CN114813671A

CN114813671A - Analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof and application

Info

Publication number: CN114813671A
Application number: CN202210357743.9A
Authority: CN
Inventors: 陈飘飘; 孟妍明; 刘堂喻亨; 应斌武
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-04-06
Filing date: 2022-04-06
Publication date: 2022-07-29
Anticipated expiration: 2042-04-06
Also published as: CN114813671B

Abstract

The invention provides an analysis method and application for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nano particles thereof, and relates to the technical field of biomedical diagnosis and analysis methods. The invention uses the metal ions and the corresponding nano particles as bridges, and selectively identifies calcein and QDs for the double fluorescence and binary visualization analysis of the Mycobacterium tuberculosis LAM, thereby providing more choices for clinical diagnosis and providing guidance and laying a foundation for developing various biochemical and medical inspection methods.

Description

Analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof and application

Technical Field

The invention relates to the technical field of biomedical diagnosis and analysis methods, in particular to an analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nano particles thereof and application thereof.

Background

Tuberculosis is the leading cause of death by a single infectious microorganism, with mycobacterium tuberculosis (Mtb) infecting approximately 23% of the world's population. Tuberculosis is associated with other diseases, such as human immunodeficiency virus infection, making treatment more difficult. Early diagnosis of active tuberculosis is essential for effective treatment of tuberculosis, and the discovery of Mtb and its components provides direct evidence for rapid diagnosis of tuberculosis. Although bacterial culture is still the gold standard for tuberculosis diagnosis, the detection period is often long, and professional personnel are required to operate, and the detection sensitivity is low. The Xpert-MTB/RIF detection is a high-sensitivity and high-specificity active tuberculosis diagnosis method, can detect the bacteria of the Mycobacterium tuberculosis complex within 2 hours, and simultaneously determine the mutation condition of common rifampicin resistance related genes of tuberculosis. However, such detection is relatively expensive and requires technical investment. Mtb-specific antigen detection has become an attractive alternative to aid in the diagnosis of active tuberculosis.

Lipoarabinomannan (LAM), a 17.5kDa glycolipid, is a major polysaccharide antigen of the outer surface of the cell wall of mycobacterium tuberculosis. LAM is a heterogeneous lipid sugar consisting of three parts: phosphatidylinositol mannoside anchor, polysaccharide backbone and end capped structures. LAM is classified into phosphatidylinositol LAM (PILAM), mannose LAM (ManLAM) and arabinose LAM (arabinase-capped LAM) according to the cap structure of the end of LAM. The three kinds of LAM exist in different mycobacteria respectively and play different roles in the growth and pathogenic processes of the bacteria. The pathogenic mycobacteria are rich in ManLAM, can cause strong immune response of a host, and are important immune targets of tuberculosis.

The prior art about the analysis and detection method of LAM has low sensitivity, and limits the clinical application of LAM. For the laboratory diagnosis index of the mycobacterium tuberculosis, the culture method is a gold standard, but the culture method is limited due to time consumption and low sensitivity, X-pert is greatly influenced by the quality of a sputum sample and has higher cost, the acid-fast staining sensitivity of a sputum smear is only 50%, and the omission phenomenon is easy to occur.

Disclosure of Invention

In view of the above, an objective of the present invention is to provide an analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof, so as to solve the technical problems that the detection method using LAM in the prior art has a single signal reading, the accuracy thereof is easily affected by media and the like, and the signal amplification technology participates in a strategy, the analysis sensitivity is still low, and the cost is greatly increased when the signal amplification is introduced.

The invention also aims to provide application of an analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nano particles thereof.

In order to achieve one of the above objects, the present invention provides an analysis method for selectively regulating and controlling fluorescence signals of QDs and calcein based on metal ions and nanoparticles thereof, the method comprising selectively regulating and controlling fluorescence signals of QDs and calcein based on metal ions and nanoparticles corresponding thereto, the metal ions and nanoparticles corresponding thereto effectively change the fluorescence signals of QDs and the fluorescence signals of calcein, and quantifying a single target based on the fluorescence signals of QDs and the fluorescence signals of calcein, the target being mycobacterium tuberculosis.

According to an alternative embodiment, the metal ion is Cu ²⁺ The nano particles are CuNPs.

According to an alternative embodiment, the metal ion is Ag ⁺ And the nano particles are Ag NPs.

According to an alternative embodiment, the QDs comprise CdTe QDs or CdSe QDs.

According to an alternative embodiment, the excitation wavelength of said QDs and said calcein is 495 nm.

According to an alternative embodiment, the fluorescence signals of QDs and calcein are detected by a fluorometer, the difference in color shade of the reaction solution in the test tube under an ultraviolet lamp, and/or the diffusion distance of the reaction solution on the paper strip into which the ink jet printed test strip is to be inserted.

According to an alternative embodiment, the detectable concentration of LAM in the dual signal mode ranges from 10fg/mL to 1pg/mL, wherein the linear equation for calcein is: y-98 LogC +486, R ² 0.991, where the linear equation for QDs is: -71LogC +304, R ² ＝0.983。

In order to achieve the second object, the present invention provides an application of an analysis method based on metal ions and nanoparticles thereof for selectively regulating QDs and calcein fluorescence signals, the application comprising applying the analysis method based on metal ions and nanoparticles thereof for selectively regulating QDs and calcein fluorescence signals according to any one of claims 1 to 7 to a mycobacterium tuberculosis LAM analysis.

According to an alternative embodiment, the application further comprises a plurality of metal ions in combination with different analytical instruments and signal molecules for constructing analytical strategies for detection diagnosis of different molecules.

The analysis method and the application of selectively regulating and controlling QDs and calcein fluorescence signals based on the metal ions and the nano particles thereof have the following technical effects:

the analysis method for selectively regulating and controlling QDs and calcein fluorescence signals based on metal ions and nano particles thereof is characterized in that the metal ions and the nano particles corresponding to the metal ions selectively regulate and control the QDs fluorescence signals and the calcein fluorescence signals, the metal ions and the nano particles corresponding to the metal ions effectively change the QDs fluorescence signals and the calcein fluorescence signals, then a single target is quantified based on the QDs fluorescence signals and the calcein fluorescence signals, the target is Mycobacterium tuberculosis LAM, and the analysis method provided by the invention is used as a novel biochemical and medical diagnosis technology and has the characteristics of low cost, high sensitivity and accuracy. By using metal ions and corresponding nano particles as bridges, the selective recognition of calcein and QDs on the two ions is used for double-fluorescence and binary visualization analysis of Mycobacterium tuberculosis LAM, more choices are provided for clinical diagnosis, and guidance and foundation are provided for developing various biochemical and medical inspection methods.

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 graph of a dual fluorescence signal visualization analysis of LAM based selective recognition reaction for selective recognition mechanism;

FIG. 2 is a schematic diagram of a dual fluorescent signal immunoassay of LAM based on selective recognition reaction;

FIG. 3 is a material characterization and LAM analysis feasibility;

FIG. 4 is LAM analysis condition optimization;

FIG. 5 LAM analysis performance;

FIG. 6 is a clinical specimen test.

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 principle of the analytical method of the present invention is as follows:

as shown in FIG. 1, the present invention describes the fluorescence signal of calcein and QDs vs Cu ²⁺ And selective recognition phenomenon of Cu NPs, i.e. Cu ²⁺ Calcein and QDs fluorescence signals can be more effectively inhibited compared with Cu NPs. And the difference can be reflected on the color depth difference of the reaction liquid in the test tube under the ultraviolet lamp and the diffusion distance of the reaction liquid on the paper tape after the ink-jet printing test strip is inserted for the same time, so that the quantification can be realized, and the quantification can be realizedVisual detection independent of instruments.

As shown in FIG. 2, by combining the above phenomenon with immune reaction, a diagnostic system using Mycobacterium tuberculosis LAM as a target substance was constructed. The selective recognition of LAM by primary and secondary biotinylated antibodies immobilized at the bottom of the reaction wells can produce different amounts of diabody sandwich complexes at different LAM concentrations, while the binding between streptavidin and biotin can allow biotinylated T30 to be bound to the bottom of the plate, allowing the concentration of free T30 to be inversely related to the LAM concentration.

The higher the LAM concentration in the sample, the more anti-LAM-anti-biotin-streptavidin-biotin-T30 complexes formed and the less free T30, which subsequently reacted to produce T30 template Cu NPs and free Cu ²⁺ The lower the concentration ratio of (a). Thus, upon selective recognition, the fluorescence signals of calcein and QDs will be lower, the weaker the tube color, the longer the diffusion distance on the paper tape. The quantification of LAM can be performed by observation and measurement of these values.

The technical scheme of the invention is specifically verified by combining the embodiment.

Synthesis of CdTe QDs

CdTe QDs are synthesized by a one-pot method:

first, 0.5mmol of CdCl ₂ And 0.20g trisodium citrate dissolved in 50 ml water, 52. mu.L mercaptopropionic acid (MPA) was added;

then, after the pH of the mixed solution was adjusted to 10.5 using NaOH solution, 0.1mmol of Na was added ₂ TeO ₃ And 50mg KBH ₄ Adding into the above solution, refluxing for 1 hr until the solution is red and strong red fluorescence appears under ultraviolet lamp;

finally, the CdTe QDs solution is purified by n-propanol precipitation and 11000rpm, 30 min centrifugation. The synthesized MPA-CdTe QDs are stored at 4 deg.C before use.

Step of LAM analysis

2.1 double fluorescence Signal fluorescence detection mode for calcein and QDs

Prior to testing, 200. mu.L of urine sample was transferred to a 3kDa ultrafiltration tube. After centrifugation at 8000rpm for 15 minutes, the inner tube solution was collected and transferred to a 30kDa ultrafiltration tube and centrifuged again at 8000rpm for 15 minutes.

The outer tube solution was collected and replenished with ultrapure water to a final volume of 200. mu.L. To each well, 40. mu.L of the treated sample and 60. mu.L of MOPS buffer (10mM MOPS, 100mM NaNO) were added ₃ pH 7.1), incubated at 37 ℃ for 1 hour.

Thereafter, 200. mu.L of washing buffer (10mM MOPS, 100mM NaNO) was used ₃ pH 7.1, 0.5% tween 20) for one minute and poured over and repeated three times.

Subsequently, 40. mu.L of 0.4. mu.g/mL biotin-labeled secondary LAM antibody was added to the wells, incubated at 37 ℃ for 50 minutes, and washed three times as described above. To each well was added 30 μ L of a 1 μ g/mL solution of Streptavidin (SA), incubated at room temperature for 40 minutes, and washed three times as described above.

To each well 25. mu.L of 0.9. mu.M biotin-labeled T30 solution and 25. mu.L of MOPS buffer were added and incubated at room temperature for 30 minutes to form Ab ₁ -LAM-Ab ₂ After the biotin-SA-biotin-T30 complex, 20. mu.L of the supernatant was added with 60.5. mu.L of CuCl ₂ (100. mu.M) and 20. mu.L ascorbic acid (4mM) incubated at room temperature for 4min to form CuNPs, resulting in Cu ²⁺ The concentration is reduced.

Subsequently 0.8 μ L calcein (10 μ M) was added and incubated for 105s, 1.8 μ L QDs stock solution was added and incubated for 45s at room temperature to complete the complexation and cation exchange reactions.

Finally, fluorescence detection was performed on the reaction solution having an excitation wavelength of 495nm to obtain a fluorescence value, which was taken into the standard curve to perform concentration calculation.

2.2 calcein and QDs double fluorescence signal paper tape visualization mode

Both QDs and calcein were printed on chromatography paper with an ink jet printer and the paper was then cut into 2.5X 110mm sizes.

Before the immune incubation, a 2mL test tube is added with 70 mu L of MOPS buffer solution, and the supernatant fluid after the reaction of the specimen is 50 mu L of Cu ²⁺ 100μM 60.5μL，AA 4mM 20μL。

After incubation for 2min at room temperature, QDs and calcein dipsticks were inserted into the aspirated supernatant solution and incubated for 2min at room temperature to complete the cation exchange and complexation reactions, respectively. The LAM test results were obtained by comparing the diffusion distance of the sample with the diffusion distance of the calibration curve under the uv lamp.

2.3 calcein and QDs double fluorescence signal color visualization mode

Before the immune incubation, 20 mu L of reaction supernatant is taken and added into 20 mu L of MOPS buffer solution and Cu after the incubation is finished ²⁺ 100 mu M60.5 mu L and AA 4mM 20 microliter, after incubating for 4min, adding 3.2 mu L of quantum dot stock solution, after shaking uniformly 95 mu L of 100 mu M calcein, immediately observing the fluorescence color under an ultraviolet lamp. And obtaining the LAM concentration of the sample by comparing with the standard curve.

3. Material characterization and LAM analysis feasibility

After the QDs are successfully synthesized (shown in FIG. 3C, E as TEM and UV-visible absorption peak pattern of QDs), FIG. 3A, B show that CuNPs are generated in the reaction system. The uv-visible absorption peaks of the copper nanoparticles and quantum dots were located at about 340nm and 625nm, respectively (fig. 3B, E). The TEM image of FIG. 3D shows Cu ²⁺ The cation exchange reaction with QDs causes the QDs to generate CuTe through agglomeration.

With Cu of different concentrations ²⁺ And T30 examined selective recognition phenomena. The results show that fluorescence quenching of quantum dots as well as calcein is effectively suppressed after formation of Cu NPs (fig. 3G and 3I) (fig. 3F and 3H). In addition, concentration differences can be identified by reading the solution color and the distance the test strip is moved under an ultraviolet lamp with the naked eye (fig. 3F and 3H inset).

Comparing fluorescence signal values of the quantum dot and the calcein at different excitation wavelengths in the dual-signal molecular solution (fig. 3J), it was found that when the excitation wavelength is 365nm, the fluorescence signal of the calcein is too low to obtain an obvious peak shape, and the fluorescence signals of the quantum dot and the calcein at the excitation wavelength of 495nm are both significant, so that the excitation wavelength of 495nm is used in subsequent experiments.

Finally, calcein, quantum dots and Cu NPs were used as signal reporters to monitor the fluorescence signal of the solution under different conditions. When CuNPs were used as signal reporter, the fluorescence signal of the solution gradually decreased with increasing LAM concentration (0,1,10,100pg/mL) (FIG. 3K). When calcein and quantum dots were used as signal reporter molecules, the fluorescence signal of the solution also decreased gradually with increasing LAM (0,0.1,1,10pg/mL) concentration (fig. 3L), but the sensitivity was higher than that of Cu NPs. The above results indicate that the dual signal fluorescent system can be successfully used for LAM quantitative analysis.

Optimization of LAM analysis conditions

In order to obtain better analytical performance, the conditions involved in the experiment were examined.

The recognition reaction between primary antibody and LAM was completed within 60 minutes (fig. 4A). Ab formation when 0.4. mu.g/mL secondary antibody (FIG. 4B) was incubated for 50 minutes (FIG. 4C) ₁ -LAM-Ab ₂ Maximum difference in fluorescence signal is obtained with biotin sandwich complexes. With increasing Streptavidin (SA) content, the fluorescence signal of the system gradually decreased, and the difference of the fluorescence signal reached the maximum when 30. mu.L of 1. mu.g/mL SA was used (FIG. 4D).

The fluorescence signal of the system gradually increased as the concentration of biotin-labeled T30 increased. The difference between the fluorescence signals of the experimental group and the blank group was the largest when the concentration was 0.9. mu.M (FIG. 4E). For the formation reaction of copper nanoparticles, when 60.5. mu.L of 100. mu.M Cu is used ²⁺ The greatest signal difference was obtained when incubated for 4 minutes (FIG. 4H) with 20. mu.L of 4mM AA (FIG. 4F). The fluorescence signal of the system gradually increased with increasing amounts of calcein and quantum dots, reaching a maximum difference in fluorescence signal upon addition of 0.8 μ L of 10 μ M calcein (fig. 4I) and 1.8 μ L of QDs stock (fig. 4J).

The fluorescence signal decreased with increasing reaction time after calcein and QDs were added to the system, and the maximum difference in fluorescence signal was obtained if calcein and QDs were added at 105 seconds and quantum dot reaction time was 45 seconds (fig. 4K and 4L).

LAM analytical Properties

The performance of the LAM assay was then examined and the sensitivity and selectivity of the LAM assay method was evaluated using different signal reporter systems under optimized experimental conditions.

The fluorescence signal of CuNPs is in the range of 100fg/mL to 100pg/mL and is good at the LAM concentrationLinear relation, linear equation is Y ═ 75LogC +355 (R) ² 0.991), the LOD was 30fg/mL (fig. 5A and 5B, based on 3-fold signal-to-noise ratio).

When calcein was used as the single-signal report, the log of the fluorescence signal of calcein to LAM concentration showed a good linear relationship in the range of 10fg/mL to 10pg/mL (fig. 5C and 5D). The linear equation is-71 LogC +534 (R) ² 0.992), LOD 2.5 fg/mL.

When using only quantum dots as signal reporters, a similar linear range (10fg/mL-10pg/mL) can be obtained with a linear equation of-162 LogC +811(R ═ 162LogC + 811) ² 0.993), the LOD was 3fg/mL, as shown in fig. 5E and 5F.

The detectable concentrations of the dual signal patterns (QDs and calcein) ranged from 10fg/mL to 1pg/mL (FIGS. 5G and 5H). The results show that as the concentration of LAM increases, the fluorescence signals of quantum dots and calcein both decrease, and the linear equation is-98 LogC +486 (R) ² 0.991) had an LOD of 2.5 fg/mL. In the dual signal detection mode, the detection sensitivity of the method is similar to that in the single signal mode, and no significant interference exists between the dual signals. The sensitivity of this binary visual reading method is significantly higher than commercial kits.

Whereas visual performance analysis using color or distance changes as readings showed that in a single signal system, fluorescence intensity decreased with increasing LAM concentration, LAMs as low as 100fg/mL could be visually identified by color readings (fig. 5I and 5J).

When visualized using dual fluorescence signals, the color changed from orange to yellow as the LAM concentration increased (fig. 5K), which is more pronounced than the strong and weak contrast of the same color fluorescence. The resolving power is similar to that of a single signal, and the LOD is 100 fg/mL. The diffusion distance on the test strip can be used as a visual basis for quantification. As the LAM concentration increases, the diffusion distance of the solution on the QDs or calcein inkjet printing test strip becomes longer. The diffusion distance on the calcein or QDs bands was longer when the LAM concentration was increased from 10fg/mL to 10pg/mL (FIGS. 5L and 5M). The sensitivity of the visual test paper (100fg/mL) is slightly lower than that of the fluorescence detection method, but is similar to the sensitivity of the color pattern of the solution observed by the human eye.

This visualization method has many advantages, such as the long time that the tape results can be stored, the low instrument dependence, the lower cost, and the suitability for remote and poor areas.

In addition, the results of the specificity analysis indicated that the strategy had good selectivity (fig. 5N). Changes in fluorescence signal caused by potential interferents at a concentration of 1pg/mL are almost negligible, while LAM at a concentration of only 25fg/mL can result in a significant decrease in fluorescence signal.

6. Clinical sample analytical Performance

The clinical applicability of the method was verified by examining urine samples from clinically confirmed tuberculosis patients and non-tuberculosis subjects (fig. 6A). The results of the assay are compared with clinical diagnostic results and with laboratory results relating to Computed Tomography (CT), tuberculosis DNA, Mycobacterium tuberculosis interferon-gamma release test (TB-IGRA) and sputum culture tuberculosis. The LAM concentration in urine of healthy people and non-tuberculosis patients is lower than 30fg/mL, the detection result of the positive sample is higher than 30fg/mL, and the detection result is consistent with the clinical diagnosis result and the imaging CT result, which shows that the detection system has good specificity to tuberculosis.

For the clinical applicability of the test strip, clinical urine samples are used for verification, and the method is used for detecting urine samples of healthy people and tuberculosis positive patients. There was a significant difference in the band spread distance between the healthy and tuberculously infected samples (fig. 6C, D), indicating that the visualization method can accurately distinguish between tuberculously infected and healthy people. The spreading distance of the test strip for the patient with tuberculosis is longer than that of the patient without tuberculosis.

In conclusion, the strategy can be successfully used for high-sensitivity, simple and low-cost double-fluorescence visualization analysis of LAM in clinical urine samples, and can assist in tuberculosis diagnosis of patients. Selective recognition of Cu by QDs and calcein reported in the strategy ²⁺ And Cu NPs, are not only useful for LAM detection as described in this patent. If different biological reactions, signal molecules and detectors are combined, analysis of different analysis target objects can be realized, and the method has a wide application scene.

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

1. An analysis method for selectively regulating and controlling fluorescence signals of QDs and calcein based on metal ions and nano particles thereof is characterized by comprising the steps of selectively regulating and controlling fluorescence signals of QDs and calcein based on the metal ions and the nano particles corresponding to the metal ions, effectively changing the fluorescence signals of QDs and the calcein by the metal ions and the nano particles corresponding to the metal ions, and quantifying a single target based on the fluorescence signals of QDs and the calcein, wherein the target is Mycobacterium tuberculosis LAM.

2. The method of claim 1, wherein the metal ion is Cu ²⁺ The nano particles are CuNPs.

3. The method of claim 1, wherein the metal ion is Ag and the metal ion is selected from the group consisting of QDs and calcein ⁺ And the nano particles are Ag NPs.

4. The analytical method for selectively modulating QDs and calcein fluorescence signals based on metal ions and nanoparticles thereof according to claim 1, wherein the QDs comprise CdTe QDs or CdSe QDs.

5. The analytical method for selectively modulating fluorescence signals of QDs and calcein based on metal ions and nanoparticles thereof according to claim 1, wherein the excitation wavelength of QDs and calcein is 495 nm.

6. The method according to claim 5, wherein the QDs and the calcein are detected by a fluorometer, the color shade difference of the reaction solution in the test tube under an ultraviolet lamp, and/or the diffusion distance of the reaction solution on the paper strip when the ink jet printing test strip is inserted into the reaction solution.

7. The analytical method for selectively modulating QDs and calcein fluorescence signals based on metal ions and their nanoparticles according to claim 5, wherein the detectable concentration of LAM in dual signal mode is in the range of 10fg/mL to 1pg/mL, wherein the linear equation of calcein is: y-98 LogC +486, R ² 0.991, where the linear equation for QDs is: -71LogC +304, R ═ Y ² ＝0.983。

8. An application of an analysis method based on metal ions and nano-particles thereof for selectively regulating and controlling QDs and calcein fluorescence signals, which is characterized in that the application comprises applying the analysis method based on metal ions and nano-particles thereof for selectively regulating and controlling QDs and calcein fluorescence signals according to any one of claims 1 to 7 to Mycobacterium tuberculosis LAM analysis.

9. An application of an analysis method for selectively regulating QDs and calcein fluorescence signals based on metal ions and nano particles thereof is characterized by further comprising the step of combining a plurality of metal ions with different analysis instruments and signal molecules to construct an analysis strategy for detecting and diagnosing different molecules.