CN116338171A - Washing-free homogeneous detection method - Google Patents
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Abstract
The invention belongs to the field of medical inspection, food safety and environmental monitoring detection, and particularly relates to a washing-free homogeneous detection method. The invention uses nano ZnO coupling antibody as immune probe, captures target object with immune magnetic bead to form double-antibody sandwich compound to separate target object, then uses HNO 3 ZnO in the cleavage complex releases a large amount of Zn 2+ The invention provides a wash-free homogeneous detection strategy which uses ZnO as a beacon carrier for the first time and cleaves ZnO to release Zn by gathering AIE dye molecules containing carboxyl to generate strong fluorescence and recording the fluorescence signal intensity to quantitatively detect the concentration of a target object in a sample 2+ Initiating AIE to achieve signal releaseLarge and no-wash. Compared with the traditional ELISA, the method has the advantages of simpler steps, easy operation, elimination of the plate washing step and higher sensitivity.
Description
Technical Field
The invention belongs to the field of medical inspection, food safety and environmental monitoring detection, and particularly relates to a washing-free homogeneous detection method.
Background
Fluorescence is a type of photoluminescence phenomenon induced by self-electronic transition after a substance receives an external light source. Generally, the concentration of the fluorescent dye in the system is increased, the fluorescence intensity is also increased, however, most of the traditional fluorescent dyes are in an aggregation state, and a fluorescence quenching phenomenon called an aggregation-induced quenching effect (ACQ) occurs, so that scientists have to reduce the concentration of the fluorescent dye for a long time to prevent the ACQ effect, and the wide application of the fluorescent dye in the fluorescence field is affected. The AIE effect is contrary to the ACQ effect, and AIE dye has weak fluorescence or even no fluorescence at low concentration in a system, and shows extremely strong fluorescence at high concentration or even pure product. Compared with the traditional dye, the AIE dye has lower background, higher signal output and extremely high signal-to-noise ratio, and is greatly beneficial to the application in various industrial fields.
Homogeneous phase detection is a common detection means for clinical diagnosis, food safety, environmental monitoring and the like, wherein an enzyme-linked immunosorbent assay (ELISA) is one of classical representatives. Compared with an instrument method, the homogeneous detection does not need expensive instruments, and the detection mode is flexible. However, the conventional homogeneous phase detection requires a complex and professional operation flow, especially the plate washing operation is more time-consuming and labor-consuming, and has high requirements on operators. Therefore, a simple and rapid homogeneous detection strategy is one of the development trends.
Disclosure of Invention
Based on the above situation, the inventor constructs a washing-free homogeneous phase rapid detection strategy based on the AIE initiated by ZnO nano probe cleavage, takes ZnO as a probe, takes magnetic enrichment as a means for separating a target object, and releases a large amount of Zn after ZnO is cleaved 2+ The fluorescent dye reacts with AI E dye molecules to trigger AIE to generate strong fluorescence, thereby achieving the function of signal amplification, improving the detection sensitivity and simultaneously realizingThe whole process has no washing effect. At present, no similar report exists. The technical scheme is as follows:
a washing-free homogeneous detection method comprises the following steps:
(1) Adding a sample solution to be detected into a solution containing ZnO immune probes and immune magnetic beads, magnetically separating after incubation, discarding supernatant, and adding water to disperse and precipitate;
(2) Adding HNO 3 Digesting the solution, then adjusting the pH value to be neutral, adding an AIE dye solution containing carboxyl, and calculating the concentration of the to-be-detected object in the to-be-detected sample solution by recording the fluorescence signal intensity;
the ZnO immune probe is obtained by coupling nano ZnO with an antibody of a to-be-detected object; the immunomagnetic beads are obtained by coupling superparamagnetic ferrite with an antibody of a to-be-detected object.
The invention takes nano ZnO coupling antibody as immune probe, adds the immune magnetic bead into the sample to be detected, captures the target object to form double-antibody sandwich compound, then magnetically separates the target object, and takes HNO 3 ZnO in the cleavage complex releases a large amount of Zn 2+ ，Zn 2+ And the fluorescent signal intensity is recorded to quantitatively detect the concentration of the target in the sample.
Wherein the object to be detected is mainly macromolecular substance, and is cell, bacteria or protein.
Preferably, the ZnO immuno-probe is prepared by the following process: adding nano ZnO into borate buffer solution, adding an antibody of a to-be-detected object for marking, adding casein solution for sealing, and finally centrifuging, wherein the lower layer of sediment is the ZnO immune probe. Wherein the marking time is 2h; the closing time was 1h.
Preferably, the particle size of the nano ZnO is 0.05-10 mu m, and the morphology is nano flower, nano sphere or nano rod. In the ZnO immune probe, the antibody coupling amount is 4 mug/mg-200 mug/mg.
Preferably, the immunomagnetic beads have a particle size of 0.1 μm to 5. Mu.m.
Preferably, the carboxyl group-containing AIE dye is a tetraphenyl ethylene, a silole or a triphenyl amine.
Preferably, the incubation time is 10min, the magnetic separation time is 3min, and the digestion time is 1min.
Preferably, the solution comprising ZnO immunoprobes and immunomagnetic beads is obtained by the following procedure: dispersing ZnO immune probes in PBS (phosphate buffer solution) to obtain a ZnO immune probe solution, dispersing immune magnetic beads in the PBS solution to obtain an immune magnetic bead solution, and then uniformly mixing the ZnO immune probe solution and the immune magnetic bead solution; in step (2), HNO 3 The concentration of the solution is 0.1M-2M, and the concentration of the AIE dye solution containing carboxyl is 0.1mM-10mM; znO immune probe solution and HNO 3 The volume ratio of the solution to the carboxyl group-containing AIE dye solution was (1. Mu.L-10. Mu.L): (2. Mu.L-20. Mu.L): (1. Mu.L-10. Mu.L).
The beneficial effects of the invention are as follows: the washing-free homogeneous phase detection strategy provided by the invention takes ZnO as a beacon carrier for the first time, and cleaves ZnO to release Zn 2+ AIE is initiated to achieve the purposes of signal amplification and no-clean. Compared with the traditional ELISA, the method has the advantages of simpler steps, easy operation, elimination of the plate washing step and higher sensitivity.
Drawings
FIG. 1 is a schematic flow chart and a schematic diagram of a detection method of the invention, wherein 1 is ZnO immuno-probe, 2 is immunomagnetic beads, 3 is target, and 4 is Zn 2+ 5 is AIE dye containing carboxyl;
FIG. 2 shows Zn 2+ A hydrated particle size diagram of an aggregate formed after coordination with a carboxyl-containing AIE dye TCBPE (tetracarboxyl-biphenyl);
FIG. 3 is a diagram showing the feasibility verification of the detection method of the invention;
FIG. 4 is a diagram showing the detection object of the detection method of the present invention;
FIG. 5 is a standard curve of the detection method of the present invention;
FIG. 6 is a diagram showing the detection of an actual sample by the detection method of the present invention;
FIG. 7 is a diagram showing a conventional fluorescent ELISA detection object;
FIG. 8 shows a standard curve of a conventional fluorescent ELISA.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention.
Example 1:
the principle of the detection of E.coli O157:H27 based on a washing-free homogeneous detection strategy of AIE initiated by ZnO nano probe cleavage is shown as figure 1, and the homogeneous detection strategy comprises the following steps: incubation, magnetic separation, digestion and luminescence. Based on the principle of double antibody sandwich, after capturing and separating the target object, the target object and ZnO form a positive correlation relation of quantity, and based on the principle of action of acid and metal oxide, znO is digested, and a large amount of Zn is released 2+ The AIE molecule aggregation is initiated, the signal output is greatly amplified, and the sensitivity is improved.
As shown in FIG. 2, zn 2+ After coordination with carboxyl groups in the carboxyl-containing AIE molecules, the AIE molecule aggregate is initiated to form, and the aggregate has an average hydration particle size of about 220nm, which proves that Zn 2+ Aggregation of carboxyl-containing AIE molecules may be initiated.
The method comprises the following specific steps:
(1) Preparation of ZnO immune probe: mu.L of the freshly prepared anti-E.coli O157:H7 monoclonal antibody solution (100. Mu.g/mL) was added to 2mL of boric acid-sodium borate buffer (0.02M, pH 8) with 20. Mu.L of nano ZnO (10 mg/mL,200nm spheres), labeled for 2H, then 200. Mu.L of casein solution (0.5%) was added, and blocked for 1H. After the reaction, centrifuging at 4deg.C at 5000r/min for 10min, discarding supernatant, dispersing the lower precipitate with 200 μl PBS (0.01M, pH 7.4), and preserving at 4deg.C;
(2) And (3) standard curve establishment: e.coli O157:H7 was diluted with PBS (0.01M, pH 7.4) to 0, 5X 10 concentrations, respectively 1 ，1×10 2 ，5×10 2 ，10 3 ，10 4 ，10 5 ，10 6 CFU/mL. 1mL of this mixture was added to a centrifuge tube to which 5. Mu.L of ZnO immunoprobe and 10. Mu.L of immunomagnetic beads were previously added. After incubation for 10min, the mixture was magnetically separated by a magnetic rack for 3min, the supernatant was discarded, 200. Mu.L of water was added for precipitation, and 5. Mu.L of HNO was added 3 (1M) digestion for 1min, neutralization of pH by adding 5. Mu.L NaOH (1M), addition of 3. Mu.L TCBPE (0.01 mM, carboxyl-containing AIE dye), fluorescent microplate readerRecording fluorescent signals (excitation: 365nm; emission: 510 nm), and establishing a standard curve between the concentration of the target and the fluorescent signals;
(3) Sample detection: 1mL of a sample to be tested of unknown concentration was added to a centrifuge tube previously charged with 5. Mu.L of ZnO immunoprobe and 10. Mu.L of immunomagnetic beads. After incubation for 10min, the mixture was magnetically separated by a magnetic rack for 3min, the supernatant was discarded, 200. Mu.L of water was added for precipitation, and 5. Mu.L of HNO was added 3 (1M) digestion for 1min, adding 5 mu L NaOH (1M) to neutralize the pH value, adding 3 mu L TCBPE (0.01 mM), recording fluorescent signals by a fluorescent enzyme-labeling instrument, substituting the fluorescent signals into a standard curve, and calculating the concentration of the to-be-detected object.
According to the above specific steps (1) and (3), 4 experimental groups were set up, and each of the groups was performed in 4 wells, and the information of each well was shown in Table 1.
TABLE 1
Wherein "+" indicates addition and "-" indicates no addition. As shown in Table 1 and FIG. 3, only (4) the E.coli O157:H27 (E.coli O157:H27), which is added with all the required reagents and targets, showed strong fluorescence, confirming the feasibility of the strategy.
According to the step (2), 4 parallel sets are arranged for each concentration, and the final experimental physical result is shown in fig. 4, and as the concentration of the target object E.coli O157:H27 in the sample increases, the fluorescence intensity correspondingly increases. The fluorescence signals are read by a fluorescence multi-well plate reader, the specific data are shown in fig. 5, the data are processed and a corresponding standard curve is established as follows: y= 1.0763ln (x) -3.7188, the linear range is: 50-10 3 CFU/mL, the lowest limit of detection is: 31CFU/mL.
According to the step (3), after adding any amount of E.coli O157:H27 into milk samples purchased in supermarket, detecting according to the given steps, wherein the final result physical diagram is shown in fig. 6, and the concentration of the detected objects is calculated from left to right after the reader reads fluorescence: 872 917,1.24 ×10 5 ，429，396，<50CFU/mL。
Example 2: comparison with conventional fluorescent ELISA methods
Routine fluorescent ELISA assay procedure:
(1) Fluorescent dye TCBPE labeled secondary antibody: 10 mu L of TCBPE (1 mg/mL) is added into 1000 mu L of goat anti-mouse secondary antibody solution (0.1 mg/mL), after labeling for 2 hours, the dialysis bag is dialyzed for 3 days to remove the superfluous unlabeled TCBPE;
(2) And (3) multi-antibody coating: 50. Mu.L of anti-E.coli O157:H2 7 rabbit polyclonal antibody solution (0.02 mg/mL) was added to ELIS A plate wells, incubated for 30min, and plates were washed 3 times after plating. The plates were then washed 3 times with BSA solution (1%) for 30 min.
(3) And (3) standard curve establishment: e.coli O157:H7 was diluted with PBS (0.01M, pH 7.4) to 0, 5X 10 concentrations, respectively 1 ，1×10 2 ，5×10 2 ，10 3 ，10 4 ，10 5 ，10 6 CFU/mL. 200 mu L of the mixture is added into ELISA plate holes which are coated with the polyclonal antibody in advance, after incubation for 30min, the excessive polyclonal antibody solution is removed, and the mixture is washed for 2-3 times by using a washing liquid. Then 50. Mu.L of anti-E.coli O157:H2 7 mab solution (0.01 mg/mL) was added, after 30min incubation, the excess mab solution was removed and washed 3 times with wash solution. After further addition of 50. Mu.L of TCBPE-labeled secondary antibody solution (0.01 mg/mL) and incubation for 30min, the excess TCBPE-labeled secondary antibody solution was removed and washed 3 times with a washing liquid. Finally, 200 mu LPBS (0.01M, pH 7.4) is added, and the mixture is sent to a fluorescence microplate reader to read fluorescence signals (excitation: 365nm; emission: 510 nm), and a standard curve between the concentration of the target and the fluorescence signals is established.
According to the above steps, 4 parallel sets are arranged for each concentration, and the final experimental physical result is shown in fig. 7, and as the concentration of the target object e.coli O157: H7 in the sample increases, the fluorescence intensity correspondingly increases. The fluorescence signals are read by a fluorescence multi-well plate reader, the specific data are shown in fig. 8, the data are processed and a corresponding standard curve is established as follows: y=0.467 ln (x) -2.6504, linear range: 10 3 -10 6 CFU/mL, the lowest limit of detection is: 291CFU/mL.
As can be seen from the comprehensive examples 1 and 2, compared with the traditional fluorescence ELISA, the method provided by the invention has the advantages that the operation steps are simple and quick, the no-wash detection is realized, and the final sensitivity is improved by nearly one order of magnitude.
The present invention is not limited to the above embodiments, but is merely preferred embodiments of the present invention, and the present invention should be construed as being limited to the above embodiments as long as the technical effects of the present invention are achieved by the same means. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.
Claims (10)
1. The washing-free homogeneous detection method is characterized by comprising the following steps of:
(1) Adding a sample solution to be detected into a solution containing ZnO immune probes and immune magnetic beads, magnetically separating after incubation, discarding supernatant, and adding water to disperse and precipitate;
(2) Adding HNO 3 Digesting the solution, then adjusting the pH value to be neutral, adding an AIE dye solution containing carboxyl, and calculating the concentration of the to-be-detected object in the to-be-detected sample solution by recording the fluorescence signal intensity;
the ZnO immune probe is obtained by coupling nano ZnO with an antibody of a to-be-detected object; the immunomagnetic beads are obtained by coupling superparamagnetic ferrite with an antibody of a to-be-detected object.
2. The wash-free homogeneous assay of claim 1, wherein the ZnO immuno-probe is prepared by the process of: adding nano ZnO into borate buffer solution, adding an antibody of a to-be-detected object for marking, adding casein solution for sealing, and finally centrifuging, wherein the lower layer of sediment is the ZnO immune probe.
3. The wash-free homogeneous assay of claim 2, wherein the labeling time is 2 hours; the closing time was 1h.
4. The method for washing-free homogeneous phase detection according to claim 1, wherein the particle size of nano ZnO is 0.05-10 μm, and the morphology is nano flower, nano sphere or nano rod.
5. The method for wash-free homogeneous assay according to claim 1, wherein the amount of antibody coupling in the ZnO immuno-probe is 4 μg/mg to 200 μg/mg.
6. The method for wash-free homogeneous assay according to claim 1, wherein the immunomagnetic beads have a particle size of 0.1 μm to 5. Mu.m.
7. The method for washing-free homogeneous phase detection according to claim 1, wherein the carboxyl group-containing AIE dye is tetraphenyl ethylene, silole or triphenyl amine.
8. The method according to claim 1, wherein the sample is a cell, a bacterium or a protein.
9. The wash-free homogeneous assay of claim 1, wherein incubation time is 10min, magnetic separation time is 3min, and digestion time is 1min.
10. The wash-free homogeneous assay of claim 1, wherein the solution comprising ZnO immunoprobes and immunomagnetic beads is obtained by: dispersing a ZnO immune probe in a PBS (phosphate buffer solution) to obtain a ZnO immune probe solution, dispersing immune magnetic beads in the PBS to obtain an immune magnetic bead solution, and uniformly mixing the ZnO immune probe solution and the immune magnetic bead solution; in step (2), HNO 3 The concentration of the solution is 0.1M-2M, and the concentration of the AIE dye solution containing carboxyl is 0.1mM-10mM; znO immune probe solution and HNO 3 The volume ratio of the solution to the carboxyl group-containing AIE dye solution was (1. Mu.L-10. Mu.L): (2. Mu.L-20. Mu.L): (1. Mu.L-10. Mu.L).
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