CN112485236A - Homogeneous phase visualization and double-fluorescence signal analysis method based on multiple selective recognition reactions and application - Google Patents
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Abstract
The invention provides a homogeneous phase visualization and double fluorescence signal analysis method based on multiple selective recognition reactions, relating to the technical field of diagnosis and analysis2+And Cu2+A complex with PPi, and Ce3+Selectively identifying PPi and other phosphates to obtain fluorescence signals of QDs and PPi-Ce CPNs, and quantifying a single target based on the fluorescence signals of QDs and PPi-Ce CPNs. The invention uses PPi-Ce CPNs and QDs as double-fluorescence signal molecules to quantitatively analyze the target object, improves the accuracy of the method, and simultaneously uses the luminescent nano material Ce3+The visual POCT analysis is realized by introducing biochemical and medical diagnosis. And the assay method of the present invention can be used for assaying pyrophosphatase or alkaline phosphatase.
Description
Technical Field
The invention relates to the technical field of biomedical diagnosis and analysis methods, in particular to a homogeneous phase visualization and double fluorescence signal analysis method based on multiple selective recognition reactions and application thereof.
Background
In the existing biochemical and medical diagnosis systems, the quantitative analysis of a single target object is realized mainly by depending on the signal intensity of a single signal molecule. For example, enzyme-linked immunosorbent assay (ELISA) based on immune recognition reaction uses an ultraviolet-visible spectrophotometer to monitor ultraviolet signals, an electrochemiluminescence strategy to monitor electrochemical signals of ruthenium tripyridine and the like, and a fluorescence strategy to monitor fluorescence signals of fluorescent dyes (such as FITC, FAM and the like).
Although, there are few researchers using dual signal molecules to achieve target analysis, it is mainly based on one signal molecule as an internal standard and quantitative determination based on the change of another signal molecule, which is called a ratio strategy. Furthermore, in the above ratio strategy, there is no interaction between the two signal molecules, i.e. strictly speaking the system only affects a single signal molecule, and not a dual signal strategy.
Disclosure of Invention
The invention aims to provide a homogeneous phase visualization and dual fluorescence signal analysis method based on multiple selective recognition reactions, so as to solve the problems that the quantification of a target object is realized by a single signal molecule and the target object is difficult to visually read in the prior art. Furthermore, even if there are few researchers using dual signal molecules to achieve target analysis, it is a rate strategy and not a technical problem of the dual signal strategy.
The invention also aims to provide application of the homogeneous phase visualization and double fluorescence signal analysis method based on multiple selective recognition reactions.
In order to achieve one of the above objects, an embodiment of the present invention provides a homogeneous visualization and dual fluorescence signal analysis method based on multiple selective recognition reactions, the method comprising selective recognition of Cu based on QDs2+And Cu2+A complex with PPi, and Ce3+Selectively identifying PPi and other phosphates to obtain fluorescence signals of QDs and PPi-Ce CPNs, and quantifying the fluorescence signals of the QDs and PPi-Ce CPNsA single target.
According to a preferred embodiment, the Cu2+The complex formed with PPi comprises PPi-Cu2+-a PPi complex.
According to a preferred embodiment, the selective recognition of Cu based on QDs2+And PPi-Cu2+-PPi complex to obtain the fluorescence signal of QDs. Involving the use of QDs and Cu2+And PPi-Cu2+-PPi complexes for selective cation exchange reactions, based on Cu2+And PPi-Cu2+The PPi complexes quench the QDs fluorescence signal to different degrees, respectively, so that each system produces a distinct and visible color change.
According to a preferred embodiment, the Cu2+Can remarkably quench the fluorescence signal of QDs, the PPi-Cu2+-PPi complexes inhibit Cu2+Quenching of the QDs fluorescence signal.
According to a preferred embodiment, said Ce is3+PPi and other phosphates were selectively identified to obtain fluorescent signals for PPi-Ce CPNs. Involving the use of Ce3+The PPi-Ce CPNs formed by the selective coordination polymerization reaction with PPi and other phosphates can emit fluorescent signals.
According to a preferred embodiment, the fluorescence signal of the PPi-Ce CPNs peaks at 348nm and the fluorescence signal of the QDs peaks at 670 nm.
According to a preferred embodiment, said QDs comprise CdTe QDs or CdSe QDs.
In order to achieve the second object, an embodiment of the present invention provides an application of the homogeneous visualization and dual fluorescence signal analysis method based on multiple selective recognition reactions, which includes applying any one of the homogeneous visualization and dual fluorescence signal analysis methods based on multiple selective recognition reactions to an assay of pyrophosphatase or alkaline phosphatase, which hydrolyzes the PPi after incubation at room temperature or 37 ℃.
According to a preferred embodiment, the use further comprises a method for reactions involving PPi, including PCR reactions, RCA reactions or nucleic acid signal amplification reactions involving TdT enzymes.
According to a preferred embodiment, the application further comprises a method for containing Cu2+And PPi assay systems, which use dual fluorescent signal molecules to quantify analytes in conjunction with a variety of signal amplification strategies.
The homogeneous phase visualization and double fluorescence signal analysis method based on multiple selective recognition reactions provided by the invention has the following technical effects:
the homogeneous phase visualization and double fluorescence signal analysis method based on various selective recognition reactions selectively recognizes Cu through QDs2+And Cu2+Complexes with PPi, and by Ce3+Selectively identifying PPi and other phosphates to obtain fluorescence signals of QDs and PPi-Ce CPNs, and quantifying a single target based on the fluorescence signals of QDs and PPi-Ce CPNs. Namely, double fluorescence signal molecules are used to quantify an analyte, the accuracy of the method is improved, and meanwhile, the luminescent nano material Ce is used3+The visual POCT analysis is realized by introducing biochemical and medical diagnosis.
The application of the homogeneous phase visualization and double fluorescence signal analysis method based on multiple selective recognition reactions provided by the invention has the following technical effects:
the application of the homogeneous phase visualization and double fluorescence signal analysis method of multiple selective recognition reactions can be used for the analysis of pyrophosphatase or alkaline phosphatase.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows a diagram of pyrophosphatase homogeneous visualization and dual fluorescence signal analysis based on selective recognition reaction;
FIG. 2 is a graph showing fluorescence peaks and data from a PPi-Ce CPNs and QDs dual fluorescence analysis system;
FIG. 3 is a diagram showing the optimization of the conditions of the pyrophosphatase assay;
FIG. 4 is a graph showing the performance of the pyrophosphatase assay.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The POCT, point-of-care testing, refers to clinical testing and bedside testing performed by a patient, and is not always performed by a clinical tester, and is a new method for performing analysis immediately at a sampling site, thereby saving a complicated processing procedure of a sample during laboratory testing and obtaining a test result quickly.
The PPi is pyrophosphoric acid, and the molecular formula is H4P2O7PPi is its abbreviation in biochemistry.
The technical solution of the present invention will be explained in detail below.
The invention provides a homogeneous phase visualization and double fluorescence signal analysis method based on multiple selective recognition reactions, which comprises selective recognition of Cu based on QDs2+And Cu2+A complex with PPi, and Ce3+Selectively identifying PPi and other phosphates to obtain fluorescence signals of QDs and PPi-Ce CPNs, and quantifying a single target based on the fluorescence signals of QDs and PPi-Ce CPNs. Preferably, Cu2+The complex formed with PPi comprises PPi-Cu2+-a PPi complex. Preferably, Cu is selectively recognized based on QDs2+And PPi-Cu2+-PPi complex to obtain the fluorescence signal of QDs. Involving the use of QDs and Cu2+And PPi-Cu2+-PPi complexThe compounds being subjected to selective cation exchange reactions based on Cu2+And PPi-Cu2+The PPi complexes quench the QDs fluorescence signal to different degrees, respectively, so that each system produces a distinct and visible color change. Preferably, Cu2+Can obviously quench the fluorescence signal of QDs, PPi-Cu2+-PPi complexes inhibit Cu2+Quenching of the QDs fluorescence signal. Preferably, Ce3+PPi and other phosphates were selectively identified to obtain fluorescent signals for PPi-Ce CPNs. Involving the use of Ce3+The PPi-Ce CPNs formed by the selective coordination polymerization reaction with PPi and other phosphates can emit fluorescent signals.
The principle is as follows:
FIG. 1 shows a homogenous visualization and dual fluorescence signal analysis diagram of pyrophosphatase based on selective recognition reactions, as shown in FIG. 1, based on two selective recognition reactions:
(1) selective recognition of Cu by CdTe QDs2+And PPi-Cu2+-PPi complex (PPi, pyrophosphate), as shown in figure 1A;
(2)Ce3+selective recognition of PPi and other phosphates is shown in figure 1B.
(3) Hydrolysis of PPi by pyrophosphatase to yield Pi (phosphate) to Cu2+No complexing force, and Pi to Ce3+The phenomenon that the fluorescence signal generates negligible influence can easily construct a double-fluorescence signal homogeneous visualization analysis strategy of the PPase, and as shown in FIG. 1C, an analysis schematic diagram of the PPase is shown.
Cu2+Can remarkably quench the fluorescence signal of QDs, and PPi and Cu2+After the complex is formed, the quenching effect on QDs is obviously inhibited, as shown in FIG. 2C. Ce3+Can be coordinated and polymerized with PPi to form nanometer materials (PPi-Ce CPNs) and emit fluorescence signals at 348 nm; and other phosphates (Pi, etc.) to Ce3+Produces negligible effects as shown in figure 2D.
Based on the above phenomena, the PPi-Ce CPNs are combined to generate a peak at 348nm, the CdTe QDs generate a peak at 670nm, and no spectrum overlapping exists between the PPi-Ce CPNs, so that the PPi-Ce CPNs and the CdTe QDs are independent from each other and do not influence each other. I.e.the quantification of a single target using the above two signal molecules is feasible. After the signal molecules are selected, PPi is used as a common reaction reagent, pyrophosphatase (PPase) is selected as a typical target, and the feasibility and the applicability of the dual-signal analysis strategy are verified.
The technical solution of the present invention will be described in detail with reference to examples.
Example 1:
this example provides a method for the synthesis of CdTe QDs.
First, 0.5mmol of CdCl2And 0.20g trisodium citrate in 50 ml water, to the above solution was added 52. mu.L mercaptopropionic acid (MPA). Adjusting the pH value of the mixture solution to 10.5 by using NaOH solution;
then, 0.1mmol of Na2TeO3And 50mg KBH4Adding into the above solution, refluxing for 1 hr until the solution is red, and under the irradiation of ultraviolet lamp, showing strong red fluorescence;
finally, the CdTe QDs solution is purified by precipitation (using n-propanol) and centrifugation (11000rpm, 30 minutes).
The MPA-CdTe QDs synthesized above are stored at 4 deg.C before use.
Example 2:
this example provides the steps for analyzing PPase, which are as follows:
(1) to the centrifuge tube were added 77 μ L of Tris-HCl buffer (pH 7.4, 10mM, 500mM NaCl, 100mM MgCl) in sequence2) 10 μ L of pyrophosphoric acid (PPi, 17.5mM) and 50 μ L of pyrophosphatase (PPase) at different concentrations were incubated at 37 ℃ for 0.5h to complete the hydrolysis reaction;
(2) subsequently, 10. mu.L of CuCl was added to the centrifuge tube2The solution (100. mu.M) was incubated at 37 ℃ for a further 0.5h to complex the copper ions with PPi;
(3) next, 2. mu.L of Ce (NO) was added to the centrifuge tube3)3Reacting the solution (0.5mM) and 1 μ L of CdTe QDs stock solution at room temperature for 12min to complete cation exchange reaction and coordination and complexation reaction;
(4) the above solution was transferred to a cuvette, and data were measured and recorded using a fluorometer (excitation wavelength: 295nm, emission wavelength range: 310nm-800 nm).
Example 3:
this example provides PPase analysis-verification of excitation wavelength selectivity and selectivity recognition phenomena, as follows:
considering that the optimal excitation wavelength required by PPi-Ce CPNs is 295nm, and the optimal excitation wavelength of CdTe QDs is 365nm, the excitation wavelength of the dual fluorescence signal analysis system is selected firstly. As shown in FIG. 2A, PPi-Ce CPNs and QDs have corresponding emission peak shapes at 295nm excitation, while PPi-Ce CPNs do not peak at 348nm but show a peak shape of the excitation wavelength at 365nm when 365nm excitation is selected.
In conclusion, the final selectivity is 295nm, which is the excitation wavelength of the system.
At the same time, PPi-Cu with different concentrations is further verified2+The effect of PPi complexes on the fluorescence signal of QDs, and different PPi on Ce3+Influence of the fluorescence signal. As shown in FIG. 2B, as the PPi concentration increases, QDs and Ce3+The fluorescence signal of (a) is significantly increased.
In addition, PPi and Pi (PPi's enzymatic hydrolysis products of PPi) were compared against QDs and Ce, respectively3+The effect of fluorescence signal, as shown in FIGS. 2C and 2D, indicates that PPi is responsible for QDs and Ce3+The effect of the fluorescence signal was significantly stronger than Pi, i.e. selective cation exchange and selective coordination polymerization reactions were verified, while also confirming that the system is feasible for PPase analysis.
Example 4:
this example provides PPase analysis-condition optimization as follows:
before the analysis performance of the PPase analysis strategy is considered, the experimental conditions are optimized. FIG. 3 shows optimization of the conditions for pyrophosphatase analysis.
The results of the experiment showed that the PPi concentration was 17.5mM, as shown in FIGS. 3A and 3B; PPase hydrolyzed PPi for 30 min as shown in FIG. 3C; PPi and Cu2+The complexation reaction time was 30 minutes, as shown in fig. 3D; cu2+The concentration was 100. mu.M, as shown in FIGS. 3E and 3F; the volume of the stock solution of QDs is 1 μ L, as shown in FIG. 3G and FIG. 3H; QDs and Cu2+/PPi-Cu2+The reaction time for cation exchange between PPi was 12 minutes, as shown in FIG. 3I; ce3+The concentration was 0.5mM, as shown in FIG. 3J and FIG. 3K; and PPi/Pi and Ce3+The reaction time, 8 minutes, is the optimum experimental condition for this system, as shown in FIG. 3L.
Example 5:
this example provides the analytical performance of PPase, as follows:
after optimization of experimental conditions, the PPase assay performance of the dual fluorescence signal assay system was examined, as shown in fig. 4.
First, visual analysis using QDs as signal molecules was evaluated. As shown in FIG. 4A, 5U/L of PPase enzyme was distinguished from the blank solution with naked eyes under UV light irradiation, and the color of the solution gradually became lighter as the enzyme activity increased (1-125U/L).
When the solution fluorescence signal was monitored using a fluorometer and linearly fitted, it was found that both the PP-Ce CPNs and QDs systems exhibited a decreasing trend in fluorescence signal with increasing PPase activity, as shown in FIG. 4B.
PPi-Ce CPNs are signal molecule systems which show good linearity in the range of 1-25U/L, and the linear equation is that Y is 7300-190C (R)20.995). Similar to PPi-Ce CPNs, QDs are signal molecules which are linear well in the 5-125U/L activity range with the linear equation Y-5079-32C (R)20.993), as shown in fig. 4C.
The detection limit of the two signal molecules to the PPase analysis is 0.15U/L and 0.8U/L respectively (derived from triple signal-to-noise ratio). The detection sensitivity is similar to that of a system without the participation of the existing signal amplification strategy, and the method is simpler to operate and low in cost. In addition, the content of the PPase in normal human bodies is about several hundred U/L, namely the analysis strategy can meet the requirement of clinical detection.
Subsequently, the interference resistance was examined, and as shown in FIGS. 4D and 4E, the effect of high concentration of potential interfering protein on the fluorescence signal of the system was similar to that of the blank solution (protein concentration is 100nM, ALP is 100U/L), i.e., both of them produced significant interference.
It is noted that ALP can also hydrolyze PPi to generate Pi, but the phenomenon has no influence, and the phenomenon lays a good foundation for the application of the system in clinical serum and other samples. Low concentrations of PPase enzyme (10, 25, 100U/L) caused a significant decrease in fluorescence signal compared to high concentrations of interfering proteins.
In conclusion, the double-fluorescence signal PPase analysis system has good selectivity.
As can be seen from the above, PPi-Ce CPNs show a peak at 348nm, CdTe QDs show a peak at 670nm, and there is no spectral overlap therebetween, i.e., they can be mutually independent and do not affect, i.e., a dual-fluorescence signal molecule is used to quantify an analyte, improve the accuracy of the method, and at the same time, the luminescent nano material Ce is used3+The visual POCT analysis is realized by introducing biochemical and medical diagnosis.
The homogeneous phase visualization and double-fluorescence signal analysis method based on various selective recognition reactions can obtain an analysis result based on a single target by changing double-fluorescence signals of PPi-Ce CPNs and QDs, and can be applied to the following analysis:
(1) assays for pyrophosphatase or alkaline phosphatase that can be incubated at room temperature or 37 ℃ to hydrolyze PPi.
(2) There are reactions involving PPi, which are various nucleic acid polymerization reactions including PCR reaction, RCA reaction, or nucleic acid amplification reaction involving TdT enzyme.
(3) Containing Cu2+And PPi assay systems, which use dual fluorescent signal molecules to quantify analytes in conjunction with a variety of signal amplification strategies.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. Homogeneous phase visualization and double-fluorescence signal separation based on multiple selective recognition reactionsA method comprising selectively identifying Cu based on QDs2+And Cu2+A complex with PPi, and Ce3+Selectively identifying PPi and other phosphates to obtain fluorescence signals of QDs and PPi-Ce CPNs, and quantifying a single target based on the fluorescence signals of QDs and PPi-Ce CPNs.
2. The method for homogeneous visualization and dual fluorescence signal analysis based on multiple selective recognition reactions of claim 1, wherein the Cu is2+The complex formed with PPi comprises PPi-Cu2+-a PPi complex.
3. The homogeneous visualization and dual fluorescence signal analysis method based on multiple selective recognition reactions as claimed in claim 2, wherein the selective recognition of Cu based on QDs is performed by using a fluorescence-based method2+And PPi-Cu2+-PPi complex to obtain the fluorescence signal of QDs. Involving the use of QDs and Cu2+And PPi-Cu2+-PPi complexes for selective cation exchange reactions, based on Cu2+And PPi-Cu2+The PPi complexes quench the QDs fluorescence signal to different degrees, respectively, so that each system produces a distinct and visible color change.
4. The method for homogeneous visualization and dual fluorescence signal analysis based on multiple selective recognition reactions of claim 3, wherein the Cu is2+Can remarkably quench the fluorescence signal of QDs, the PPi-Cu2+-PPi complexes inhibit Cu2+Quenching of the QDs fluorescence signal.
5. The homogeneous visualization and dual fluorescence signal analysis method based on multiple selective recognition reactions according to claim 1, wherein the Ce is3+PPi and other phosphates were selectively identified to obtain fluorescent signals for PPi-Ce CPNs. Involving the use of Ce3+The PPi-Ce CPNs formed by the selective coordination polymerization reaction of the PPi and other phosphates can emit fluorescent signalsNumber (n).
6. The method for homogeneous visualization and dual fluorescence signal analysis based on multiple selective recognition reactions as claimed in claim 1, wherein the fluorescence signal of PPi-Ce CPNs peaks at 348nm and the fluorescence signal of QDs peaks at 670 nm.
7. The method for homogeneous visualization and dual fluorescence signal analysis based on multiple selective recognition reactions according to claim 1, wherein the QDs comprise CdTe QDs or CdSe QDs.
8. Use of a method for homogeneous visualization and dual fluorescence signal analysis based on multiple selective recognition reactions, comprising applying the method for homogeneous visualization and dual fluorescence signal analysis based on multiple selective recognition reactions according to any one of claims 1 to 7 to an assay for pyrophosphatase or alkaline phosphatase that hydrolyzes said PPi upon incubation at room temperature or 37 ℃.
9. The use of claim 8, further comprising a reaction involving PPi, said reaction comprising a PCR reaction, an RCA reaction, or a nucleic acid signal amplification reaction involving a TdT enzyme.
10. The use of claim 8, further comprising including Cu2+And PPi assay systems, which use dual fluorescent signal molecules to quantify analytes in conjunction with a variety of signal amplification strategies.
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| CN113092754A (en) * | 2021-04-09 | 2021-07-09 | 四川大学华西医院 | Immunofluorescence and two-dimensional visualization multi-mode analysis method for ultra-sensitively identifying HIV p24 antigen and application thereof |
| CN113092754B (en) * | 2021-04-09 | 2022-11-15 | 四川大学华西医院 | Detection product for multi-mode analysis of HIV p24 antigen based on immunofluorescence and two-dimensional visualization and application thereof |
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