Dissolution-enhanced time-resolved fluoroimmunoassay based on rare earth nanomaterial

Abstract

This invention relates to a rare earth nanomaterial labeled biomolecule, its labeling method and a dissolution-enhanced time-resolved fluoroimmunoassay based on the rare earth nanomaterial. The rare earth nanomaterial serves as a label having stable properties, large specific surface area, strong modifiability, low-cost and thousands of lanthanide ions contained in each nanocrystal, the labeling ratio of rare earth ions can be greatly improved. Furthermore, the rare earth nanomaterial can be less affected by exogenous rare earth ions, unaffected by anticoagulants, and has broader applicability; after the immune complex was formed by labeling the biomolecules with the nanomaterial containing rare earth, an enhancer solution was added to allow the rare earth nanomaterial to dissolve into the rare earth ions, which can in turn form new signaling molecules with the chelates in the enhancer solution to induce intramolecular and intermolecular energy transfer, thereby significantly increasing fluorescence intensity by about a million times to greatly enhance the detection sensitivity by using time-resolved fluorescence assay.

Claims

1. A method of dissolution-enhanced time-resolved fluoroimmunoassay, comprising: obtaining an immune complex comprising a rare earth nanomaterial labeled biomolecule; dissolving the immune complex in an enhancer solution to release rare earth ions into the enhancer solution, wherein rare earth ions combine with chelates in the enhancer solution to form molecules capable of emitting fluorescence signals; and detecting the molecules using time-resolved fluoroimmunoassay, and wherein the rare earth nanomaterial is nanocrystal having a formula XYF.sub.4, wherein X represents sodium, and Y represents europium.

2. The method according to claim 1, wherein the step of obtaining the immune complex comprises: 1) immobilizing a capture antibody or antigen that specifically binds to a target antigen or a target antibody on a microplate through physical adsorption or covalent coupling; 2) blocking the microplate with a blocking solution; 3) adding a sample containing the target antigen or target antibody to the blocked microplate to allow binding of the target antigen or target antibody to the immobilized capture antibody or antigen; and 4) binding the rare earth nanomaterial labeled biomolecule to the bound target antigen or the target antibody to form the immune complex.

3. The method according to claim 2, wherein the sample contains the target antibody.

4. The method according to claim 2, wherein said binding of the rare earth nanomaterial labeled biomolecule of step 4) comprises: (a) adding a biotin-labeled antibody that specifically binds to the bound target antigen or antibody; and (b) adding the rare earth nanomaterial labeled with avidin to form the immune complex.

5. The method according to claim 1, wherein the enhancer solution comprises a buffer solution, -diketone, a nonionic surfactant, and a synergistic agent.

6. The method according to claim 1, wherein the detection mode is sandwich assay, direct assay, or competitive assay.

7. The method according to claim 1, wherein the rare earth nanomaterial is labeled with the biomolecule via chemical coordination or physical adsorption to provide the rare earth nanomaterial labeled biomolecule.

8. The method according to claim 1, wherein the biomolecule is biotin, avidin, antibody, or aptamer.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic diagram of (a) the conventional dissociation-enhanced lanthanide-based time-resolved fluorescence immunoassay (DELFIA) and (b) the dissolution-enhanced luminescent bioassay (DELBA) based on the rare earth nanomaterial of the present invention.

(2) Wherein, it is shown that the antigen or antibody to be detected is labeled by (a) the rare earth chelate or (b) the rare earth nanomaterial with double antibody sandwich method, and after the formation of immune complex, the enhancer solution is added and the fluorescence signals are detected by using the time-resolved method.

(3) As shown in FIG. 1, each rare earth nanocrystal particle contains thousands of rare earth ions, and thus greatly improve the labeling ratio of rare earth ions, and upon addition of the enhancer solution a large number of molecules having strong fluorescence signals are formed, thereby significantly enhancing fluorescence signals and detection sensitivity.

(4) FIG. 2: Transmission electronmicroscopy (TEM) graph of NaEuF.sub.4 nanocrystals. Instrument: Model JEM-2010; Manufacturer: JEOL.

(5) FIG. 3: Powder X-ray diffraction patterns of NaEuF.sub.4 nanocrystals. Instrument: Model MiniFlex2; Manufacturer: Rigaku; Wave length of a copper target radiation: X=0.154187 nm.

(6) FIG. 4: Standard curve for the carcinoembryonic antigen assay using the double antibody sandwich method of the present invention.

(7) FIG. 5: Standard curve for the carcinoembryonic antigen assay using the commercial time-resolved carcinoembryonic antigen test kits.

SPECIFIC EMBODIMENTS

(8) Hereinafter, the present invention will be illustrated with reference to the figures and the examples. However, the protection scope of the present invention should not be limited to the following examples. According to the disclosure of the present invention, those skilled in the art would recognize that the many changes and modifications based on the following examples belong to the protection scope of the present invention without departing from the technical features and the scope provided in the present invention.

EXAMPLES

Example 1

(9) A process of dissolution-enhanced time-resolved fluoroimmunoassay based on the rare earth nanomaterial, comprising the following steps of:

(10) 1. Synthesis of NaEuF.sub.4 Nanocrystals

(11) 1) weighing out 1 mmol of Eu(Ac).sub.3 and adding it into the mixed solvent of 6 mL of oleic acid and 15 ml of octadecene, excluding air and stirring at 160 C. under nitrogen atmosphere for 30 min to dissolve to give a solution A;

(12) 2) weighing out 150 mg of NH.sub.4F and 100 mg of NaOH, adding them into methanol and dissolving to obtain a solution B;

(13) 3) after the solution A was cooled down to room temperature, slowly adding the solution B dropwise into the solution A with a dropper, excluding air, increasing temperature to 60 C. under nitrogen atmosphere, and stirring for 30 min to remove methanol;

(14) 4) increasing temperature to 120 C., and stirring the reaction for 10 min to remove residual water;

(15) 5) increasing temperature to 300 C. and stirring the reaction for 0.5 h;

(16) 6) after the solution cooled down to room temperature, adding two times the volume of anhydrous ethanol to precipitate the nanocrystals;

(17) 7) centrifuging, washing the nanocrystals with anhydrous ethanol for three times to be ready for use.

(18) FIG. 2 and FIG. 3 respectively show the TEM image and Powder X-ray diffraction patterns of the synthesized NaEuF.sub.4 nanocrystals.

(19) 2. Labeling Biotin or Antibody with NaEuF.sub.4 Nanocrystals

(20) A. labeling biotin with NaEuF.sub.4 nanocrystals via chemical coordination method

(21) 1) weighing out 20 mg of the NaEuF.sub.4 nanocrystals synthesized in step 1 to dissolve in 15 mL of hydrogen chloride-ethanol solution with a pH of 1.0, ultrasonicating for 30 min, collecting the nanoparticles by centrifugation, and then washing with anhydrous ethanol for three times to remove oleic acid on the surface of the nanocrystals, and adding 2 mL deionized water to dissolve to obtain 10 mg/mL water-soluble nanocrystals.

(22) 2) adding 1 mmol of biotin and two drops of ammonia water into the solution of step 1), ultrasonicating for 20 min, centrifuging washing with deionized water for three times, and finally dissolving in 1 mL of deionized water to be ready for use.

(23) B. NaEuF.sub.4 nanocrystals labeling antibody via physical adsorption method: taking 1 mL of the water-soluble nanocrystals synthesized in step A-1), adding 100 g of antibody, adding 100 l of the phosphate buffer solution with a pH of 8.0, shaking at room temperature for 1 h, collecting the nanoparticles by centrifugation, washing with water three for times, and dissolving in the buffer solution with a pH of 8.0 to be ready for use.

(24) 3. Preparation of an Enhancer Solution

(25) Weighing out 1 g of Triton X-100, 26.6 mg of naphthoyltrifluoroacetone, 193 mg of tri-n-octylphosphine oxide, adding distilled water to the volume of 1 L, and adjusting pH to 2.0 with diluted HCl to be ready for use.

(26) 4. Carcinoembryonic antigen detection using double-antibody sandwich assay based on NaEuF.sub.4 nanocrystals

(27) 1) Coating: diluting the antibody against carcinoembryonic antigen to 10 g/mL with 0.05 mol/L of carbonate buffer, which was then added to a 96-well polystyrene plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(28) 2) Blocking: formulating 2% of bovine serum albumin with 0.05 mol/L of carbonate buffer, which was then added to a 96-well plate at 300 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(29) 3) Loading: formulating a series of standard solutions of carcinoembryonic antigen having concentrations in the range of 0.00256-1000 ng/mL with PBS buffer to provide standard samples respectively having the following concentrations: 0 ng/mL, 0.00256 ng/mL, 0.064 ng/mL, 0.0128 ng/mL, 0.32 ng/mL, 1.6 ng/mL, 8 ng/mL; incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(30) 4) Adding NaEuF.sub.4 nanocrystal labeled antibody: formulating 1 g/mL of NaEuF.sub.4 nanocrystal labeled antibody (prepared in the above step 2) with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing six times with PBST washing buffer.

(31) 5) Adding the enhancer solution: adding the enhancer solution at 200 L per well, detecting fluorescence signals using time-resolved assays with the measurement parameters: excitation wavelength of 340 nm, emission wavelength of 615 nm, and delay time of 250 s.

(32) 6) Plotting standard curve: plotting the standard curve with the concentration of the carcinoembryonic antigen standard solution as abscissa and the fluorescence intensity corresponding to each concentration of the standard solution as ordinate. As shown in FIG. 4, in the range of 0.00256-8 ng/mL, a linear correlation was observed between the concentration of the carcinoembryonic antigen and the fluorescence intensity, wherein y=480.87x+425, R=0.9987, and the limit of detection is 0.1 g/mL, based on the blank mean plus three times the standard deviation (SD).

(33) 7) Testing sample: adding 100 L of the test sample in step 3), while the other steps being the same as above, and calculating the corresponding concentration value by putting the fluorescence intensity of the sample into the standard curve equation.

(34) 8) Said step 4) can also be realized by the following steps:

(35) (1) Adding the biotin-labeled antibody: formulating 1 g/mL of biotin labeled antibody with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer;

(36) (2) Adding avidin: formulating 5 g/mL of avidin with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 0.5 h; removing the liquid contained in each well, and washing three times with PBST washing buffer;

(37) (3) adding NaEuF.sub.4 nanocrystal labeled biotin: formulating 10 g/mL of NaEuF.sub.4 nanocrystal labeled biotin (prepared in the above step 2) with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 0.5 h, removing the solution contained in each well, and washing six times with PBST washing buffer.

Example 2: Comparison Between the Method According to the Invention and the Commercial Time-Resolved Carcinoembryonic Antigen Assay Kits

(38) 1) Coating: diluting the antibody against carcinoembryonic antigen to 10 g/mL with 0.05 mol/L of carbonate buffer, which was then added to a 96-well polystyrene plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(39) 2) Blocking: formulating 2% of bovine serum albumin with 0.05 mol/L of carbonate buffer, which was then added to a 96-well plate at 300 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(40) 3) Loading: formulating a series of standard solutions of carcinoembryonic antigen having concentrations in the range of 0.00256-1000 ng/mL with PBS buffer to provide standard samples respectively having the following concentrations: 0 ng/mL, 0.00256 ng/mL, 0.064 ng/mL, 0.0128 ng/mL, 0.32 ng/mL, 1.6 ng/mL, 8 ng/mL; incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(41) 4) Adding the biotin-labeled antibody: formulating 1 g/mL of biotin-labeled antibody with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.mL

(42) 5) Adding avidin: formulating 5 g/mL of avidin with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 0.5 h; removing the liquid contained in each well, and washing three times with PBST washing buffer.

(43) 6) Adding NaEuF.sub.4 nanocrystal labeled biotin: formulating 10 g/mL of NaEuF.sub.4 nanocrystal labeled biotin (prepared in the above step 2 of Example 1) with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 0.5 h, removing the solution in each well, and washing six times with PBST washing buffer.

(44) 7) Adding the enhancer solution: adding the enhancer solution at 200 L per well, detecting fluorescence signals using time-resolved assays with the measurement parameters: excitation wavelength of 340 nm, emission wavelength of 615 nm, and delay time of 250 s.

(45) 8) Plotting standard curve: plotting the standard curve with the concentration of the carcinoembryonic antigen standard solution as abscissa and the fluorescence intensity corresponding to each concentration of the standard solution as ordinate. As shown in FIG. 4, in the range of 0.00256-8 ng/mL, a linear correlation was observed between the concentration of the carcinoembryonic antigen and the fluorescence intensity, wherein y=480.87x+425, R=0.9987, and the limit of detection is 0.1 g/mL, based on the blank mean plus three times the standard deviation (SD).

(46) 9) Detecting carcinoembryonic antigen by using time-resolved carcinoembryonic antigen assay kits: according to the operation of the description, plotting the standard curve. As shown in FIG. 5, in the range of 0.1-800 ng/mL, a linear correlation was observed between the concentration of the carcinoembryonic antigen and the fluorescence intensity, wherein y=12.732x+223.98, R=0.9989, and the limit of detection is 90 pg/mL, based on the blank mean plus three times the standard deviation (SD). The detection sensitivity of the present invention is 900 times higher than that of commercial time-resolved carcinoembryonic antigen assay kits.

Example 3

Comparison of the Recovery of Different Samples Determined by the Method According to the Present Invention and the Commercial Time-Resolved Carcinoembryonic Antigen Assay Kits

(47) 1) Coating: diluting the antibody against carcinoembryonic antigen to 10 g/mL with 0.05 mol/L of carbonate buffer, which was then added to a 96-well polystyrene plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(48) 2) Blocking: formulating 2% of bovine serum albumin with 0.05 mol/L of carbonate buffer, which was then added to a 96-well plate at 300 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(49) 3) Loading: formulating a series of standard solutions of carcinoembryonic antigen having concentrations in the range of 0.00256-1000 ng/mL with PBS buffer to provide standard samples respectively having the following concentrations: 0 ng/mL, 0.00256 ng/mL, 0.064 ng/mL, 0.0128 ng/mL, 0.32 ng/mL, 1.6 ng/mL, 8 ng/mL; incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing three times with PBST washing buffer.

(50) 4) Adding NaEuF.sub.4 nanocrystal labeled antibody: formulating 1 g/mL of NaEuF.sub.4 nanocrystal labeled antibody (prepared in the above step 2 of Example 1) with PBS buffer, which was then added to a 96-well plate at 100 L per well, incubating at 37 C. for 1 h, removing the liquid contained in each well, and washing six times with PBST washing buffer.

(51) 5) Adding the enhancer solution: adding the enhancer solution at 200 L per well, detecting fluorescence signals using time-resolved assays with the measurement parameters: excitation wavelength of 340 nm, emission wavelength of 615 nm, and delay time of 250 s.

(52) 6) Plotting standard curve: plotting the standard curve with the concentration of the carcinoembryonic antigen standard solution as abscissa and the fluorescence intensity corresponding to each concentration of the standard solution as ordinate. In the range of 0.00256-8 ng/mL, a linear correlation was observed between the concentration of the carcinoembryonic antigen and the fluorescence intensity, wherein y=480.87x+425, R=0.9987.

(53) 7) Determining the recovery of serum and plasma matrices according to the present invention: dividing the same serum and the plasma containing EDTA anticoagulant into two portions respectively, one of which was added with 2 ng/mL of the carcinoembryonic antigen standard solution, adding 100 L of the test samples in step 3), the other steps are the same as described above, testing each sample three times respectively, and calculating the corresponding concentration value of the test samples by putting the fluorescence intensity of the samples into the standard curve equation.

(54) 8) Determining the recovery of serum and plasma matrices by using the commercial time-resolved carcinoembryonic antigen assay kits: according to the operation of the description, plotting the standard curve. In the range of 0.1-800 ng/mL, a linear correlation was observed between the concentration of the carcinoembryonic antigen and the fluorescence intensity, wherein y=12.732x+223.98, R=0.9989, and determining the recovery of serum and plasma matrices as described in step 7).

(55) 9) Conclusion: as shown in Table 1, the recoveries of the serum matrices by the method of the present invention and by using the commercial time-resolved carcinoembryonic antigen assay kits were both above 95%, while regarding the plasma matrix containing EDTA anticoagulant, the recoveries were 95.2% and 85% respectively. Therefore it is indicated that the samples containing EDTA anticoagulant were negatively interfered by using the commercial time-resolved chelate labeled carcinoembryonic antigen assay kits, while they were not negatively interfered by the method of the present invention, therefore the method of the present invention has broader applicability.

(56) TABLE-US-00001 TABLE 1 Comparison of the recovery of different samples determined by the method of the present invention and by using the commercial time-resolved carcinoembryonic antigen assay kits. Back- Theo- ground Spiked retical Measured Average value value value value recov- Method Sample (ng/mL) (ng/mL) (ng/mL) (ng/mL) ery(%) Present Serum 5.8 2 7.8 7.6 0.2 97.4 invention Plasma 7.7 2 9.7 14.0 0.3 95.2 Commer- Serum 6.9 2 8.9 8.6 0.2 96.2 cial kits Plasma 8.9 2 10.9 9.3 0.4 85.0