NL2031621B1 - A eugenol time-resolved fluorescence immunochromatographic test strip and a preparation method thereof - Google Patents

Abstract

The present invention provides a eugenol time-resolved fluorescence immunochromatographic test strip and a preparation method thereof. Eu3+ is coupled on nanofluorescent microspheres, and coupled with eugenol monoclonal antibody and then is sprayed on the binding pad, a test line is coated and coupled with the eugenol carrier protein, a control line is coated with a goat anti-mouse IgG antibody and immobilized on a reaction membrane to form an immunochromatographic test strip, and the production parameters of the test strip are optimized, including concentration of a coupling reagent, activation time of microspheres, pH of a buffer, coating amount of the test line and the quality control line, and pore size selection of a nitrocellulose membrane, etc., so that the detection sensitivity of the prepared test strip is up to 0.1 ug/mL, which fits well with the HPLC test results. Due to good accuracy and precision, the test strip is applicable for rapid detection of eugenol residues in aquatic products, providing a method suitable for on-site rapid detection by regulatory authorities and testing organizations.

Description

A EUGENOL TIME-RESOLVED FLUORESCENCE IMMUNOCHROMATOGRAPHIC TEST STRIP AND A PREPARATION METHOD THEREOF

TECHNICAL FIELD The present invention belongs to the technical field of rapid safety detection of aquatic products, in particular to a eugenol time-resolved fluorescence immunochromatographic test strip and a preparation method thereof.

BACKGROUND Eugenol, with chemical name as 2-methoxy-4-propenyl phenol. Since its strong anesthetic effect on fish was detected in 1972, eugenol has been widely used in the field of aquatic transportation for its good effect, extensive source, high cost performance and short residual period. At present, however, there is still a great dispute with regard to the safety eugenol and its similar compounds as well as other fishing anesthetics worldwide. Relevant studies have shown that eugenol has liver toxicity, the US National Toxicology Program (NTP) believes that eugenol compounds are carcinogens or potential carcinogens to rodents, the World Agency for Research on Cancer classifies eugenol as a Group 3 carcinogen. Therefore, supervision on quality and safety of eugenol should be strengthened and especially centered on research and application of eugenol detection technology, and development of high-sensitivity, accurate and rapid detection technology, in order to effectively guarantee the quality safety of aquatic products.

Currently, eugenol detection is dependent on instrumental analysis and immunoassay technology. However, it is not feasible to achieve rapid monitoring and promotion of the instrumental analysis at the grassroots and on-site due to the high price of analytical equipment, high technical operation requirements, engagement of professional and technical personnel in operation, long analysis cycles, and complex sample pretreatment processes.

DETAILED DESCRIPTION OF THE INVENTION In order to solve the defects of the prior art, the present invention provides a eugenol time- resolved fluorescence immunochromatographic test strip and a preparation method thereof. Eu** is coupled on nano-fluorescent microspheres, and coupled with eugenol monoclonal antibody and then is sprayed on the binding pad; a test line is coated and coupled with the eugenol carrier protein, a control line is coated with a goat anti-mouse IgG antibody and immobilized on a reaction membrane to form an immunochromatographic test strip, and the production parameters of the test strip are optimized, including concentration of a coupling reagent, activation time of microspheres, pH of a buffer, coating amount of the test line and the quality control line, and pore size selection of a nitrocellulose membrane, etc., so that the detection sensitivity of the prepared test strip is up to 0.1 ug/mL, which fits well with the HPLC test results; the coefficient of variation within and between batches is respectively 2%-7.33% and 2.73%-6.97%, and the recovery rate is within 94.16%-109.27%. Due to good accuracy and precision, the test strip is applicable for rapid detection of eugenol residues in aquatic products, providing a method suitable for on-site rapid detection by regulatory authorities and testing organizations.

In one aspect, the present invention provides a time-resolved fluorescence immunochromatographic test strip for rapid detection of eugenol, and the test strip comprises a binding pad, a test line (T) and a control line (C); wherein the binding pad comprises an Eu’*- labeled nano-fluorescent microspheres-eugenol monoclonal antibody; the test line is coated with a eugenol-coupled carrier protein; the quality control line is coated with a goat anti-mouse IgG antibody.

Through numerous studies, the present invention indicates that when it is used for eugenol detection, compared with the Eu** labeled nanofluorescent microspheres prepared from other rare earth elements, such as La?’ Ce**, Er**, Yb*, Tm?’ and Pr’* labeled nano-fluorescent microsphere, the Eu®* labeled nano-fluorescent microsphere-eugenol monoclonal antibody containing rare earth element can be better coupled with the eugenol monoclonal antibody, which is conductive to significantly improving the performance of the chromatographic test strip and also the detection sensitivity of eugenol. The reason may lie in that the rare earth element Eu'*-labeled nano- fluorescent microspheres have better spatial structure to enable coupling with the eugenol monoclonal antibody. The prepared Eu’’-labeled nano-fluorescent microspheres-eugenol monoclonal antibody is characterized by more excellent luminescence properties and stronger binding ability to the eugenol monoclonal antibody (Eugenol-mAb).

In the other aspect, the present invention provides a preparation method of nano-fluorescent microspheres for rapid detection of eugenol, wherein the method comprises the following steps: (1) Prepare Eu**-labeled nano-fluorescent microspheres; (2) Wash the Eu*"-labeled fluorescent nanospheres with MES buffer; (3) Activation of carboxyl group: adding NHS solution and EDC solution to the Eu**- labeled nano-fluorescent microsphere solution obtained in the step (2), incubating it, and then adding MES buffer to resuspend the precipitation; (4) Protein coupling: adding a eugenol monoclonal antibody to the Eu°*-labeled nano- fluorescent microsphere solution obtained in the step (3), mixing it uniformly, and performing ultrasonic processing, and rotating at room temperature for 1-3 h in the dark; (5) Microspheres are enclosed.

The present invention has the following beneficial effects:

1. Strong specificity and high sensitivity. Eu?” is coupled to nano-fluorescent microspheres to obtain time-resolved fluorescent microspheres, and it is then coupled with eugenol monoclonal antibody to obtain the test strip, and the production parameters of test strips are optimized, including concentration of a coupling reagent, activation time of microspheres, pH of a buffer, coating amount of the test line and the quality control line, and pore size selection of a nitrocellulose membrane, etc., so that the detection sensitivity of the prepared test strip is up to 0.1 ng/mL, which fits well with the HPLC test results; the coefficient of variation within and between batches is respectively 2%-7.33% and 2.73%-6.97%, the recovery rate is within 94.16%-109.27%, and good accuracy and precision are provided.

2. Rapid detection, simple operation, easy to carry. When the test strip of the present invention used, no other reagents are required, it only needs to drop about 80 uL of the tested sample on the sample pad, and the test result can be determined within 20 minutes. This enables to achieve the rapid, large quantity and convenient site detection of eugenol, solving the existing problems of long and expensive instrument cycle, high operation requirements and complex sample pretreatment process in the detection;

3. The results are intuitive and accurate. The test strip obtains the fluorescence signal intensity of the T line and C line of the test strip by a portable fluorescent reading device to determine the presence of eugenol in the sample according to the T/C ratio value. The detection limit obtained by the instrument is more sensitive and objective than that by the naked eye, which will not easily cause judgment of false negatives and false positives;

4. Low cost and low investment. When the rapid detection test strip 1s used, no additional reagents are required but a fluorescence reader, and the detection can be carried out at low cost regardless of time and place, which is favorable for saving a lot of expensive instruments and equipment cost;

5. Wide range of application, and convenience in application and popularity. The rapid detection test strip disclosed by the present invention is convenient to operate, and able to meet the needs of personnel at different levels, ranging from professional testing, customs quarantine, health and disease prevention, quality monitoring, animal product processing, intensive breeding to individual breeding. Hence, it has broad market prospects, and good economic and social benefits.

DESCRIPTION OF THE ATTACHED DRAWINGS FIG. 1 is a detection result reading diagram of a time-resolved fluorescence immunochromatographic test strip for rapid detection of eugenol according to Embodiment 1.

DETAILED DESCRIPTION OF EMBODIMENTS

Preparing the time-resolved fluorescence immunochromatographic test strip for rapid detection of eugenol described in the embodiment comprises the following steps:

1. Coupling of eugenol and carrier protein The eugenol is coupled with bovine serum albumin BSA (or chicken ovalbumin OVA, preferably bovine serum albumin in this embodiment) through a carbodiimide method, to obtain a coating antigen (for immobilization on the reaction membrane) and an immunogen (for preparation of monoclonal antibodies) antigen. The specific method is described as follows: dissolve 0.2 mmol eugenol in I mL N N-dimethylformamide (DMF), add 0.25 mmol N,N-dicyclohexylcarbodiimide (DCC), and stir it for 15 min's reaction, then add 0.25 mmol N-hydroxysuccinimide (NHS), stir it at room temperature for reaction overnight, centrifuge it at 4000 rpm for 10 min, and add 300 pL supernatant slowly to 6 mL CBS (carbonate buffer) solution containing 120 mg BSA (or OVA), and stir to react for 4 h. After the reaction is over, place the solution to dialysis bag with a molecular weight of 14,000 and dialyze it with 0.01 mol/L PBS for 3 d, and change the medium 3-4 times a day. Take it out and store in a -20°C refrigerator.

2. Preparation of eugenol monoclonal antibody (1) Animal immunization: inject the immunogen into Balb/c mice at a dose of 100 ug/mouse to produce antiserum.

(2) Cell fusion and cloning: if the mouse serum is tested high, take out the spleen cells and fuse it with SP2/0 myeloma cells according to 7:1 (quantitative proportion), and determine the cell supernatant by indirect competitive ELISA, and screen positive wells. Clone the positive pores by limiting dilution method until a monoclonal antibody-secreting hybridoma cell line is obtained.

(3) Cryopreservation and recovery of cells: add cryopreservation solution to the monoclonal hybridoma cell line to produce 110° cells/mL cell suspension, and store it in liquid nitrogen for a long time. When resuscitated, take out the cryovial, immediately place it into a 37°C water bath for fast melting, remove the cryopreserved solution by centrifugation action, and transfer it into a culture bottle for culturing.

(4) Production and purification of monoclonal antibodies: inject sterilized paraffin oil to the Balb/mouse intraperitoneally according to 0.5 mL/mouse, and 7 days later, inject monoclonal hybridoma cell lines with eugenol intraperitoneally by 5x107/mouse, and 7 d later, collect the ascites. Purify the ascites by the octanoic acid-saturated ammonium sulfate method to obtain a eugenol monoclonal antibody (Eugenol-mAb), and store it at -20°C.

3. Preparation of fluorescent microspheres (1) Preparation of Eu**-labeled nano-fluorescent microspheres: take carboxylated polystyrene nano-microspheres, add 100 ml of a mixture of deionized water and acetone (the volume ratio of deionized water and the acetone is 1:1) so that the content of carboxylated polystyrene microspheres is 1wt%; stir it evenly, add 0.1 mol/L Eu(NOs); solution 60 uL, 0.1 mol/L B- naphthoyl trifluoroacetone 150 ul, 0.1 mol /L trioctylphosphine oxide 150 pL successively, make it react at room temperature for 16 h, remove the organic solvent by vacuum distillation, and use centrifugation.

5 (2) Wash the Eu**-labeled fluorescent nanospheres with MES buffer: conduct ultrasonic processing on the Eu*'-labeled nanofluorescent microspheres (solid content 1%) for 2 min and shaking it well, take 100 uL into a 2 mL centrifuge tube, centrifuge at 13000 rpm under 4°C for min to remove the supernatant, add 400 uL MES buffer (0.05 mol/L, pH 6.0), start ultrasonic processing again, mix it evenly and use centrifugation; repeat this step to remove the supernatant, 10 and then add 800 uL MES buffer (0.05 mol/L, pH 6.0), and make it evenly by ultrasonic processing; (3) Carboxyl activation: add 45 pL. 100 mg/mL NHS solution, mix it well, add 25 uL 100 mg/mL EDC solution, mix it well and incubate at room temperature for 30 min in the dark, centrifuge at 13000 rpm and 4 °C for 10 min to remove the supernatant, add 1 mL of MES bufter to resuspend the precipitation, make ultrasonic processing evenly, and use a fluorescent flashlight to detect the presence of any particles visible to the naked eye in the solution, if any, start ultrasonic processing again, (4) Protein coupling: add 100 ug Eu**-labeled eugenol monoclonal antibody in the activated 100 uL microsphere solution, use a vortex to mix it up, after reaction for 1-2 min, apply ultrasonic processing for 30 s, and rotate it for reaction at room temperature in the dark for 2 h; (5) Blocking of microspheres: after protein coupling, remove the sonicator for 30 s, then add pL blocking solution (15% BSA), use a vortex to mix, and rotate for 1 h at room temperature in the dark, 13000 rpm, 4°C, centrifuge for 10 min to remove the supernatant, remove the uncoupled antibody, add 100 uL microsphere reconstituted solution, apply ultrasonic processing for 2 min to resuspend the precipitation, and then store it at 4°C in the dark for later use.

25 4. Assembly of the test strip Paste the sample pad, bonding pad, and water-absorbing pad on the PVC bottom plate that is pasted with a drying NC membrane, and install it. The assembly sequence is as follows: (DPaste NC membrane: paste a NC membrane on the relative position of the 60 mm x 300 mm PVC base plate, and then carry out membrane scribing, make sure the C line is above the T line, and coat the goat anti-mouse secondary antibody and immobilized antigen; (2)paste the water-absorbing pad: cut the water-absorbing pad to a size of 17 mm*300 mm, and paste it on the top of the NC membrane near the C line and keep overlapped with the upper edge of the NC membrane by 1.8-2 mm; (3paste the binding pad: cut the binding pad sprayed with fluorescent microsphere labeled antibody along the edge of the fluorescent strip and paste it under the NC membrane near the T line, and make it overlapped with the lower edge of the NC membrane by 1-

1.5mm; @ paste the sample pad: paste a 17 mm x 300 sample pad under the binding pad; Gut the strips: cut the pasted PVC base plate into a size of 60 mm x 4 mm by a strip cutter, place it into the corresponding groove, and put it in a sealed bag with a desiccant at 4°C for storage in the dark.

5. Reaction principle of an immunochromatographic test strip for rapid detection of eugenol Take 80 uL negative and positive samples respectively, and add them to the sample feeding hole of the groove, let it react for 15 min reaction time, and wait for color development. Its color development principle: the sample solution moves upward along the test strip under the action of capillary, and performs competitive reaction on the reaction membrane. For the negative sample without eugenol in the solution, the eugenol antibody coated with fluorescent label on the binding pad specifically binds to the immobilized complete antigen on the T line by virue of chromatography, and the remaining antibody binds to the goat anti-mouse IgG immobilized on the C line, under the irradiation of ultraviolet light, both T and C lines develop color, forming a bright orange-red strip; positive samples containing eugenol in the solution, first specifically binds to the fluorescently labeled antibody coated on the binding pad, then the remaining unbound antibody binds to the T-line immobilized antigen. with the increasing of eugenol concentration, the fluorescent probe antibody bound to the T-line antigen is decreased, and the fluorescent strip becomes lighter in color until colorless, then the antigen-antibody conjugate and the unbound antibody continues to bind to the goat anti-mouse secondary antibody immobilized on the C line by the upward chromatography, and the C line develops color.

6. Preparation of test sample solution: It is simply prepared by collecting aquaculture water in the aquatic product testing and transportation process, and centrifuging it to remove solid impurities.

7. Detection operation method: take out the test strip from the packaging bag, take the tested solution with a dropper, add 3 drops (about 80 pL) into the sample feeding hole and start counting, and read the result within 20 minutes.

8. Qualitative judgment of test results: Take 80 pL negative and positive samples respectively, and add them into the sample feeding hole on the groove. Illuminated by the fluorescent flashlight, it can be clearly seen that the Eu" labeled nano-fluorescent microspheres makes quick tomographic movement upward under the action of capillaries, and reacts with the coated antigen and goat anti-mouse secondary antibody immobilized thereon when passing by the NC membrane pass, so that the T line and the C line develop color, and the color development result is judged after reaction for 15 minutes. The color development result of the test strip is shown in Figure 1: if the T line on the cellulose membrane is lighter than the C line (two bright orange marks "II") or only C line is shown (one bright orange mark "I") under the fluorescent or ultraviolet irradiation, two bright orange marks "II" are shown,

and the it reads T/C < 1, the test result is positive, indicating that there is presence of eugenol drug in the tested sample liquid, if two bright orange marks "II" are displayed on the cellulose membrane on the test strip for rapid detection of eugenol drugs, and the T line is darker than or the same as the C line, and it reads T/C > 1, the test result is negative, indicating that eugenol drug is not contained in the tested sample liquid, it C line is not shown, it may be caused by improper operation or invalid reagent plate.

9. Quantitative judgment of test results: the concentration of standard eugenol sample has a linear relationship with the T/C value, and the concentration of eugenol is quantitatively detectable by developing a standard curve.

Selection of immunization time In the embodiment, the test strip is prepared according to the method stated in embodiment

1. Negative samples and positive samples are added respectively for spotting test, and T line and C line fluorescence intensity and T/C ratio of test strips are recorded at 5, 10, 15, 20, 25 and 30 min after starting the immune response, the fluorescence signal intensity of T line and C line are recorded every 5 minutes according to the reaction time by using a fluorescence immunochromatographic test strip reader, and with the immunochromatographic reaction time of the strip as horizontal axis, the fluorescence signal intensity of T and C lines and the T/C ratio as the vertical axis, a kinetic curve of the test strip is drawn.

With the progress of the immunochromatographic reaction time, there are more antibodies and antigen-antibody complexes flowing to the T and C lines, the immobilized antigens and goat anti-mouse IgG coated on the lines are captured, and the fluorescence signal intensity gradually increases. When the immunization time is more than 20 min, the change slows down and tends to be stable, so 20 min after the reaction started is considered as the optimal detection time.

Verification of test strip sensitivity In the embodiment, for the eugenol immunochromatographic test strips prepared according to the optimized conditions of the above embodiments, standard eugenol samples with different concentrations (0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1.0 ng/mL) are respectively added to the negative sample to prepare standard eugenol solutions with different concentrations. Add the solution on the test strip, repeat the detection of each concentration for 5 times, and take the T/C ratio of the test strip as the vertical axis and the exponential value of the phenol standard concentration as the horizontal axis to draw a standard curve.

Between 0.05 pg/mL and 0.4 pg/mL, the logarithm of the standard eugenol concentration basically keeps a linear relation with the T/C value, and the linear equation is y=-2.415Inx+2.5894, R?=0.994. As learned from the reading results of the instrument, when the concentration of standard eugenol sample is greater than 0.1 pg/mL, and the T/C ratio is < 0.94, it is concluded as a positive result according to the judgment standard. When the concentration of standard eugenol sample is 0.3 ug/mL, under UV light irradiation, it is possible to clearly see the difference from the negative sample by naked eye; when the concentration of standard eugenol sample is 1.0 ng/mL, the T line will not be shown.

To sum up, when the elimination concentration of eugenol immunofluorescence chromatography test strip is 1.0 pg/mL, the detection limit of naked eye can be 0.3 pg/mL, and the detection limit of instrument is 0.1 pg/mL.

Verification of test strip precision and accuracy In the embodiment, an HPLC standard curve for eugenol concentration is developed.

Respectively, the fluorescent immunochromatographic test strips provided by embodiment 1 and the HPLC instrument are used to jointly detect the negative samples with spike concentration of

0.1,0.15,0.2, 0.3 and 0.4 pg/mL, and calculate the recovery rate of the test strip, thus to verify the detection accuracy of eugenol by the test strip. Verification is made to the spike recovery of the test strip, and compared with the test results of instruments with high accuracy, the recovery rate is required to be generally within +15% of the positive and negative deviations, that is, the difference between the measured value and the true value should not exceed 15%.

Errors within and between batches are represented by the coefficient of variation within and between batches of the test strips, in order to demonstrate the precision of test strips. The coefficient of variation within batches is the T-line and C-line signal values as well as the T/C value of the same batch of fluorescent immunochromatographic test strips for quantitative detection of the spike samples with 5 concentrations (0.1, 0.15, 0.2, 0.3 and 0.4 pg/mL), and such values are subject to analysis of dispersion degree, and each concentration is determined for 5 times; the coefficient of variation between batches is the T-line and C-line signal values as well as the T/C value of the different batches of fluorescent immunochromatographic test strips for quantitative detection of the spike samples with 5 concentrations (0.1, 0.15, 0.2, 0.3 and 0.4 ug/mL), and such values are subject to analysis of dispersion degree, and each concentration is determined for 5 times.

The test results are shown in Table 1. As seen from Table 1, the recovery rate of the test strip is basically kept between 94% and 109%, and both the coefficient of variation within and between batches are below 8%, showing good precision. At the same spike concentration, a coordinate axis is established with the detection result of the test strip as the horizontal axis, and the detection result of HPLC as the vertical axis, and linear fitting is performed to obtain the equation is y=-

1390.1x+1767.9, R?=0.995. From the above, it can be judged that the test strip is perfectly consistent with the HPLC detection results.

Table 1 Comparison of fluorescent immunochromatographic test strips and HPLC methods

Spike Test strip (n=5) HPLC (n=5) content Recovery rate (%) Within batch Between Recovery rate (%) CV (%) pg/mL +SD CV (%) batches CV +5D (%)

0.10 94.1641.68 2.00 5.19 95.53+£1.57 1.64

0.15 98.89+2 49 2.82 441 102.98+41.14 1.11

0.20 98.4242 34 2.66 6.97 108.24+1.51 1.39

0.30 98.09+5.94 6.76 2.73 106.77+0.74 0.69

0.40 109.27+7.16 7.33 5.55 94.89+1.15 1.21 Stability verification of test strips The eugenol immunofluorescence test strip prepared according to the embodiment mainly comprises two parts, namely a test strip and a fluorescent probe; wherein the test strip is stored at room temperature, and the fluorescent probe needs to be stored at 4-8°C. To verify the storage period and stability of the test strip, the test strips in the same batch is divided into 2 bags and sealed up for storage. One bag is placed in a 50°C oven for one month, which has the same effect as storage for one year at room temperature; then an accelerated destruction stability experiment is carried out, and the T/C ratio is determined every 7 days; and another bag is placed in a 37°C oven for one month for control. The test is conducted respectively with negative samples spiked with medium, low and high concentrations, and the T/C values in different time periods are recorded, with the results as shown in Table 2. Table 2 Stability test of immunochromatographic test strips Spike content pg/mL 0.2 0.5 0.8 37°C 7 0.6246+0.0229 0.1585+0.0337 0.0541+0.0057 acceleration 14 0.6022+0.0207 0.1722+0.0237 0.0491+0.0014 28 0.5814+0.0218 0.1622+0.0412 0.0519+0.0052 50°C 7 0.5764+0.0416 0.1519+0.0511 0.0499+0.0065 acceleration 14 (.5988+0.0388 0.1692+0.0348 0.0505+0.0029 28 0.5867+0.0470 0.1794+0.0431 0.0514+0.0030 According to the results given in Table 2, it can be concluded through analysis that basically there is no major change seen in T/C values of the test strips stored at 37°C and 50°C for 7 d, 14 d and 28 d, and the values are relatively stable, which meet the normal use requirements of 1 year at room temperature.

Claims (2)

CONCLUSIESCONCLUSIONS 1. Een teststrip om de tijd opgeloste fluorescentie-immuniteit chromatografie van syringol snel te testen, met kenmerken van combinatiepad, testlijn en kwaliteit controleren lijn. De combinatiepad hierboven genoemd bevat de nano fluorescerende microsferen die gemerkt van Eu" - monoklonaal syringol; de testlijn bevat de drager eiwit van de geconjugeerde syringol; de kwaliteit controleren lijn bevat geit anti-muis IgG antilichaam.1. A test strip to quickly test the time-resolved fluorescence immunity chromatography of syringol, with characteristics of combination path, test line and quality control line. The combination pad mentioned above contains the nano fluorescent microspheres labeled with Eu" - monoclonal syringol; the test line contains the carrier protein of the conjugated syringol; the quality check line contains goat anti-mouse IgG antibody. 2. Een manier om nano fluorescerende microsferen van syringol snel te testen met kenmerken in volgende stappen: Om nano fluorescerende microsferen die met Eu** wordt gemerkt te produceren: Om nano fluorescerende microsferen schoon te maken met MES bufferen was vloeistof, Activering van hydroxylgroep: Voeg NHS oplossing en EDC oplossing in nano fluorescerende microsferen oplossing met Eu*” merk die in de vorige stap gekregen wordt toe om te incuberen. Voeg MES bufferen oplossing toe om te resuspenderen en neerslagen; Eiwit conjugeren: Voeg de monoklonale antilichamen van syringol toe in de nano fluorescerende microsferen oplossing met Eu’ merk die in de vorige stap gekregen wordt. Dan meng het en gebruik echografie. Plaats het in de omgeving van binnentemperatuur en gebruik rotatie reactie voor 1 uur tot 3 uur; Microsferen is gesloten.2. A way to quickly test nano fluorescent microspheres of syringol with characteristics in following steps: To produce nano fluorescent microspheres labeled with Eu**: To clean nano fluorescent microspheres with MES buffer wash liquid, Activation of hydroxyl group: Add NHS solution and EDC solution in nano fluorescent microspheres solution with Eu*” label obtained in the previous step to incubate. Add MES buffering solution to resuspend and precipitate; Conjugate protein: Add the monoclonal antibodies of syringol in the Eu' labeled nano fluorescent microsphere solution obtained in the previous step. Then mix it and use ultrasound. Place it in the environment of indoor temperature and use rotational reaction for 1 hour to 3 hours; Microspheres is closed.