CN115791717A - Detection reagent for detecting reducing salt based on competitive selective recognition and binary visualization and application thereof - Google Patents
Detection reagent for detecting reducing salt based on competitive selective recognition and binary visualization and application thereof Download PDFInfo
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Abstract
The invention provides a detection reagent for detecting reducing salt based on competitive selective recognition and binary visualization and application thereof, and relates to the technical field of biomedical diagnosis. Based on the strong reducibility of the reducing salt, the invention 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/the low-valence metal ions, so as to successfully construct a homogeneous dual-fluorescence and binary visual analysis system for diagnosing related diseases containing the reducing salt.
Description
Technical Field
The invention relates to the technical field of biomedical diagnosis, in particular to a detection reagent for detecting reducing salt 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 widely noticed because of their high incidence rate (about 5% -10%), high recurrence rate within 5 years, and high cost. Hyperoxaluria is the most common type of stone (about 85%), consisting of insoluble calcium oxalate, and increases the risk of chronic kidney disease or renal failure. Therefore, detection of oxalate levels is important for early diagnosis and monitoring of stone formation.
The existing diagnosis method of calcium oxalate urolithiasis mainly comprises clinical imaging examination (such as ultrasound, CT and the like), a chromatographic instrument and a quantitative detection method of oxalate based on a colorimetric method, a fluorescence method, an electrochemical method, a high performance liquid chromatography, an ion chromatography and the like.
As for the prior art: (1) the clinical imaging examination cannot distinguish the types of calculi, such as oxalate, uric acid, phosphate and other types of calculi, and the price is high; (2) analytical methods based on chromatography and the like of oxalates require relatively expensive instruments, specialized personnel or cumbersome operating procedures; (3) it is difficult to achieve a point-of-care testing (POCT) with high sensitivity, and a visual reading: (4) the cost is high, and the popularization in primary hospitals and the self-monitoring of patients are difficult.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a detection reagent for detecting a reducing salt based on competitive selective recognition and binary visualization, so as to solve the defects of the prior art that clinical imaging examination and analysis methods based on chromatography and other oxalate methods are adopted.
The invention also aims to provide application of the detection reagent for detecting the reducing salt based on competitive selective recognition and binary visualization.
In order to achieve one of the above objects, the present invention provides a detection reagent for detecting a reducing salt based on competitive selective recognition and binary visualization, the reagent comprising a high-valence metal ion reduced by the reducing salt into a low-valence metal ion, the high-valence metal ion and the corresponding low-valence metal ion selectively modulating fluorescence signals of QDs and calcein, the high-valence metal ion and the corresponding low-valence metal ion effectively changing fluorescence signals of QDs and calcein, and quantifying the reducing salt in a binary visualization manner based on the fluorescence signals of QDs and the fluorescence signals of calcein.
According to an alternative embodiment, the lower valent metal ions quench the fluorescence signals of QDs and calcein to a greater extent than the higher valent metal ions.
According to an alternative embodiment, the binary visualization comprises a color visualization and a distance visualization.
According to an alternative embodiment, the color can be visualized as calcein and QDs appearing green and red, respectively, under ultraviolet light.
According to an alternative embodiment, the distance is visualized by preparing a two-color test strip by using an inkjet printing technology, and the visual reading is realized by reading the moving distance on the test strip.
According to an alternative embodiment, the reducing salt is an oxalate salt or a pathogenic marker with strong reduction.
According to an alternative embodiment, the high valent metal ion is Cu 2+ The low valence metal ion is Cu + 。
According to an alternative embodiment, the high valent metal ion is Fe 3+ The low-valence metal ion is Fe 2+ 。
According to an alternative embodiment, the excitation wavelength of said QDs and said calcein in the co-presence is 486nm.
In order to achieve one of the above objects, the present invention provides a use of a detection reagent for detecting a reducing salt based on competitive selective recognition and binary visualization, the use comprising applying any one of the detection reagents for detecting a reducing salt based on competitive selective recognition and binary visualization to the detection of oxalate in urolithiasis.
The detection reagent for detecting the reducing salt based on competitive selective recognition and binary visualization and the application thereof have the following technical effects:
the invention relates to a detection reagent for detecting reducing salt based on competitive selective recognition and binary visualization, which comprises high-valence metal ions, wherein 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 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 is a schematic diagram of an oxalate analysis strategy based on competitive selective recognition and binary visualization;
FIG. 2 is Cu of calcein and QDs 2+ /Cu + A competitive identification phenomenon verification map;
FIG. 3 is 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 should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention uses reducing salt as oxalate and high valence metal ion as Cu 2+ The low-valence metal ion is Cu + The technical means of the present invention will be described in detail.
The principle analysis of the invention is as follows:
as shown in figure 1, the oxalate analysis strategy principle based on competition selective recognition and binary visualization described in the invention, oxalic acid is a diagnostic marker commonly found in urine of calcium oxalate urolithiasis patients, has strong reducibility, and can rapidly and rapidly analyze Cu in a short time 2+ Reduction to Cu + . In the presence of oxalates, cu 2+ Is reduced to Cu + The rapid coordination and cation exchange reactions with calcein and QDs, respectively, quench the fluorescence of calcein and QDs. In contrast, when oxalate is not present, although Cu 2+ React with calcein and QDs, but Cu 2+ The fluorescence quenching effect of (A) is far less than that of Cu + . Therefore, oxalate can be rapidly quantified by measuring the decrease in calcein and QDs fluorescence signals.
Furthermore, in view of the two signal reporters employed by the present invention: the high luminescence properties of calcein and QDs allow for the preparation of portable test strips using ink jet printing technology for visual reading. When the strip is immersed in the reaction solution, the solution is drawn across the strip surface by capillary action and reacts with the calcein or QDs on the surface. The increase of the oxalate concentration in the reaction system allows Cu to be produced + The concentration is increased, the fluorescence quenching effect on calcein and QDs is enhanced, and the quenching distance on the test strip is increased. Therefore, the oxalate content can be detected by observing the color change of the solution and measuring the fluorescence quenching distance on the test strip.
The technical scheme of the invention is specifically verified by combining the embodiment.
Synthesis of QDs
CdTe QDs are synthesized according to a one-pot method:
first, cdCl is prepared 2 (0.5 mmol) and trisodium citrate (0.2 g) in 50 mL;
then, 52 μ L of MPA was immediately added to the above solution, and the pH of the solution was adjusted to 10.5 using NaOH;
subsequently, na is added 2 TeO 3 (0.1 mmol) and KBH 4 (50 mg) was added to the prepared solution and refluxed for 1h to obtain CdTe quantum dots;
finally, the CdTe QDs are obtained in high purity by precipitation with n-propanol and centrifugation (11000 rpm). The purified red CdTe QDs are dispersed in high purity water before use.
2. Oxalate analysis procedure:
2.1 homogeneous double fluorescence Signal analysis procedure for oxalate
First, 50. Mu.L of 10mM 3- (N-morpholino) propanesulfonic acid (MOPS) buffer (100 mM NaNO) 3 ，pH 7.3)、7μL CuCl 2 The solution (50 μ M) was mixed with various concentrations of oxalate (40 μ L), and the mixed solution was incubated at Room Temperature (RT) for 1.5min for reduction;
then, 4.8 μ L of calcein (10 μ M) and 3 μ L of CdTe QDs (5 times diluted solution) are added into the above solution, and reacted at room temperature for 1.5min to complete coordination reaction and cation exchange reaction;
finally, the fluorescence signals of calcein and QDs were detected at an excitation wavelength of 486nm.
2.2 distance reading test strip analysis procedure
Test strips of QDs and calcein were printed separately on chromatography paper (Whatman) using a common printer and cut to a specification of 7.5cm × 0.3 mm.
Using the QDs test strip, 80. Mu.L of 10mM MOPS buffer (100 mM NaNO) 3 ，pH7.3)、8μL CuCl 2 The solution (50. Mu.M) was mixed with different concentrations of oxalate (40. Mu.L) and incubated for 1.5min at room temperature. The QDs test strip was gently inserted into the reaction solution and waited for 1.5min. And taking out the test strip, drying and carrying out visual observation under an ultraviolet lamp.
When using a calcein strip, 80. Mu.L of 10mM MOPS buffer (100 mM NaNO) 3 ，pH 7.3)、4μL CuCl 2 Solution (80. Mu.M) and various concentrations of oxalate (40. Mu.L) inIncubate at room temperature for 2min. The calcein test strip was gently inserted into the solution and reacted for 1min. And taking out the test strip, drying and carrying out visual observation under an ultraviolet lamp.
3. Calcein and QDs vs Cu 2+ And Cu + To verify the competitive identification phenomena
Calcein and QDs on Cu before oxalate analysis 2+ /Cu + The competitive identification phenomenon was verified as shown in fig. 2.
As shown in FIGS. 2A, 2B and 2C, transmission Electron Microscope (TEM) images showed that the synthesized QDs were uniformly dispersed, appeared spheroidal, and had a particle size of about 4nm; at a certain concentration of Cu 2+ When present, the particle size of QDs increases; when Cu + When present, the degree of aggregation of QDs is further increased.
As shown in FIGS. 2D and 2E, the characteristic UV absorption peaks for QDs and calcein are at 630nm and 480nm, respectively.
As shown in FIGS. 2F and 2G, the mono-or divalent copper ions showed significantly different degrees of quenching effect on the fluorescence of calcein and QDs, i.e., cu + Is superior to Cu 2+ 。
In addition, as shown in fig. 2F, 2G and the interpolation chart, this phenomenon can also be visually recognized by the color change of the centrifuge tube and the reading of the strip quenching distance. Cu 2+ And Cu + The higher the concentration, the darker the solution color, the longer the quenching distance on the test strip, and Cu can be observed + Response to calcein and QDs compared to Cu 2+ And is more sensitive.
4. Oxalate analysis feasibility verification
The excitation wavelengths of the dual fluorescence signals were first screened. As shown in FIG. 2H, calcein and QDs both showed good fluorescence signal values at an excitation wavelength of 486nm, and thus 486nm was selected as the excitation wavelength.
As shown in fig. 2I and 2J, the principle of the assay system was verified using Ascorbic Acid (AA) with reducing properties, and the two fluorescence signal values gradually decreased as the concentration of AA increased.
As shown in c-f and i-l of FIG. 2K, the signal values for calcein and QDs are quantitatively decreased with increasing oxalate concentration in the dual signal assay system (0.1-1000 nM).
The results show that the dual-signal analysis system can realize quantitative detection of nanomolar oxalate concentration and can simultaneously realize reading of visual results of naked eyes.
5. Oxalate assay condition optimization
After the experimental feasibility is verified, the experimental conditions involved in the experiment were examined.
As shown in fig. 3A, with Cu 2+ The reaction time with oxalate is increased, the fluorescence values of calcein and QDs are gradually reduced, and the reaction is finished after 90 s.
As shown in fig. 3B and 3C, with Cu 2+ Volume increase, oxalate-containing solution (high oxalate + Cu concentration) 2+ + calcein + QDs) and blank solution (Low concentration oxalate + Cu) 2+ + calcein + QDs) increases and then decreases. When Cu 2+ The maximum difference was reached at a volume of 7. Mu.L. The results show that when Cu is used 2+ When the content of (B) is 7. Mu.L, 50. Mu.M, cu 2+ Reduced to Cu to the maximum extent by oxalate + 。
As shown in FIGS. 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. Mu.L and 3. Mu.L, respectively.
In addition, oxalate and Cu as shown in FIG. 3F 2+ Reduced product of Cu + Has rapid quenching effect on calcein and QDs, and can be finished within 90 s.
6. Examination of oxalate analytical Properties
In the prior art, quantum Dots (QDs) are used as signal reporter molecules for detecting calcium oxalate urolithiasis. Oxalate reaction of Cu 2+ Reduction to Cu + ，Cu 2+ /Cu + Can be selectively identified by cadmium telluride quantum dots (CdTe QDs), and the fluorescence signals of the quantum dots are detected by a fluorescence meter, so that the oxalate can be quantified within 6 min. However, this strategy only achieved a sensitivity of nM- μ M and a reading mode developed using tube color visualization was usedThe method is subjective and is not beneficial to the use of patients with color blindness/color weakness.
As shown in FIG. 4, the present invention examined the sensitivity of oxalate analysis in multiple modes.
As shown in FIG. 4A, when calcein was used as the signaling reporter, the fluorescence signal of calcein was linear with oxalate in the concentration range of 10nM to 10mM, with a detection limit of 4nM (based on three signal-to-noise ratio).
As shown in FIG. 4B, when QDs were used as signaling reporters, the detectable oxalate concentrations ranged from 10nM to 1mM, with a limit of detection of 3nM.
As shown in FIGS. 4C and 4D, the double fluorescent signal analysis using calcein and QDs, where the fluorescent signals of calcein and QDs were observed to quantitatively decrease with increasing oxalate concentration in the range of 10pM to 10nM, were detected at 3pM and 4pM, respectively.
The results indicate that the dual-fluorescence signal analysis system has excellent sensitivity, which is 3 orders of magnitude higher than that of the single-signal mode, probably due to competitive recognition of Cu by calcein and QDs + The disproportionation reaction rate is reduced, and the reaction efficiency is improved. Then, a visual reading mode of the color of the centrifugal tube and the distance between the test strips under an ultraviolet lamp is further developed, so that the analysis system can be suitable for POCT.
As shown in fig. 4E, 4F and 4G, as the oxalate concentration increased, the tube color became darker and the strip quenching distance became longer. Notably, oxalate was visually distinguishable at concentrations as low as 100nM in the single signal mode.
However, as shown in FIGS. 4H and 4I, color changes as low as 100pM of oxalate were seen in the dual signal mode, with more pronounced color contrast. By reading the distance of the test strip, a more visual and sensitive visual result is obtained, and a good linear relation exists between the test strip quenching distance 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, independence of instruments, realization of naked eye reading in multiple modes and the like. In addition, the assay system has good target selectivity. As shown in FIG. 4J, high concentrations (100 nM) of interfering species such as ions and small molecules had no significant effect on the fluorescence signal, while low concentrations of oxalate (1 and 10 nM) significantly reduced the fluorescence signal.
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 think of the changes or substitutions within the technical scope of the present invention, and shall cover 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. A detection reagent for detecting a reducing salt based on competitive selective recognition and binary visualization is characterized in that the reagent comprises a high-valence metal ion, the high-valence metal ion is reduced into a low-valence metal ion by the reducing salt, the high-valence metal ion and the corresponding low-valence metal ion selectively regulate and control fluorescence signals of QDs and calcein, the high-valence metal ion and the corresponding low-valence metal ion effectively change fluorescence signals of QDs and calcein, and the reducing salt is quantified in a binary visualization mode based on the fluorescence signals of QDs and the fluorescence signals of calcein.
2. The detection reagent for detecting a reducing salt based on competitive selective recognition and binary visualization according to claim 1, wherein the lower-valent metal ions quench the fluorescence signals of QDs and calcein to a greater extent than the higher-valent metal ions.
3. The reagent for detecting a reducing salt based on competitive selective recognition and binary visualization of claim 1, wherein the binary visualization comprises color visualization and distance visualization.
4. The detection reagent for detecting a reducing salt based on competition selective recognition and binary visualization according to claim 3, wherein the colors can be visualized as that calcein and QDs respectively appear green and red under ultraviolet light irradiation.
5. The detection reagent for detecting a reducing salt based on competition selective recognition and binary visualization as claimed in claim 3, wherein the distance visualization is realized by preparing a two-color test strip by adopting an ink-jet printing technology and reading the moving distance on the test strip to realize visual reading.
6. The detection reagent for detecting a reducing salt based on competition selective recognition and binary visualization according to claim 1, wherein the reducing salt is oxalate or a pathogenic marker with strong reducibility.
7. The detection reagent for detecting reducing salts based on competitive selective recognition and binary visualization as claimed in claim 1, wherein the high-valence metal ion is Cu 2+ The low valence metal ion is Cu + 。
8. The detection reagent for detecting reducing salts based on competitive selective recognition and binary visualization as claimed in claim 1, wherein the high-valence metal ion is Fe 3+ The low-valence metal ion is Fe 2+ 。
9. The detection reagent for detecting a reducing salt based on competition selective recognition and binary visualization according to claim 1, wherein the excitation wavelength of the QDs and the calcein in the co-presence is 486nm.
10. Use of a reagent for the detection of a reducing salt based on competitive selective recognition and binary visualization, which comprises applying the reagent for the detection of a reducing salt based on competitive selective recognition and binary visualization according to any one of claims 1 to 9 to the detection of oxalate in urolithiasis.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0534354A (en) * | 1991-07-29 | 1993-02-09 | Nissan Gosei Kogyo Kk | Measuring method for calcium |
| CN105866117A (en) * | 2016-06-03 | 2016-08-17 | 四川大学 | Detection method for organophosphorus pesticide dimethoate residue based on test paper colorimetry |
| US20160363535A1 (en) * | 2014-02-28 | 2016-12-15 | Sysmex Corporation | Method for urine sample analysis, reagent for urine sample analysis, and reagent kit for urine sample analysis |
| CN107110851A (en) * | 2014-11-05 | 2017-08-29 | 尼尔米达斯生物技术公司 | Metal composite for Enhanced Imaging |
| CN112444515A (en) * | 2020-11-10 | 2021-03-05 | 湖南柿竹园有色金属有限责任公司 | Method for rapidly determining calcium in wolframite concentrate by EDTA titration |
| 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 |
| CN113156128A (en) * | 2020-01-23 | 2021-07-23 | 四川大学华西医院 | Method for detecting oxalic acid by quantum dots and application |
| CN114813671A (en) * | 2022-04-06 | 2022-07-29 | 四川大学华西医院 | Analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof and application |
| CN114859058A (en) * | 2022-04-26 | 2022-08-05 | 四川大学华西医院 | Homogeneous phase double fluorescence and binary visualization analysis method for assisting IFN-gamma in enzyme-free nucleic acid amplification and selective recognition reaction and application |
| US20240024521A1 (en) * | 2022-07-20 | 2024-01-25 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Loading of human car t-cells with superparamagnetic iron-based particles for magnetic targeting |
-
2022
- 2022-09-22 CN CN202211157616.0A patent/CN115791717A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0534354A (en) * | 1991-07-29 | 1993-02-09 | Nissan Gosei Kogyo Kk | Measuring method for calcium |
| US20160363535A1 (en) * | 2014-02-28 | 2016-12-15 | Sysmex Corporation | Method for urine sample analysis, reagent for urine sample analysis, and reagent kit for urine sample analysis |
| CN107110851A (en) * | 2014-11-05 | 2017-08-29 | 尼尔米达斯生物技术公司 | Metal composite for Enhanced Imaging |
| CN105866117A (en) * | 2016-06-03 | 2016-08-17 | 四川大学 | Detection method for organophosphorus pesticide dimethoate residue based on test paper colorimetry |
| CN113156128A (en) * | 2020-01-23 | 2021-07-23 | 四川大学华西医院 | Method for detecting oxalic acid by quantum dots and application |
| CN112444515A (en) * | 2020-11-10 | 2021-03-05 | 湖南柿竹园有色金属有限责任公司 | Method for rapidly determining calcium in wolframite concentrate by EDTA titration |
| 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 |
| CN114813671A (en) * | 2022-04-06 | 2022-07-29 | 四川大学华西医院 | Analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof and application |
| CN114859058A (en) * | 2022-04-26 | 2022-08-05 | 四川大学华西医院 | Homogeneous phase double fluorescence and binary visualization analysis method for assisting IFN-gamma in enzyme-free nucleic acid amplification and selective recognition reaction and application |
| US20240024521A1 (en) * | 2022-07-20 | 2024-01-25 | Friedrich-Alexander-Universität Erlangen-Nürnberg | Loading of human car t-cells with superparamagnetic iron-based particles for magnetic targeting |
Non-Patent Citations (4)
| Title |
|---|
| LIN CHANG: "determination of L-cysteine base on the reversion of fluorescence quenching of calcein by copper（II）ion", MICROCHIM ACTA, vol. 177, 31 December 2012 (2012-12-31), pages 295 - 300, XP035055372, DOI: 10.1007/s00604-011-0759-0 * |
| PIAOPIAO CHEN等: "rapid binary visual detection of oxalate in urine samples of urolithiasis patients via competitive recognition and distance reading test strips", JOURNAL OF MATERIALS CHEMISTRY, vol. 11, 31 December 2023 (2023-12-31), pages 2530 - 2537 * |
| 景蓓蓓: "基于钙黄绿素的荧光分光光度法测定谷胱甘肽", 现代生物医学进展, vol. 11, no. 7, 30 April 2011 (2011-04-30), pages 1359 - 1362 * |
| 李媛媛: "家庭因素对成都市2~6岁儿童饮奶行为的影响", 中国学校卫生, vol. 37, no. 7, 31 December 2016 (2016-12-31), pages 977 - 979 * |
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