CN110261353A - A kind of zero background fluorescence detection method and its application of hydrazine - Google Patents

Abstract

The invention discloses a kind of zero background fluorescence detection method of hydrazine and its applications, the present invention promotes the condensation reaction between 4- lignocaine salicylide and hydrazine by cetyltriethylammonium bromide (CTAB) micella, indicator of the generated in-situ fluorescent material with intramolecular hydrogen bond as hydrazine proposes a kind of new method of hydrazine detection.This method constructs the detection pattern of zero background, and avoids the use of organic synthesis and organic solvent cumbersome during testing.Furthermore, in actual water sample, serum sample, living cells even mouse be imaged in it is extensive studies have shown that probe 4- lignocaine salicylide (DEASA) and original position sensing solutions are in sensitivity, anti-interference ability and imaging side face is with good stability and broad application prospect.

Description

A zero-background fluorescence detection method for hydrazine and its application

technical field

The invention belongs to the technical field of hydrazine detection, and relates to a method strategy for detecting hydrazine. More specifically, it relates to a zero-background fluorescence detection method for hydrazine and its application.

Background technique

As we all know, hydrazine is a widely used rocket propellant. Due to its inherent strong alkalinity and reducing properties, it is widely used in industrial fields, mainly for the manufacture of chemical catalysts, pharmaceutical intermediates and agricultural chemicals. However, the World Health Organization defines hydrazine as a highly toxic and carcinogenic environmental pollutant during production and transportation, and the threshold limit is set at 10ppb. In addition, hydrazine has excellent water solubility and is easily absorbed through oral and skin contact, causing serious damage to human organs such as kidneys, lungs, liver and even the central nervous system. Therefore, for the detection of hydrazine, it is very important to monitor its trace concentration to meet the demand of high sensitivity and good sensing performance in aqueous solution.

At present, the commonly used analytical methods for the detection of hydrazine mainly include electrochemical analysis, HPLC/GS-MS method and capillary electrophoresis, but these methods usually need to be carried out on high-cost experimental instruments, the sample preparation is cumbersome, and compared with fluorescent probes , not suitable for analysis in biological systems.

Fluorescent probes, as a compound with special optical properties in the sensing of targets, can meet the growing needs of solving environmental pollution and human health problems, and fluorescent probes have made great achievements in sensitivity and selectivity. Great progress. So far, a series of fluorescent probes suitable for actual samples, living cells and even organisms have been developed. These probes are simple and easy to use for labeling or biological imaging, making them have great potential in early diagnosis of diseases and detection of environmental pollution. .

In addition, in order to improve the analytical sensitivity and signal-to-noise ratio of fluorescent probes, although currently reported in the literature, there have been a variety of methods to design and construct molecular structures to improve the response signal or reduce background interference. For example, probe structures are designed through photophysical processes such as twisted intramolecular charge transfer (TICT), fluorescence resonance energy transfer (FRET), and photoinduced electron transfer (PET). However, the analysis method that uses probes and analytes to generate chromophores in situ can completely avoid the interference of background fluorescence, and such probes usually have ingenious molecular structures, which themselves are not luminescent, and can be self-generated when combined with analytes The luminophore realizes the detection of the analyte, and the simple molecular structure can effectively avoid the tedious synthesis and purification process. It is currently a relatively valuable method for the detection of hydrazine in water, through the specific reaction between the probe and the target analyte The analytical method of generating chromophores provides a new idea for the high-sensitivity and zero-background detection of target substances.

Contents of the invention

In view of this, the purpose of the present invention is to address the problems in the prior art, provide a CTAB micelles to promote the condensation reaction of 4-diethylamino salicylaldehyde and hydrazine, the chromophore generated in situ as a hydrazine detection indicator Novel sensing strategies for agents.

In order to achieve the above object, the technical scheme of the present invention is as follows:

A zero-background fluorescence detection method for hydrazine, the condensation reaction between 4-diethylaminosalicylaldehyde and the analyte hydrazine is promoted by cetyltriethylammonium bromide (CTAB) micelles, and the in situ generated A new chromophore for intramolecular hydrogen bonding serves as an indicator for hydrazine detection.

In the present invention, through the condensation reaction of 4-diethylamino salicylaldehyde (DEASA) with hydrazine in CTAB micelles, the new chromophore generated in situ is used as a fluorescent indicator for hydrazine detection, and the zero-background detection of hydrazine in the aqueous phase is realized. , avoiding the cumbersome organic synthesis and the use of organic solvents.

The specific detection method disclosed above includes the following steps:

Step 1: Add PBS buffer as a solvent in the colorimetric tube;

Step 2: Add probe DEASA and specific concentration of CTAB to the solvent in step 1.

Step 3: adding the analyte hydrazine to the solution in step 2, DEASA and hydrazine undergo a condensation reaction to generate the luminescent substance HDBM.

It should be noted that the reaction temperature of the above condensation reaction is room temperature, and the reaction is rapid.

Preferably, the synthesis method of the new chromophore specifically includes the following steps:

(1) Using PBS buffer as the reaction medium, add 4-diethylamino salicylaldehyde and cetyltriethylammonium bromide (CTAB);

(2) The analyte hydrazine is added to the solution obtained in step (1), and the DEASA and hydrazine undergo a condensation reaction to finally generate the new chromophore disclosed in the present invention—a luminescent substance HDBM.

It should be noted that, referring to the accompanying drawing 1 of the specification, the present invention conducts structural characterization of the generated luminescent substance HDBM by hydrogen nuclear magnetic resonance spectroscopy, to show that the luminescent substance HDBM is successfully generated by the above synthesis method.

In particular, the specific scheme for preparing the luminescent material HDBM disclosed in the present invention is as follows:

The binding ratio of hydrazine and DEASA was determined to be 1:2 by the equimolar continuous change method (Job method). By detecting the spectrum of the reaction solution in the presence of different concentrations of CTAB, the optimized CTAB concentration was 1×10 ^-3 mol·L ^-1 , so add 2mL of PBS buffer as a solvent in a 5mL colorimetric tube, add 20μL DEASA DMSO solution (5× ^10-3 mol L ^-1 ), and then add 2μL CTAB aqueous solution (1mol· L ^-1 ), after adding 0.5 equivalent of hydrazine, the luminescent substance HDBM is obtained.

Wherein, the structure of the luminescent substance HDBM generated in the present invention is:

Preferably, the molar ratio of the analyte hydrazine to 4-diethylamino salicylaldehyde is 1:2.

It is worth noting that in the present invention, the molar ratio of the analyte hydrazine and 4-diethylaminosalicylaldehyde is 1:2, which has the best luminescent effect, so the fluorescence detection method disclosed in this application is selected from both. The molar ratio is 1:2.

Preferably, the concentration of cetyltriethylammonium bromide (CTAB) is controlled between 0.1˜5×10 ⁻³ mol·L ⁻¹ .

Among them, in the present invention, controlling the concentration of cetyltriethylammonium bromide (CTAB) within the range of 0.1 to 5×10 ^-3 mol·L ^-1 can promote the formation of the luminescent substance HDBM, and the preferred concentration of the present invention is is 1×10 ^-3 mol·L ^-1 . And the applicant's investigation range of the concentration of CTAB is 0.1～5×10 ^-3 mol·L ^-1 , as shown in Figure ³ , when the concentration of CTAB is 1×10 ^-3 mol·L ^-1 , the luminescent substance HDBM The fluorescence intensity is the highest, so the concentration of CTAB is preferably 1×10 ^-3 mol·L ^-1 for subsequent experiments.

Another object of the present invention is to provide an application of a zero-background fluorescence detection method for hydrazine in the selective identification and quantitative detection of hydrazine in a solvent system.

Preferably, the concentration of cetyltriethylammonium bromide (CTAB) in the solvent system is 1×10 ^-3 mol·L ^-1 .

In some application scenarios, it also includes the application of the detection method in the detection and imaging of hydrazine as a marker.

It is worth noting that the present invention discloses a hydrazine sensing strategy for the chromophore generated by the condensation reaction between 4-diethylaminosalicylaldehyde (DEASA) and hydrazine. Among them, cetyltrimethylammonium bromide (CTAB) micelles provided stable hydrophobic cavities, which facilitated the condensation reaction of DEASA and hydrazine at the micellar interface. At the same time, the generated luminescent substance has better luminescent performance under the protection of CTAB micelles, so that the detection sensitivity can be improved. The specific sensing principle is shown in Figure 2 of the specification.

It can be known from the above-mentioned technical scheme that, compared with the prior art, the present invention discloses a zero-background fluorescence detection method for hydrazine and its application. First, the present invention provides a 4-diethylamino salicylaldehyde in CTAB micelles A new sensing strategy for the reaction of hydrazine with hydrazine to generate a new chromophore as an indicator for hydrazine detection. This zero-background detection mode has ultra-high sensitivity and is suitable for the detection of trace hydrazine in actual samples and organisms;

Secondly, the detection scheme disclosed in the present invention is based on the condensation reaction of the non-fluorescent substance DEASA and hydrazine. By introducing CTAB micelles to provide suitable reaction sites for hydrophobic probes and hydrophilic hydrazine, the reaction efficiency has been greatly improved. Great improvement, using the fluorescent product HDBM containing intramolecular hydrogen bonds as a fluorescent signal indicator;

Then, the CTAB micelles used in the present invention make the detection of hydrazine convenient and feasible in aqueous solution without adjustment by organic solvent;

Finally, the sensing detection analysis disclosed in the present invention successfully detects hydrazine in actual samples, and is quite sensitive in live cell imaging. The research on hydrazine in vivo further proves that it is not disturbed by complex environments and durable Good imaging performance.

In summary, the detection method disclosed in the present invention can efficiently and selectively identify hydrazine, and has high sensitivity to hydrazine. The invention provides a new detection method for hydrazine, which has the advantages of simple preparation, good biocompatibility, etc., and has market application and promotion value for industry, life and biological fields.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only It is an embodiment of the present invention, and those skilled in the art can also obtain other drawings according to the provided drawings on the premise of not paying creative efforts.

Fig. 1 is the proton nuclear magnetic resonance spectrum of HDBM of the present invention in deuterated DMSO.

Fig. 2 is a hydrazine sensing schematic diagram of the condensation reaction between 4-diethylaminosalicylaldehyde (DEASA) and hydrazine to generate a chromophore in the present invention.

Fig. 3 is a diagram of the fluorescence intensity of DEASA of the present invention and 0.5 mM hydrazine when containing different concentrations of CTAB.

Fig. 4 is the fluorescence spectrogram of the DEASA solution containing CTAB and different concentrations of hydrazine in the present invention.

Fig. 5 is a fluorescence spectrogram of hydrazine and other analytes contained in DEASA and CTAB solutions of the present invention.

Fig. 6 is the determination table of hydrazine in the actual sample of the present invention.

Fig. 7 is the confocal fluorescence images of HeLa cells and different concentrations of hydrazine after being treated with DEASA in the present invention.

Fig. 8 is the fluorescence imaging of hydrazine in mice of the present invention.

Fig. 9 is a bar graph of the survival rate of HeLa cells of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The embodiment of the present invention discloses a sensing strategy in which 4-diethylamino salicylaldehyde reacts with hydrazine in CTAB micelles to generate a new chromophore as a hydrazine indicator.

For a better understanding of the present invention, the present invention will be further specifically described below through the following examples, but it should not be construed as a limitation of the present invention. For some non-essential improvements and adjustments made by those skilled in the art according to the above-mentioned content of the invention, It is also considered to fall within the protection scope of the present invention.

The invention discloses a zero-background fluorescence detection method for hydrazine, which promotes the condensation reaction between 4-diethylamino salicylaldehyde and the analyte hydrazine through cetyltriethylammonium bromide (CTAB) micelles. A new chromophore with intramolecular hydrogen bonding generated at the site was used as an indicator for the detection of hydrazine.

The generation of the new chromophore-luminescent substance HDBM of the present invention is to add PBS buffer solution as a solvent in the colorimetric tube, then add probes DEASA and CTAB, and finally add analyte hydrazine, and condensation reaction occurs in CTAB micelles, finally forming Luminescent material HDBM.

Concrete preparation scheme is as follows:

Add 2 mL of pure aqueous solution as a solvent to a 5 mL colorimetric tube, add 20 μL of DEASA in DMSO (5×10 ^-3 mol·L ^-1 ), and then add CTAB in water (1×10 ^-3 mol·L ^-1 ), then add 0.5 equivalent of hydrazine to obtain the luminescent substance HDBM.

In order to further verify the excellent effect of the present invention, the inventor has also carried out the following experiments:

Experiment 1: Synthesis and structural characterization of luminescent material HDBM

1. Synthesis of luminescent material HDBM

Add 2 mL of pure aqueous solution as a solvent to a 5 mL colorimetric tube, add 20 μL of DEASA in DMSO (5×10 ^-3 mol·L ^-1 ), and then add CTAB in water (1×10 ^-3 mol·L ^-1 ), then add 0.5 equivalent of hydrazine to obtain the luminescent substance HDBM.

2. Test analysis

The peak value of the ¹ HNMR spectrum of the luminescent substance HDBM is:

¹ H NMR (500MHz, DMSO-d6) δ (ppm) 11.47 (s, 2H), 8.59 (s, 2H), 7.26 (d, J = 8.8Hz, 2H), 6.30-6.28 (m, 2H), 6.10 (d, J=1.7Hz, 2H), 3.36(q, J=6.9Hz, 4H), 2.48(t, J=7.0Hz, 4H);

¹³ CNMR spectrum peak is:

¹³ C NMR (126MHz, DMSO-d6) δ (ppm) δ 161.08, 133.44, 106.85, 104.49, 97.50, 44.29, 12.99.

According to the H spectrum and C spectrum of the luminescent substance prepared by the synthesis method disclosed in the present invention, the spectrum peaks in the drawings correspond to HDBM one by one, which proves that the synthesis of the luminescent substance HDBM is successful.

Experiment 2: A specific application of a hydrazine-based zero-background fluorescence detection method for the detection of hydrazine in a solvent system

1. Test experiment

In the aqueous solution containing 1.0mM CTAB and 50μM DEASA, add hydrazine, K ⁺ , Na ⁺ , Mg ²⁺ , Ca ²⁺ , F ^- , Cl ^- , NO ₃ ^- , Glu to each colorimetric tube in sequence, Aqueous solutions of Cys, Gly, and aniline (1×10 ^-4 mol·L ^-1 ), after mixing, tested their fluorescence spectra, see Figure 5(A).

In contrast, in an aqueous solution containing 1.0 mM CTAB, 100 μM hydrazine and 50 μM DEASA, hydrazine, K ⁺ , Na ⁺ , Mg ²⁺ , Ca ²⁺ , F ^- , Cl ^- , The aqueous solution (1×10 ^-4 mol·L ^-1 ) of NO ₃ ^- , Glu, Cys, Gly, and aniline was mixed well, and its fluorescence spectrum was tested, see Figure 5(B).

2. Test analysis

Probe DEASA is relatively selective to hydrazine, and a variety of potential interfering compounds are involved in the experiment, such as cations (K ⁺ , Na ⁺ , Mg ²⁺ and Ca ²⁺ ), anions (F ^- , Cl ^- and NO ₃ ^- ) , amino acids (Glu, Cys, and Gly) and even analogues (aniline), the fluorescence response can only be elicited by hydrazine, while potentially interfering species cause little spectral change. Further comparative experiments showed that the coexistence of interfering substances had almost no effect on the response of DEASA to hydrazine.

The above results indicate that the probe DEASA has strong specificity for the detection of hydrazine and is suitable for detection in a physiological environment, which may be provided by a specific reaction-based sensing strategy that provides a blank background and in situ generated signal system.

In summary, the present invention can realize the selective recognition of hydrazine in aqueous solution.

Experiment 3: Determination of the minimum detection limit of the probe DEASA for the quantitative detection of hydrazine

At 25°C, using fluorescence emission spectroscopy, according to the titration experiment of hydrazine with 1.0mMCTAB and 50μM DEASA, through 3σ/k calculation, the lowest detection limit of probe DEASA for hydrazine is 0.42nM, indicating that the method can detect hydrazine The high sensitivity indicates that the probe DEASA has potential application value in the high-efficiency detection of hydrazine in CTAB micelles.

Experiment 4: Specific application of the above-mentioned zero-background fluorescence detection method for hydrazine in real water and serum samples, live cells, and even mouse imaging

(1) Specific application of the detection method to detect hydrazine in actual water samples and serum samples

First, the proposed sensing analysis method was applied to the detection of tap water, and the hydrazine content in seawater, sewage and diluted human serum collected from five places were detected respectively, and the test results are shown in Fig. 6. From the determination table of hydrazine in the actual sample shown in Figure 6, the recoveries and relative standard deviation (RSD) values are within the normal range, the other two high levels of hydrazine content (respectively 2μM and 10μM), revealing that the sewage hydrazine residues. The above results demonstrate that the probe DEASA and the proposed in situ detection design can be applied to the quantitative analysis of real samples.

(2) Specific application of the detection method to detect hydrazine in living cells

HeLa cells were incubated with 50 μM probe DEASA for 1 hour, washed with PBS buffer, and then added with different concentrations of hydrazine (increased from 10 μM to 150 μM) for another 2 hours. Cells that were not further treated with hydrazine were used as control samples, as shown in the figure 7. 7(a) to 7(e) show the fluorescence imaging of cells when the hydrazine concentrations are 0 μM, 10 μM, 50 μM, 100 μM, and 150 μM, respectively.

From Fig. 7, cells not further treated with hydrazine showed completely dark fluorescence, indicating that the probe DEASA together with the reaction medium CTAB micelles could not trigger any fluorescent reaction. This zero response affirms the zero-background nature of the sensing assay and rules out the possibility of interference from different components in living cells. In addition, obvious green fluorescence emission could be captured after HeLa cells were treated with 10 μM hydrazine, which verified the feasibility of in situ sensing for bioimaging.

(3) The specific application of the detection method in the detection of hydrazine in organisms

The probe DEASA (40 μL, 50 μM in DMSO solution) was injected through the skin into nude mice, and the first two imaging pictures were taken at 0 and 2 hours after the injection, respectively, as shown in FIG. 8 . The mouse itself does not emit light, while the fluorescent image still does not emit light within 2 hours, proving the relative stability of DEASA in complex biological environments. Shortly after taking the second picture, additional hydrazine (2 μL, 0.1 M) was injected at the same site, and fluorescence images were captured according to the planned schedule, followed by observation of fluorescence imaging 6 hours after hydrazine injection, which was derived from DEASA with The luminescent product HDBM produced during the hydrazine reaction indicates that the in situ sensing strategy proposed by the present invention can also be applied to organisms.

In addition, the emission intensity obtained during the 24-hour real-time detection period can also be normalized and quantified, and strong fluorescence can still be obtained after 24 hours without intensity loss, which indicates that the in situ generated luminescent species in the assay has the advantage of long-lasting and effective imaging. . Therefore, the good response characteristics and high stability exhibited by this scheme enable the detection method disclosed in the present invention to realize the detection of hydrazine through in-situ condensation reaction even at the biological level.

In summary, the detection method disclosed in the present invention can be successfully applied to live cell imaging and mouse body imaging, which shows that the above-mentioned zero-background fluorescence detection method has good biocompatibility. And in order to further prove the biocompatibility of the detection method, the applicant also performed a characterization analysis on HeLa cytotoxicity, and the specific experimental results are shown in Experiment 5.

Experiment 5: Cytotoxicity experiment of the zero-background fluorescence detection method for hydrazine above

HeLa cells were seeded in a 96-well plate, incubated for 24 hours, added 50 μM probe DEASA and incubated for 0, 1, 2, 4, 8, 12, and 24 hours, respectively, and then washed the cells with phosphate buffer, and used the CCK-8 method To detect cytotoxicity, the bar graph of the survival rate of HeLa cells is shown in FIG. 9 .

It can be seen from Figure 9 that the cell survival rate remains above 90% after 24 hours of incubation with 50 μM DEASA, which proves that DEASA has relatively low cytotoxicity. This result shows the bioimaging study of the probe DEASA on hydrazine in living cells Has good biocompatibility.

In conclusion, the present invention proposes a multifunctional and direct hydrazine detection strategy with background-free detection function, suitable for real samples and in vivo detection. The advantages of CTAB micelles make the detection feasible in aqueous solution without adjustment by organic solvents, and through the in situ detection scheme, the probe DEASA has relatively high sensitivity and selectivity for hydrazine, and the background-free property can also be used for intracellular imaging and even biological analyze. The excellent hydrazine fluorescence detection and sensing scheme disclosed in the present invention provides a new detection approach for high-sensitivity and zero-background detection of target objects, which is extremely valuable for market application and promotion in the fields of industry, life and biology.

The above descriptions of the disclosed embodiments and experimental examples enable those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (7)

1. A zero-background fluorescence detection method for hydrazine, characterized in that, the condensation reaction between 4-diethylamino salicylaldehyde and analyte hydrazine is promoted by cetyltriethylammonium bromide micelles, in situ The resulting new chromophore with intramolecular hydrogen bonding acts as an indicator for hydrazine detection. 2. the zero-background fluorescence detection method of a kind of hydrazine according to claim 1, is characterized in that, the synthetic method of described new chromophore specifically comprises the steps: (1) taking PBS buffer as the reaction medium, adding 4-diethylaminosalicylaldehyde and cetyltriethylammonium bromide; (2) The analyte hydrazine is added to the solution obtained in step (1), and DEASA and hydrazine undergo a condensation reaction to finally generate the new chromophore disclosed by the present invention—a luminescent substance HDBM. 3. A zero-background fluorescence detection method for hydrazine according to claim 1 or 2, wherein the molar ratio of the analyte hydrazine to 4-diethylaminosalicylaldehyde is 1:2. 4. A zero-background fluorescence detection method for hydrazine according to claim 1 or 2, characterized in that the concentration of cetyltriethylammonium bromide is controlled at 0.1 to 5×10 ^-3 mol· Between L ^-1 . 5. The application of a zero-background fluorescence detection method of hydrazine as claimed in claim 1 in the selective identification and quantitative detection of hydrazine in a solvent system. 6. The application of a zero-background fluorescence detection method for hydrazine according to claim 5, further comprising the application of the detection method in the detection and imaging of hydrazine as a marker. 7. The application of a zero-background fluorescence detection method for hydrazine according to claim 5, wherein the concentration of cetyltriethylammonium bromide in the solvent system is 1× ^10-3 mol ·L ^-1 .