-step method is used to couple the energy acceptor and the donor to two monoclonal antibodies of the antigen, and the energy acceptor TQPS and the donor SA- When the PS is close, when the distance between the photosensitive microsphere SA-PS and the quantum dot nano-hydrosol TQPS is less than 200nm, the oxygen signal transmission can occur, and then the fluorescence resonance energy transfer between the QDS and the thiazine compound in the TQPS is excited (as shown in Figure 2 Show).

In TQPS, according to the principle of fluorescence resonance energy transfer, it is necessary to realize the fluorescence resonance energy transfer between the luminescent antenna material thiazine compound and the quantum dots, and to realize the resonance energy transfer requires the emission spectrum of the antenna material to match the excitation wavelength of the quantum dots ( As shown in Figure 3). According to the luminescence spectrum of the selected luminescent material thiazine compound and the molar extinction spectrum of quantum dots, the theoretical calculation of spectral overlap is:

$\mathrm{<span\; class="notranslate">\; J</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; \&lambda;</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{\underset{\mathrm{<span\; class="notranslate">\; 0</span>}}{\overset{\mathrm{<span\; class="notranslate">\; \&infin;</span>}}{<span\; class="notranslate">\; \&Integral;</span>}}{\mathrm{<span\; class="notranslate">\; \&epsiv;</span>}}_{\mathrm{<span\; class="notranslate">\; A</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; \&lambda;</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&CenterDot;</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; \&lambda;</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&CenterDot;</span>{\mathrm{<span\; class="notranslate">\; \&lambda;</span>}}^{\mathrm{<span\; class="notranslate">\; 4</span>}}<span\; class="notranslate">\; \&Center\; Dot;</span>\mathrm{<span\; class="notranslate">\; d</span>}\mathrm{<span\; class="notranslate">\; \&lambda;</span>}}{\underset{\mathrm{<span\; class="notranslate">\; 0</span>}}{\overset{\mathrm{<span\; class="notranslate">\; \&infin;</span>}}{<span\; class="notranslate">\; \&Integral;</span>}}{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; D.</span>}}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; \&lambda;</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; \&CenterDot;</span>\mathrm{<span\; class="notranslate">\; d</span>}\mathrm{<span\; class="notranslate">\; \&lambda;</span>}}$

Here A represents the quantum dot energy acceptor, and D represents the luminescent antenna material. ε _A is the excitation spectrum function of the quantum dot, λ is the wavelength, and ID is the fluorescence emission spectrum function of the energy donor _. It is calculated that J(λ)=2.86×10 ¹⁶ .

Light-emitting antenna material close to quantum dots needs Distance theory formula:

R ₀ =(8.79×10 ^-5 ·κ ² ·Φ _D ·n ^-4 ·J(λ)) ^1/6

Wherein, Φ _D is the quantum yield (2.5%) of the light-emitting antenna material, к ² is a kinetic parameter generally 2/3, and n is the solution refractive index. Calculated by this formula, the fluorescence resonance energy transfer between the luminescent antenna material and the quantum dots The distance is 2.3nm, which is the average distance at which 50% energy transfer efficiency occurs between the luminescent antenna material and the quantum dots.

And the fluorescence resonance energy transfer efficiency:

$\mathrm{<span\; class="notranslate">\; E.</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}^{\mathrm{<span\; class="notranslate">\; 6</span>}}}{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}^{\mathrm{<span\; class="notranslate">\; 6</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; r</span>}}^{\mathrm{<span\; class="notranslate">\; 6</span>}}}$

Where r is the spatial distance between the energy donor and the acceptor; in practical applications, the energy transfer efficiency E is generally 1.5%-98.5% as the effective transfer range, and the distance between the luminescent antenna material and the quantum dot can be obtained as 1.6nm<R<4.6 nm. Therefore, in terms of spatial structure, the thiazine compound and quantum dots used in the present invention can theoretically meet the conditions of fluorescence energy transfer by simply embedding them in polystyrene microspheres.

Because the molar excitation spectrum of the red quantum dots (fluorescence emission peak 605nm, quantum yield 48%) has a large overlap with the emission spectrum of the thiazide compound, the photosensitive microspheres and TQPS are respectively labeled with streptavidin and CEA. Cloned antibody S001 constitutes a quantum dot sensor based on singlet oxygen channel luminescence, and another monoclonal antibody 6F11 from CEA is biotinylated. Therefore, when the singlet oxygen channel-based luminescent quantum dot sensor, biotinylated 6F11, and CEA antigen undergo an immune reaction, the photosensitive microspheres and TQPS are drawn closer. Become high-energy singlet oxygen, and the singlet oxygen is transferred to the interior of the quantum dot nano-hydrosol, and then stimulates the fluorescence resonance energy transfer between the thiazide compound and the quantum dot, and the quantum dot emits signal fluorescence (as shown in Figure 3). When the two monoclonal antibodies are in excess of the antigen CEA, the detected fluorescence resonance energy transfer signal will be proportional to the amount of the antigen (as shown in Figure 4).

Compared with the prior art, the present invention has the following beneficial effects:

The sensor provided by the invention is a novel homogeneous fluorescent immunoassay sensor based on singlet oxygen transfer, which changes the one-to-one energy ligand mode in the prior art, and breaks through the spatial detection distance of 20nm. In addition, the singlet oxygen channel luminescent quantum dot is a kind of energy acceptor with novel optical properties: the stock shift of its spectrum is relatively wide, the fluorescence emission peak is symmetrical and has no obvious tailing, and more importantly, it has strong photobleaching resistance , and the energy acceptor does not rely on expensive red-sensitive detectors. This type of sensor does not need to wash excess markers, reacts and detects in a homogeneous phase, is suitable for the detection of biomolecules such as macromolecular immunoglobulins or small molecular haptens, and has potential huge advantages in the field of in vitro immunodiagnostics.

The invention uniquely prepares a multifunctional singlet oxygen channel-based luminescent quantum dot sensor. The TQPS used in this sensor has the advantages of good water solubility, uniform size distribution, and high quantum yield, which improves the stability and sensitivity of the analysis, and also overcomes the problems of non-specific adsorption. It innovatively combines quantum dots with oxygen transfer technology. combined. Using a novel homogeneous oxygen transfer fluorescence resonance energy transfer analysis method based on a multifunctional singlet oxygen channel luminescent quantum dot sensor can not only improve the energy transfer efficiency and thus improve the analysis sensitivity, but also reduce the dependence on expensive red-sensitive photon detectors, It is of great significance in biochemical analysis such as clinical molecular diagnosis and food testing.

Description of drawings

Figure 1 is the particle size distribution diagram of the nano-hydrosol before (CPS) and after (QPS) functionalization.

Fig. 2 is a schematic diagram of a homogeneous analysis based on a singlet oxygen channel luminescent quantum dot sensor.

Fig. 3 is the molar excitation spectrum and the fluorescence emission spectrum of the thiazine compound of the water-soluble quantum dot.

Figure 4 shows the preliminary application of the homogeneous immune sandwich method for the analysis of CEA based on the singlet oxygen channel luminescent quantum dot sensor.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The polystyrene-divinylbenzenecarboxylic acid modified latex used in the following examples was purchased from Thermo Fisher Scientific (product number 8300-0520100390), fat-soluble quantum dots (product number Q21701MP) were purchased from Invitrogen, and the coupling agent Chemical reagents such as EDAC, Sulfo-NHS, and trioctylphosphine oxide were purchased from Sigma-Aldrich in the United States, and the carcinoembryonic antigen monoclonal antibodies S001 and 6F11 and carcinoembryonic antigen antigen standards were purchased from Sun Yat-sen University Daan Gene Company , other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

Example 1

The core technology of this step is to obtain symmetrical water-soluble quantum dots with high quantum yield and narrow maximum half-maximum width of the fluorescence emission peak. The concrete implementation of the present invention used is the core/shell type cadmium selenide/zinc sulfide quantum dot purchased from U.S. Invitrogen Company, and its solvent is decane, and its spectral Stock shift length is from 592nm to the wider range between short wavelength Therefore, the quantum dot can be excited with a wavelength shorter than 592nm; the fluorescence emission peak is 605±0.5nm, the maximum half-peak width is 27±1.5nm, and the quantum yield is 76%; Oxygen-receiving "antenna" thiazine compound has a matching fluorescence emission spectrum, overcomes the shortcomings of organic dyes in all aspects, and is suitable for replacement to reduce measurement errors, improve fluorescence resonance energy transfer (FRET) efficiency, and improve analytical sensitivity.

A luminescent quantum dot sensor based on a singlet oxygen channel, comprising quantum dot nano hydrosol and functionalized photosensitive microspheres; the preparation method of quantum dot nano hydrosol and functionalized photosensitive microspheres is as follows:

1. Preparation of high-performance water-soluble quantum dots: put 50 μL of purchased fat-soluble quantum dots in a centrifuge tube, add 200 μL of anhydrous methanol; mix well, seal the centrifuge tube, centrifuge at 3000 rpm, 4 ° C for 5 min; discard Add 50 μL of chloroform to the supernatant to fully dissolve; add 25 mg of mercaptosuccinic acid stabilizer and 20 μL of 0.1M sodium hydroxide aqueous solution, mix well, incubate on a shaker, and store at room temperature for 3 hours; add 100 μL of deionized Water and 1.8 mL of acetone, precipitated, centrifuged at 6000 rpm for 5 min; dissolved in 0.2 mL of deionized water to obtain water-soluble quantum dots for use. The emission spectrum of the obtained water-soluble quantum dots was analyzed by a fluorescence spectrophotometer, and the fluorescence emission peak was 605nm.

2. Preparation of luminescent quantum dot nano-emulsion spheres (TQPS) based on singlet oxygen channels:

1. Pretreatment of nano latex spheres (CPS): 100 μL polystyrene-divinylbenzenecarboxylic acid modified latex (particle diameter 205 ± 3nm, mass concentration 100mg/mL, solid concentration 10%) at 14000rmp, Centrifuge at 4°C; discard the supernatant and resuspend in 100 μL deionized water;

2. Pretreatment of water-soluble quantum dots: Add 2mg of trioctylphosphine oxide to the water-soluble quantum dots prepared in the above step 1; heat to 60°C, stir vigorously to dissolve completely, and cool to room temperature; successively Add 2 mL of benzyl alcohol, 2.8 mL of ethylene glycol and 100 μL of 0.1 M sodium hydroxide solution and stir to mix evenly;

3. Then add the quantum dot aqueous solution and thiazine compound (4 mg dissolved in 100 μL of benzyl alcohol) in step 2 dropwise to the nano-emulsion balls in step 1, and stir vigorously at 105 ° C for 15 minutes; cool to room temperature, at 12000 rpm The speed of the pellet was centrifuged for 25 minutes; dissolved in 200 μL of 2-(N-phenoline)ethanesulfonic acid buffer (MES, 50mM, pH6); the obtained high-performance water-soluble quantum dot nano latex ball (QPS), its size distribution is as follows As shown in Figure 1, the square and triangle solid lines represent the size distribution of the nano-emulsion balls before and after coating the quantum dots, respectively. The preparation method of the thiazide compound is as follows: 10-15g of p-bromoaniline is dissolved in 100mL of dimethyl sulfoxide solution, and 40mL of 1-bromotetradecane and 30mL of N,N'-diisopropyl Ethylamine. Under the protection of nitrogen, the reaction solution was continuously heated to 90° C. and cooled to room temperature after 16 hours of reaction. Add 20mL of 1-tetradecane bromide and 15mL of N,N'-diisopropylethylamine to the reaction liquid again. The reaction mixture was heated to 90°C again for 15 hours. The reaction solution was cooled to room temperature, dried in vacuo and the residue was diluted with 200 mL of dichloromethane. It was washed with 1N NaOH (2×) aqueous solution, water and brine, and finally dried with Na ₂ SO ₄ . The reaction solution was concentrated to obtain about 55 g of brown-black oil. Use Agilent 1200HPLC to prepare the liquid phase separation, mobile phase is n-hexane, obtain yellow oil, most of them are product 4-bromo-N, N'-dimethyl-p-aniline and a small amount of 1-bromomethane. The latter is obtained by decompression It can be removed by distillation (bp 105～110°C, 0.6mmH), and finally the target product is brown oil.

3. Preparation of TQPS: TQPS can be prepared by surface modification of quantum dot nano-hydrosol, and the steps are as follows: Weigh 10 mg of aminodextran and dissolve it in 100 μL of MES buffer, and adjust the pH to 6.0 again with 1M hydrochloric acid; Add dropwise to the quantum dot nanoemulsion ball (QPS) in the above step 2, stir at a speed of 300rmp; add 300 μL of EDAC solution (80mg/mL) and 1.4mL of MES buffer, and continue stirring at room temperature for 2 hours; 700 μL of 1 mM ethanolamine was incubated for 30 min; centrifuged at 17,000 rpm, discarded the supernatant, dissolved the precipitate in 20 μL of deionized water, and sonicated; repeated the above steps three times, and finally obtained quantum dot nano-hydrosol photosensitive latex balls (TQPS ).

4. Preparation of functional photosensitive microspheres:

Add 0.1mL of surface carboxylated polystyrene microspheres to 0.7mL of ethylene glycol, dissolve 5mg of BTHS phthalocyanine compound in 0.1mL of benzyl alcohol, mix the two, add 0.1mL of deionized water, and mix well Then heat up to 110°C, react for 10 minutes, add ethanol after cooling to room temperature, and size-selectively centrifuge. The collected photosensitive donor microspheres (0.1 g) were preserved in 10% ethanol until use.

Example 2

A luminescent quantum dot sensor based on a singlet oxygen channel, comprising quantum dot nano hydrosol and functionalized photosensitive microspheres; the preparation method of quantum dot nano hydrosol and functionalized photosensitive microspheres is as follows:

1. Preparation of high-performance water-soluble quantum dots: put 50 μL of fat-soluble quantum dots in a centrifuge tube, add 200 μL of anhydrous methanol; mix well, seal the centrifuge tube, centrifuge at 3000 rpm, 4 ° C for 5 min; discard the supernatant Add 40 μL of chloroform to fully dissolve; add 21 mg of glutathione stabilizer and 10 μL of 0.1M sodium hydroxide aqueous solution, mix well, shake and incubate on a shaker, and store at room temperature for 2 hours; add 100 μL of deionized water and 1.8 mL of acetone, precipitated, centrifuged at 6000 rpm for 5 min; dissolved in 0.5 mL of deionized water to obtain water-soluble quantum dots. The emission spectrum of the prepared water-soluble quantum dots was analyzed by a fluorescence spectrophotometer, and the fluorescence emission peak was 605nm.

2. Preparation of luminescent quantum dot nano-emulsion spheres (TQPS) based on singlet oxygen channels:

3. Preparation of TQPS:

4. Preparation of functional photosensitive microspheres:

Application example

Based on the application of singlet oxygen channel luminescent quantum dot sensor in the analysis of homogeneous oxygen transfer fluorescence resonance energy transfer, the principle of homogeneous analysis of singlet oxygen channel luminescent quantum dot sensor is shown in Figure 3, and the application steps are as follows:

1. Conjugate TQPS with anti-carcinoembryonic antigen monoclonal antibody, the steps are as follows: prepare 0.5M MES buffer, adjust pH to 6.1, take 100 μL for use; prepare 50 mg/mL Sulfo-NHS solution with deionized water, take 230 μL Stand-by; prepare 50 mg/mL EDAC solution with deionized water, take 240 μL for use; mix the above solution for use, dilute to 1 mL with deionized water, add to the TQPS prepared in the above step 3, and resuspend; the mixture is at room temperature Shake the reaction for 30 minutes; centrifuge at 16000rmp to remove unreacted Sulfo-NHS and EDAC, wash with deionized water, resuspend, and centrifuge three times; resuspend the activated TQPS in deionized water, and the final mass concentration is 1% , the volume is 1 mL; take 500 μL and add 500 μL monoclonal antibody S001 (1 mg/mL); gently shake and incubate for 2 hours, add 1.5 μL ethanol, and incubate at room temperature for 30 minutes; centrifuge to remove uncoupled protein and ethyl alcohol Amino alcohol; add 1mL of 50mM phosphate buffer (containing 0.05% Proclin-300), obtain the quantum dot nanometer hydrosol photosensitive latex ball coupled with monoclonal antibody S001, and adjust its concentration to 100 μg/mL, its The molar excitation spectrum is shown in the red curve on the left side of Figure 2.

2. Coupling of photosensitive microspheres and streptavidin. The surface of the prepared donor microspheres was coupled with streptavidin through EDAC and Sulfo-NHS, and the concentration was adjusted to 16 μg/mL.

3. Biotinylation labeling of anti-carcinoembryonic antigen monoclonal antibody 6F11: According to the instructions of Sigma Company, the volume ratio of antibody solution and biotin solution was 10:1 and fully mixed. A centrifuge tube with a filter membrane removes excess biotin and adjusts the antibody concentration to 5 μg/mL.

4. Prepare the CEA antigen to a series concentration of 0ng/mL, 2ng/mL, 5ng/mL, 20ng/mL, 100ng/mL, and 500ng/mL; add 25μL of the sample to be tested, 25μL of biotinylated antibody, and 25 μL of quantum dot nano-hydrosol, and affixed to cover; the microwell reaction strip was shaken and incubated in a 37°C constant temperature shaker for 15 minutes; 175 μL of streptavidin-coated donor microspheres were added under dark conditions; the microwell reaction The strips were shaken and incubated for 15 minutes in a constant temperature shaker at 37°C; the signal value was detected on a PerkinElmer 2300EnSpireTM detector. The logarithm of the reference standard substance concentration is the abscissa, and the logarithm of the signal value count is the ordinate, processed by the log-log mathematical model Log-Log function, and the linear correlation coefficient of the dose-response curve of the CEA kit measured is r=0.9918, As shown in Figure 3.

During the two hours after the above detection, the same fluorescence detection was performed every 10 minutes. The results of all samples showed that the TQPS used was extremely resistant to photobleaching, and the log-log relationship graphs of standard concentration and fluorescence values almost overlapped.

The zero reference standard (point A) was used as the specimen to measure repeatedly 20 times, and the fluorescence mean χ and standard deviation SD were calculated, and the fluorescence value obtained by χ+2SD was substituted into the standard curve equation to calculate its sensitivity, that is, the lowest detection limit of CEA 0.7ng/mL.

Hemoglobin, triglycerides, and bilirubin were used to measure the cross-reactivity of CEA samples, and the results showed no cross-reaction.

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.