Mass spectrometer imaging target plate based on n,s-mqds and applications thereof

Abstract

The application discloses a mass spectrometer imaging target plate based on N, S-MQDs and application thereof, the mass spectrometer imaging target plate is cured by light-cured resin containing N, S-MQDs, and the doping amount of N, S-MQDs is 0.1%-20%; the preparation steps of N, S-MQDs are as follows: quantum dots are prepared by using MAX phase material as raw material and DMSO as intercalation agent; the quantum dot solution, nitrogen source dopant and sulfur source dopant are subjected to reduction reaction under ultrasonic condition; and the reaction product is washed and dried in sequence to obtain N, S-MQDs. The imaging target plate containing N, S-MQDs can absorb laser energy and transfer the laser energy to a biological sample to be measured, so that high-sensitivity mass spectrum imaging detection of molecules in biological tissues can be realized without needing to spray a matrix on the biological tissues before imaging, so that the operation steps are simplified, the sample preparation time is saved, and the detection efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometer imaging, in particular to a mass spectrometer imaging target plate based on N, S-MQDs, and to the use of the mass spectrometer imaging target plate. BACKGROUND

[0002] MALDI mass spectrometry imaging (Matrix-Assisted Laser Desorption / Ionization Imaging) is an advanced mass spectrometry imaging technique for spatially resolved analysis of biomolecules. It allows for visualizing and quantitatively analyzing the distribution of molecules in complex biological samples and exploring the heterogeneity of molecular spatial distribution in biological tissues. MALDI mass spectrometry imaging technique mainly has two key steps: sample preparation and mass spectrometry imaging. In MALDI imaging, the sample is usually a biological tissue section, cells or other complex biological samples, which is first mixed with an auxiliary matrix (matrix). After sample preparation, a laser beam with a specific wavelength and intensity is used to irradiate the sample surface. The laser energy dissociates the molecules in the matrix and sample into ions, which are guided into a mass spectrometer such as a time-of-flight mass spectrometer (TOF-MS). In the mass spectrometer, the ions are accelerated and fly to the detector according to their mass-to-charge ratio (m / z). Different m / z ions reach the detector at different times, so the ions can be separated and detected according to the arrival time. The mass spectrometer records the ion signal of each pixel point and generates a two-dimensional molecular mass spectrum. This mass spectrum shows the spatial distribution of molecules on the sample surface, allowing the distribution of different molecules in biological tissues to be quantitatively and visually analyzed. Through MALDI imaging technology, researchers can explore the distribution and quantitative information of complex molecules in biological samples and understand the spatial relationship of molecules in biological processes and disease development. This provides important tools and insights for the fields of biology, medicine and drug research and development.

[0003] The matrix used in mass spectrometer imaging is usually an organic compound that helps absorb laser energy, such as horseradish peroxide or cyanine. When the laser irradiates the sample, the matrix absorbs energy, causing evaporation and photolysis, which converts the molecules in the sample from a solid state to a gaseous state and forms ions. In the MALDI imaging technology, the process of spraying the matrix plays a crucial role in MALDI imaging, and it is one of the key factors for sample analysis. Spraying the matrix may cause some problems: first, uneven spraying can lead to uneven distribution of the matrix on the sample surface, reducing spatial resolution and accuracy of molecular localization. Second, some matrixes may combine with molecules in the sample to form crystals, affecting ionization of molecules and mass spectrometry signals. In addition, some matrixes may volatilize excessively under laser irradiation, leading to a decrease in signal intensity. Finally, most MALDI uses solid (crystalline) organic matrixes, but these matrixes produce corresponding background peaks in the low mass region, interfering with the characterization of small molecule compounds and affecting the qualitative and quantitative analysis of molecules. SUMMARY

[0004] Therefore, the present application provides a mass spectrometer imaging target plate based on N, S-MQDs, which can realize mass spectrometry imaging detection without spraying matrix on the biological tissue sample, thereby simplifying the operation steps, saving sample preparation time and greatly improving the detection efficiency.

[0005] A second object of the present application is to provide an application of the mass spectrometer imaging target plate based on N, S-MQDs, which can realize visual detection of DNPD, DPPD and other phenylenediamine antioxidants in zebrafish.

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] The mass spectrometer imaging target plate based on N, S-MQDs is solidified by a photocurable resin containing N, S-MQDs, and the doping amount of N, S-MQDs is 0.1% to 20%; wherein the N, S-MQDs are prepared by the following steps:

[0008] S1, using acid etching MAX phase material as raw material, DMSO as intercalation agent, quantum dots are prepared;

[0009] S2, the quantum dot solution, nitrogen source dopant and sulfur source dopant are subjected to reduction reaction under ultrasonic condition, and the reaction product is washed and dried in sequence to obtain N, S-MQDs.

[0010] The beneficial effect is that: compared with MXene, the electronic structure of N, S-MQDs in the application is modified, nitrogen and sulfur doping can introduce new energy levels in the band gap, so that the laser absorption of 355nm wavelength is enhanced. Therefore, the imaging target plate containing N, S-MQDs in the application can itself absorb laser energy and transfer laser energy to the biological sample to be tested, thereby promoting the desorption and ionization of the biological sample to be tested. In actual use, high-sensitivity mass spectrometry imaging detection of molecules in biological tissues can be realized without spraying matrix on the biological tissues before imaging, thereby simplifying the operation steps, saving sample preparation time and greatly improving the detection efficiency.

[0011] Preferably, the preparation step S1 of N, S-MQDs in the application comprises the following specific contents:

[0012] S11, the MAX phase material is added to an acid solution, and the MAX phase material is stably suspended by constant temperature stirring at 40-80 DEG C. After etching, the precipitate is separated, washed to pH>7.0, dried, and compound I is obtained;

[0013] The MAX phase material is Ti3AlC2; the acid solution is hydrofluoric acid solution;

[0014] S12, compound I is diluted with DMSO, stirred for 12-36h, the precipitate is separated, washed, and the washed precipitate is dispersed in deionized water, and quantum dot solution is obtained by ultrasonic in nitrogen environment.

[0015] Preferably, the mass fraction of the hydrofluoric acid solution used in the application is 47%-51%.

[0016] Preferably, the reduction reaction in the preparation step S2 of N, S-MQDs in the application is sodium borohydride as a reducing agent, and the reduction reaction temperature is 35-55 DEG C; wherein the nitrogen source dopant is ammonia water (ammonia water concentration is preferably 25%-30%), and the sulfur source dopant is sodium thiosulfate.

[0017] Preferably, the ultrasonic conditions in the preparation step S2 of N, S-MQDs in the application are as follows: ultrasonic frequency≤300W, ultrasonic time≥3.0h.

[0018] Preferably, the mass spectrometer imaging target plate in the application is prepared by first dispersing N, S-MQDs uniformly in deionized water, then stirring N, S-MQDs with photocuring resin overnight to make N, S-MQDs uniformly dispersed in photocuring resin, finally pouring into the trough of the mass spectrometer, and then using 3D printing technology to prepare and irradiate under ultraviolet light until solidification to obtain an imaging target plate consistent with the shape of the target holder of the mass spectrometer.

[0019] The beneficial effects are that the imaging target plate of the application is consistent with the outer contour of the commercial target holder, which not only ensures the stable fixation of the application in the mass spectrometer, avoids sample displacement or shaking, but also ensures the positioning accuracy of the sample, avoids the distortion or deviation of the imaging signal, and also makes the placement position and angle of the sample consistent in each experiment, further ensuring the repeatability of the sample detection.

[0020] In actual preparation, the resin containing N, S-MQDs is ultrasonically treated for 10 minutes before imaging and then stirred again, so as to ensure the uniform dispersion of N, S-MQDs in the imaging target plate, effectively avoid the uneven problem of the traditional matrix spraying method, and improve the imaging effect and the repeatability of imaging.

[0021] The imaging target plate of the application is prepared by means of DLP type 3D printing technology, which has fast printing speed and low printing cost.

[0022] Preferably, the photocurable resin is Formlabs transparent resin, 3D Systems transparent resin or SOMOS transparent resin.

[0023] Preferably, the thickness of the mass spectrometer imaging target plate is 50 μm.

[0024] Compared with the prior art, the advantages of the application are embodied in the following points:

[0025] 1. Compared with MXene, the electronic structure of N, S-MQDs in the application is modified, and nitrogen and sulfur doping can introduce new energy levels in the band gap, thereby enhancing the absorption of laser with a wavelength of 355 nm. Therefore, the imaging target plate containing N, S-MQDs in the application can itself absorb laser energy and transfer laser energy to the biological sample to be tested, thereby promoting the desorption and ionization of the biological sample to be tested.

[0026] The N, S-MQDs of the present application have low baseline interference generated as a matrix, can obtain a cleaner mass spectrum, and improve the accuracy and reliability of the analysis results. The N, S-MQDs have high chemical stability and thermal stability, have a long service life, and have excellent electrical conductivity, which can accelerate the ionization process of molecules in the sample and improve the efficiency of mass spectrum signal generation.

[0027] In addition, compared with undoped MXenes, the introduction of N and S can reduce the cytotoxicity of MXenes. Cytotoxicity is usually related to the release of metal ions in the material. Doping non-metallic elements such as N and S can reduce the release of potentially harmful metal ions, thereby improving biocompatibility. Nitrogen and sulfur are common biocompatible elements in biological molecules, such as amino acids and proteins. These elements can make them more compatible with biological tissues after doping, thereby making them more suitable for mass spectrometric analysis of biological samples, such as imaging analysis of biological molecules.

[0028] 2、The N, S-MQDs of the present application can be uniformly dispersed in a sample target plate, effectively avoiding the unevenness of the traditional matrix spraying method, and improving the imaging effect and the repeatability of imaging.

[0029] 3、The imaging target plate of the present application is prepared by means of 3D printing technology, which is simple and convenient to prepare; the outer contour of the imaging target plate is consistent with the commercial target holder of the mass spectrometer, which can be stably fixed in the mass spectrometer, not only avoiding sample displacement or shaking, but also ensuring the positioning accuracy of the sample, avoiding distortion or deviation of the imaging signal, and also ensuring that the placement position and angle of the sample in each experiment are consistent, further ensuring the repeatability of the sample detection.

[0030] 4、The imaging target plate of the present application has a thickness of 50 μm and is light in weight; the cost of each imaging target plate can be controlled within 10 yuan, which is helpful for commercialization and promotion, and has great economic significance. In addition, the imaging target plate of the present application can be used for imaging and quantitative detection of phenylenediamine antioxidants in biological tissue samples, providing a new idea for the quantitative detection of phenylenediamine antioxidants, and having important significance. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the ultraviolet absorption spectrum of N, S-MQDs in Example 1 of the present application.

[0032] Figure 2 is the imaging target plate after curing in Example 2 of the present application.

[0033] Figure 3 is the TEM characterization diagram of the imaging target plate in Example 2 of the present application.

[0034] Figure 4 is the imaging diagram of DNPD in zebrafish in Application Example 1 of the present application.

[0035] Figure 5 This is an image of DPPD in zebrafish from Application Example 1 of this invention. Detailed Implementation

[0036] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the scope of protection of the present invention is not limited to the following embodiments.

[0037] I. Preparation of N,S-MQDs in this invention

[0038] Example 1

[0039] In this embodiment, N,S-MQDs are manufactured using the following steps:

[0040] S1, 2.0 g Ti3AlC2 was added to 20 mL of HF with a mass concentration of 49%, and stirred at 60℃ for 24 h (using magnetic stirring) to stabilize Ti3AlC2 in suspension; centrifugation was used to obtain a precipitate, which was washed with deionized water until pH > 7.0, and then dried under vacuum at 60℃ overnight to obtain compound I (i.e., Ti3C2 powder).

[0041] S2, Ti3C2 powder was diluted and dispersed with DMSO and stirred at room temperature for 24 h, centrifuged, and washed with deionized water; the washed precipitate was dispersed in deionized water, first sonicated in a nitrogen environment for 5 h (sonication power of 120 W), and then sonicated at 300 W for 10 h to obtain a quantum dot solution.

[0042] S3, 30 mL of ammonia water (25%-30% by mass), 0.05 g of sodium thiosulfate, sodium borohydride, and 20 mL of quantum solution were mixed and placed in an ultrasonicator. The mixture was sonicated at 200 W for 3 h at 45 °C, centrifuged, and the precipitate was washed with deionized water and dried under vacuum at 60 °C overnight to obtain N,S-MQDs. Its spectrum is shown in […]. Figure 1 .

[0043] Combination Figure 1 It is known that, compared with MXene, the electronic structure of the N,S-MQDs of the present invention is modified. Nitrogen and sulfur doping can introduce new energy levels in the band gap, thereby enhancing their absorption of laser light with a wavelength of 355 nm and transferring it to the biological tissue sample to be tested, thereby promoting the desorption and ionization of the analyte in the biological tissue sample to be tested.

[0044] II. Preparation of the mass spectrometer imaging target plate based on N,S-MQDs as described in this invention

[0045] Example 2

[0046] The N, S-MQDs (from Example 1) were uniformly dispersed in deionized water (magnetic stirring was used), and then mixed with Formlabs transparent photocuring resin, and stirred vigorously overnight (magnetic stirring speed was 13000 rpm) to make the N, S-MQDs uniformly distributed in the Formlabs transparent resin; wherein the mass fraction of N, S-MQDs in the resin mixture was 0, 1%, 2%, 5% and 10%;

[0047] Before 3D printing, the resin mixture was ultrasonically treated at room temperature for 10 min and then stirred again to ensure that the N, S-MQDs were uniformly distributed in the photocuring transparent resin; the uniformly mixed resin mixture was poured into the trough of the 3D printer, and was cured by ultraviolet light to obtain a mass spectrometer imaging target plate with a thickness of 50 μm.

[0048] In the present application, since the resin containing N, S-MQDs is directly poured into the trough of the 3D printer for curing and forming, the imaging target plate of the present application is consistent in size with the commercial target holder, and the imaging target plate of the present application can be directly fixed on the target holder during detection.

[0049] The mass spectrometer imaging target plate prepared in the present embodiment is shown in Figure 2 , and the TEM characterization is shown in Figure 3 ( Figure 3 The corresponding 5%. It can be seen from Figure 3 that the N, S-MQDs are uniformly dispersed in the imaging target plate, effectively avoiding the uneven problem of the traditional matrix spraying method, improving the imaging effect, and having imaging repeatability.

[0050] Third, the application of the mass spectrometer imaging target plate prepared in the present application

[0051] Application Example 1: Application of the mass spectrometer imaging target plate in Example 2 of the present application in visualizing benzidine anti-aging agents in zebrafish

[0052] S1, adult zebrafish of 5 months old were placed in an aerated artificial freshwater aquarium at 25℃, and were bred according to a 14-hour / 10-hour light / dark scheme, and the zebrafish were fed once a day;

[0053] S2, the zebrafish were exposed to DNPD (i.e. N, N'-di(β-naphthyl) p-phenylenediamine) and DPPD (i.e. N, N'-diphenyl-p-phenylenediamine) for 48 hours, and the zebrafish were cut to a thickness of 14 μm using a Leica CM 1850 cryostat freezing microtome; wherein if the exposed zebrafish cannot be immediately sliced for analysis, the exposed zebrafish were stored at -80℃, and were sliced for detection;

[0054] S3, the zebra fish section was placed on the mass spectrometer imaging target plate prepared in Example 2 (the mass fraction of N, S-MQDs in the resin mixture was 2% and 5%), and imaging detection was performed by using a mass spectrometer (the model of the mass spectrometer was Waters Synapt G2 MALDI-TOF), the laser power was 300, the signals of 1000 shots were averaged, and the mass spectrum signals were collected;

[0055] The determination results of DNPD in zebra fish are shown in Table 2 Figure 4 , and the determination results of DPPD in zebra fish are shown in Table 3 Figure 5 (the mass fraction of N, S-MQDs in the mass spectrometer target plate was 5%). It can be known in combination with Figures 4-5 that the distributions of DNPD and DPPD in zebra fish are successfully visualized without adding other matrixes. The results show that the imaging of biological tissue samples can be realized without adding matrixes by using the imaging target plate of the present application.

Claims

1. Use of an N, S-MQDs-based mass spectrometer imaging target plate for the visualization of a phenylenediamine-based anti-aging agent in a biological tissue sample, wherein, The mass spectrometer imaging target plate is solidified by a photocuring resin containing N, S-MQDs, and the content of the N, S-MQDs is 0.1% to 20%, and the N, S-MQDs are prepared by the following steps: S1, using MAX phase material as raw material, using DMSO as intercalation agent, quantum dots are prepared by acid etching; S2, under ultrasonic conditions, the quantum dot solution, nitrogen source dopant and sulfur source dopant are subjected to a reduction reaction, and the reaction product is washed and dried in sequence to obtain N, S-MQDs; The preparation of the mass spectrometer imaging target plate is as follows: The N, S-MQDs are uniformly dispersed in deionized water, mixed with Formlabs transparent photocuring resin, and stirred vigorously overnight to make the N, S-MQDs uniformly distributed in the Formlabs transparent resin; Before 3D printing, the resin mixture is ultrasonically treated at room temperature for 10 min, then stirred again, and the uniformly mixed resin mixture is poured into the trough of the 3D printer, irradiated with ultraviolet light to solidify, and a mass spectrometer imaging target plate with a thickness of 50 μm is obtained.

2. Use according to claim 1, characterized in that: The step S1 includes the following specific steps: S11, the MAX phase material is added to an acid solution, and the MAX phase material is stably suspended by constant temperature stirring at 40-80℃, and the precipitate is separated after etching, washed to pH >7.0, and dried to obtain compound I; Wherein, the MAX phase material is Ti3AlC2; the acid solution is hydrofluoric acid solution; S12, dilute compound I with DMSO, stir for 12-36h, separate the precipitate, wash, and disperse the washed precipitate in deionized water, and ultrasonically treat in a nitrogen environment to obtain a quantum dot solution.

3. Use according to claim 2, characterized in that: The mass fraction of the hydrofluoric acid solution is 47%-51%.

4. Use according to claim 1, characterized in that: The reduction reaction in step S2 uses sodium borohydride as a reducing agent, and the reduction reaction temperature is 35-55℃; wherein the nitrogen source dopant is 25%-30% ammonia water, and the sulfur source dopant is sodium thiosulfate.

5. The use according to claim 1, characterized in that: The ultrasonic conditions in step S2 are: ultrasonic frequency ≤200W, ultrasonic time ≥2.0h.

6. The use according to claim 1, characterized in that: The biological tissue sample includes zebrafish.

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