CN115791717B - Detection method for detecting reductive salt based on competition selective recognition and binary visualization and application thereof - Google Patents
Detection method for detecting reductive salt based on competition selective recognition and binary visualization and application thereof Download PDFInfo
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Abstract
The invention provides a detection reagent for detecting reductive salt based on competitive selective recognition and binary visualization and application thereof, and relates to the technical field of biomedical diagnosis. The invention successfully constructs a homogeneous double-fluorescence and binary visual analysis system based on the strong reducibility of the reducing salt, which can quickly reduce high-valence metal ions into low-valence metal ions and combines the competitive recognition phenomenon of calcein and QDs on the high-valence metal ions/low-valence metal ions, is used for diagnosing related diseases containing the reducing salt, and has the advantages of simple and quick operation, high sensitivity, low cost, no need of using instruments and realization of visual analysis and POCT.
Description
Technical Field
The invention relates to the technical field of biomedical diagnosis, in particular to a detection reagent for detecting reductive salts based on competitive selective recognition and binary visualization and application thereof.
Background
Urolithiasis is one of the most common diseases, with symptoms extending throughout the renal pelvis, ureter, bladder and urethra. Among them, kidney stones have been receiving a great deal of attention because of their high incidence (about 5% -10%), high recurrence rate within 5 years, and high cost. Hyperoxalic urolithiasis is the most common type of calculus (about 85%), consisting of insoluble calcium oxalate, and increases the risk of chronic kidney disease or renal failure. Thus, detection of oxalate levels is important for early diagnosis and monitoring of stone formation.
The existing diagnosis method of the calcium oxalate urolithiasis mainly comprises clinical imaging examination (such as ultrasound, CT and the like), chromatographic instruments and quantitative detection methods based on oxalate such as colorimetry, fluorescence method, electrochemical method, high performance liquid chromatography, ion chromatography and the like.
In terms of the prior art: ① Clinical imaging examination cannot distinguish the types of stones, such as oxalate, uric acid, phosphate and other stone types, and is expensive; ② Analysis methods based on oxalates such as chromatography require relatively expensive instrumentation, professionals, or cumbersome procedures; ③ It is difficult to achieve highly sensitive point-of-CARE TESTING (POCT), as well as visual reading: ④ The cost is high, and the method is difficult to popularize in primary hospitals and is used for self-monitoring of patients.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a reagent for detecting a reducing salt based on competitive selective recognition and binary visualization, which solves the drawbacks of the prior art using clinical imaging examination, analysis methods based on oxalates such as chromatography, and the like.
The second object of the invention is to provide an application of a detection reagent for detecting the reducing salt based on competition selective identification and binary visualization.
In order to achieve one of the above objects, the present invention provides a reagent for detecting a reducing salt based on competitive selective recognition and binary visualization, the reagent comprising a high-valence metal ion reduced by a reducing salt to a low-valence metal ion, the high-valence metal ion and the corresponding low-valence metal ion selectively regulating fluorescence signals of QDs and calcein, the high-valence metal ion and the corresponding low-valence metal ion effectively changing the fluorescence signals of QDs and calcein, and quantifying the reducing salt in a binary visualization manner based on the fluorescence signals of QDs and calcein.
According to an alternative embodiment, the low-valence metal ions quench the fluorescence signal of QDs and the fluorescence signal of calcein to a greater extent than the high-valence metal ions.
According to an alternative embodiment, the binary visualizations include a color visualization and a distance visualization.
According to an alternative embodiment, the color visualization is such that calcein and QDs appear green and red, respectively, under uv light.
According to an alternative embodiment, the distance visualization is implemented by preparing a bi-color test strip by using an inkjet printing technology and visually reading the distance moved on the test strip.
According to an alternative embodiment, the reducing salt is an oxalate or a pathogen marker with strong reducibility.
According to an alternative embodiment, the high-valence metal ion is Cu 2+ and the low-valence metal ion is Cu +.
According to an alternative embodiment, the high valence metal ion is Fe 3+ and the low valence metal ion is Fe 2+.
According to an alternative embodiment, the excitation wavelength of the QDs and the calcein when present together is 486nm.
In order to achieve one of the above objects, the present invention provides a use of a reagent for detecting a reducing salt based on competitive selective recognition and binary visualization, the use comprising applying any of the reagents for detecting a reducing salt based on competitive selective recognition and binary visualization described above to detection of oxalate in urolithiasis.
The detection reagent based on competition selective recognition and binary visual detection of the reducing salt and the application thereof provided by the invention have the following technical effects:
The detection reagent based on the competitive selective recognition and binary visualization detection of the reducing salt comprises high-valence metal ions, the reducing salt reduces the high-valence metal ions into low-valence metal ions, the high-valence metal ions and the corresponding low-valence metal ions selectively regulate and control fluorescence signals of QDs and calcein, the high-valence metal ions and the corresponding low-valence metal ions effectively change the fluorescence signals of the QDs and the fluorescence signals of the calcein, and the reducing salt is quantified in a binary visualization mode based on the fluorescence signals of the QDs and the fluorescence signals of the calcein.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of oxalate analysis strategy based on competitive selective identification and binary visualization;
FIG. 2 is a graph showing the verification of Cu 2+/Cu+ competitive recognition phenomena of calcein and QDs;
FIG. 3 is an oxalate analysis condition optimization;
FIG. 4 is oxalate analysis performance.
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 will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
The technical scheme of the invention is described in detail below by taking the reducing salt as oxalate, cu 2+ as high-valence metal ion and Cu + as low-valence metal ion.
The principle analysis of the invention is as follows:
As shown in fig. 1, according to the oxalate analysis strategy principle based on competition selective recognition and binary visualization described in the present invention, oxalic acid is used as a diagnostic marker commonly found in urine of patients suffering from calcitic acid, has strong reducibility, and can rapidly reduce Cu 2+ to Cu + in a short time. In the presence of oxalate, cu 2+ is reduced to Cu +, which undergoes rapid coordination and cation exchange reactions with calcein and QDs, respectively, and fluorescence of calcein and QDs is quenched. In contrast, in the absence of oxalate, cu 2+ has far less fluorescence quenching effect than Cu +, although Cu 2+ reacts with calcein and QDs. Thus, oxalate can be rapidly quantified by measuring the decrease in calcein and QDs fluorescence signals.
Furthermore, two signal reporters are employed in view of the present invention: high luminescence properties of calcein and QDs, portable test strips can be prepared using inkjet printing techniques to obtain visual readings. When the test strip is immersed in the reaction solution, the solution pulls through the surface of the test strip due to capillary action and reacts with the surface calcein or QDs. The concentration of the oxalate in the reaction system increases the concentration of the generated Cu +, and the fluorescence quenching effect on the calcein and the QDs is enhanced, so that the quenching distance on the test strip is increased. The oxalate content can thus be detected by observing the change in color of the solution and measuring the distance of fluorescence quenching on the test strip.
The technical scheme of the invention is specifically verified by the following examples.
Synthesis of QDs
CdTe QDs are synthesized according to a one-pot process:
First, a 50mL solution containing CdCl 2 (0.5 mmol) and trisodium citrate (0.2 g) was prepared;
then, 52 μl MPA was immediately added to the above solution, and the solution pH was adjusted to 10.5 using NaOH;
Subsequently, na 2TeO3 (0.1 mmol) and KBH 4 (50 mg) were added to the prepared solution and refluxed for 1h to obtain CdTe quantum dots;
finally, high purity CdTe QDs are obtained by precipitation with n-propanol and centrifugation (11000 rpm). Purified red CdTe QDs are dispersed in high purity water prior to use.
2. Oxalate analysis step:
2.1 homogeneous double fluorescence Signal analysis step of oxalate
First, 50. Mu.L of 10mM 3- (N-morpholino) propanesulfonic acid (MOPS) buffer (100 mM NaNO 3,pH 7.3)、7μL CuCl2 solution (50. Mu.M) was mixed with oxalate (40. Mu.L) at various concentrations, and the mixed solution was incubated at Room Temperature (RT) for 1.5min to perform reduction reaction;
Then, 4.8. Mu.L of calcein (10. Mu.M) and 3. Mu.L of CdTe QDs (5-fold diluted solution) are added into the solution, and the reaction is carried out for 1.5min at room temperature, thus completing the coordination reaction and the cation exchange reaction;
Finally, fluorescence signals of calcein and QDs were detected at an excitation wavelength of 486 nm.
2.2 Distance reading test strip analysis step
Test strips of QDs and calcein were printed separately on chromatographic paper (Whatman) using a conventional printer and cut into 7.5cm x 0.3mm formats.
When using the QDs strip, 80. Mu.L of 10mM MOPS buffer (100 mM NaNO 3,pH7.3)、8μL CuCl2 solution (50. Mu.M)) was mixed with different concentrations of oxalate (40. Mu.L) and incubated at room temperature for 1.5min.
When the calcein test strip is used, 80 mu L of 10mM MOPS buffer (100 mM NaNO 3,pH 7.3)、4μL CuCl2 solution (80 mu M) and oxalate (40 mu L) with different concentrations are incubated for 2min at room temperature, the calcein test strip is gently inserted into the solution for reaction for 1min, the test strip is taken out, and the test strip is observed visually under an ultraviolet lamp after drying.
3. Verification of competitive recognition phenomena of calcein and QDs for Cu 2+ and Cu +
The competitive recognition of Cu 2+/Cu+ by calcein and QDs was verified prior to oxalate analysis, as shown in FIG. 2.
As shown in fig. 2A, 2B and 2C, transmission Electron Microscope (TEM) images indicate that the QDs obtained by synthesis are uniformly dispersed, take on a spheroid shape, and have a particle size of about 4nm; in the presence of Cu 2+ at a certain concentration, the particle size of the QDs increases; and when Cu + is present, the degree of aggregation of QDs is further increased.
As shown in FIGS. 2D and 2E, the characteristic UV absorption peaks of QDs and calcein are at 630nm and 480nm, respectively.
As shown in fig. 2F and 2G, the monovalent or divalent copper ions showed significantly different degrees of quenching effect on the fluorescence of calcein and QDs, i.e., cu + was superior to Cu 2+.
In addition, as shown in figures 2F, 2G and the interpolated graph, this phenomenon can also be visually identified by the color change of the centrifuge tube and the read strip quench distance. The higher the concentrations of Cu 2+ and Cu +, the darker the solution color, the longer the quench distance on the test strip, and the more sensitive Cu + response to calcein and QDs compared to Cu 2+ can be observed.
4. Oxalate analysis feasibility verification
The excitation wavelength of the dual fluorescent signal was first screened. As shown in FIG. 2H, at an excitation wavelength of 486nm, calcein and QDs both show good fluorescence signal values, and therefore 486nm is selected as the excitation wavelength.
As shown in fig. 2I and 2J, the principle of the analysis system was verified using Ascorbic Acid (AA) having a reducing property, and the two fluorescence signal values gradually decreased as the AA concentration increased.
As shown in FIGS. 2K, c-f and i-l, the signal values of calcein and QDs quantitatively decrease (0.1-1000 nM) with increasing oxalate concentration in the dual signal analysis system.
The results show that the dual-signal analysis system can realize quantitative detection of nanomolar oxalate and can simultaneously realize reading of visual results of naked eyes.
5. Oxalate analysis condition optimization
After verifying the feasibility of the experiment, the experimental conditions involved in the experiment were examined.
As shown in FIG. 3A, as the reaction time of Cu 2+ and oxalate increases, the fluorescence values of calcein and QDs gradually decrease, and the reaction is completed after 90 seconds.
As shown in fig. 3B and 3C, as the volume of Cu 2+ increases, the difference in fluorescence signal between the oxalate-containing solution (high concentration oxalate+cu 2+ +calcein+qds) and the blank solution (low concentration oxalate+cu 2+ +calcein+qds) increases and then decreases. The maximum difference is reached when the volume of Cu 2+ is 7. Mu.L. The results showed that when the content of Cu 2+ was 7. Mu.L and 50. Mu.M, cu 2+ was reduced to Cu + to the maximum extent by oxalate.
As shown in fig. 3D and 3E, the difference in fluorescence signal values between the blank solution and the oxalate solution was greatest when the volumes of calcein and QDs were 4.8 μl and 3 μl, respectively.
In addition, as shown in FIG. 3F, the reduction products Cu + of oxalate and Cu 2+ have a rapid quenching effect on calcein and QDs, which can be accomplished within 90 s.
6. Oxalate analysis Performance investigation
In the prior art, quantum Dots (QDs) are used as signal reporter molecules for the detection of calcitia. The reduction of Cu 2+ to Cu +,Cu2+/Cu+ by oxalate can be selectively identified by cadmium telluride quantum dots (CdTe QDs), and finally, fluorescence signals of the quantum dots are detected by using a fluorometer, so that the quantification of oxalate can be realized within 6 min. However, this strategy only achieves nM- μm sensitivity and the reading patterns developed using tube color visualization are subjective and not conducive to use by achromatopsia/achromatopsia patients.
As shown in FIG. 4, the present invention examined the sensitivity of oxalate analysis in multiple modes.
As shown in FIG. 4A, when calcein is used as a signal reporter molecule, the fluorescence signal of calcein is linear with oxalate in the concentration range of 10nM-10mM, with a detection limit of 4nM (based on triple signal-to-noise ratio).
As shown in FIG. 4B, when QDs are used as signal reporter molecules, oxalate concentrations can be detected in the range of 10nM to 1mM, with a limit of detection of 3nM.
As shown in FIGS. 4C and 4D, the fluorescence signals of calcein and QDs were quantitatively decreased with the increase of the oxalate concentration in the range of 10pM-10nM, and their detection limits were 3pM and 4pM, respectively, by performing a double fluorescence signal analysis using calcein and QDs.
The results show that the dual fluorescent signal analysis system has excellent sensitivity, which is 3 orders of magnitude higher than that of the single signal mode, probably because calcein and QDs competitively recognize Cu +, the disproportionation reaction rate is reduced, and the reaction efficiency is improved. After that, we further developed a visual readout mode of the centrifuge tube color and the test strip distance under the ultraviolet lamp, so that the analysis system can be applied to POCT.
As shown in fig. 4E, 4F and 4G, the centrifuge tube darkened and the strip quenching distance increased as the oxalate concentration increased. Notably, oxalate can be distinguished visually down to 100nM concentration in the single signal mode.
However, as shown in FIGS. 4H and 4I, color changes as low as 100pM oxalate can be seen in the dual signal mode, and the color contrast is more pronounced. By means of the distance reading of the test strip, a more visual and sensitive visual result is obtained, and a good linear relation exists between the quenching distance of the test strip of calcein and QDs and the oxalate concentration.
Therefore, the dual-signal and binary visual analysis strategy has the advantages of rapidness, simplicity, high sensitivity, low cost, no dependence on instruments, capability of realizing naked eye reading under multiple modes and the like. In addition, the analysis system has good target selectivity. As shown in FIG. 4J, high concentrations (100 nM) of interfering substances such as ions and small molecules have no significant effect on the fluorescence signal, while low concentrations (1 and 10 nM) of oxalate significantly reduce the fluorescence signal.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (8)
1. The detection method is characterized in that reagents of the detection method comprise high-valence metal ions, the high-valence metal ions are reduced into low-valence metal ions by the reduction salt, the high-valence metal ions and the corresponding low-valence metal ions selectively regulate and control fluorescence signals of QDs and calcein, the high-valence metal ions and the corresponding low-valence metal ions effectively change the fluorescence signals of the QDs and the fluorescence signals of the calcein, and the reduction salt is quantified in a binary visualization mode based on the fluorescence signals of the QDs and the fluorescence signals of the calcein;
The quenching degree of the fluorescence signals of the low-valence metal ions to the QDs and the calcein is larger than that of the high-valence metal ions;
The binary visualizations include color visualizations and distance visualizations.
2. The method for detecting a reducing salt based on competitive selective identification and binary visualization according to claim 1, wherein the color visualization is that calcein and QDs respectively appear green and red under ultraviolet light irradiation.
3. The detection method for detecting the reductive salt based on the competition selectivity identification and the binary visualization according to claim 1, wherein the distance visualization is to prepare a bicolor test strip by adopting an inkjet printing technology, and visual reading is realized by reading the moving distance on the test strip.
4. The method for detecting a reducing salt based on competitive selective recognition and binary visualization according to claim 1, wherein the reducing salt is oxalate or a pathogen marker having strong reducibility.
5. The method for detecting a reducing salt based on competitive selective recognition and binary visualization according to claim 1, wherein the high-valence metal ion is Cu 2+ and the low-valence metal ion is Cu +.
6. The method for detecting a reducing salt based on competitive selective identification and binary visualization according to claim 1, wherein the high-valence metal ion is Fe 3+ and the low-valence metal ion is Fe 2+.
7. The method for detecting a reducing salt based on competitive selective identification and binary visualization according to claim 1, wherein the excitation wavelength in the co-presence of QDs and calcein is 486 nm.
8. Use of a detection method based on competitive selective recognition and binary visualization for detecting a reducing salt, characterized in that the use comprises applying the detection method based on competitive selective recognition and binary visualization for detecting a reducing salt according to any one of claims 1 to 7 to the detection of oxalate.
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| CN113030051A (en) * | 2021-03-17 | 2021-06-25 | 四川大学华西医院 | Homogeneous phase dual-fluorescence analysis method based on selective regulation and control of QDs and NMM fluorescence signals by metal ions and application thereof |
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