Application of bifunctional group-modified water-soluble melanin nanoparticles

Abstract

The present invention relates to the field of nanomaterial application technology, and more specifically, to the application of mass spectrometry analysis of water-soluble melanin nanoparticles modified with bifunctional groups. The water-soluble melanin nanoparticles modified with bifunctional groups are MNP-PEG, with a particle size of 4nm to 5nm, uniform particle size, good monodispersity in water, strong ultraviolet absorption ability at 355nm, and an absorbance value of 0.35 to 0.56. In addition, the synthesis process is simple, the preparation cost is low, it is safe, non-toxic, and biodegradable. As a new matrix for matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it can perform efficient mass spectrometry analysis. When detecting tetrabromobisphenol A series molecules, it has the advantages of low background interference peaks, high ion intensity, high sensitivity, etc., and MNP-PEG can also be used for mass spectrometry analysis of small molecules of environmental pollutants in food, which has potential significance for evaluating related health problems caused by the intake of tetrabromobisphenol A and its various derivatives through food.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterial applications, and more specifically, to the application of mass spectrometry analysis of water-soluble melanin nanoparticles modified with bifunctional groups. Background Art

[0002] Among brominated flame retardants, tetrabromobisphenol A (TBBPA) and its derivatives tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-BHEE), TBBPA diallyl ether (TBBPA-BAE), and octabromoether (TBBPA-BDBPE) are the most commonly used and are widely used in various flame retardant products such as printed circuit boards, building materials, plastics, textiles, etc. They are also often used as additives or active flame retardants for coatings, epoxy resins, polystyrene foams, etc.

[0003] With the increasing production and widespread use of TBBPA and its derivatives, these potentially hazardous chemicals are being released into the environment in large quantities. Studies have shown that TBBPA and its derivatives can be detected in environmental media such as the atmosphere, soil, and rivers, as well as in biological samples, human tissues, and even in body fluids such as breast milk. They accumulate in organisms, undergo metabolic transformation, and migrate long distances, posing a potential threat to human health. Therefore, to assess their potential impact on human health, it is essential to detect TBBPA and its derivatives and analyze dietary exposure.

[0004] Current detection methods, including gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), suffer from complex and time-consuming sample pretreatment and difficulties in spectral analysis. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a novel soft ionization biomass spectrometry technique offering advantages such as simple sample preparation, rapid analysis, high sensitivity, and excellent salt tolerance. However, conventional organic acid matrix ionization produces matrix fragments that generate numerous background interference peaks in the low molecular weight range (m / z < 1000), severely impacting small molecule detection. Summary of the Invention

[0005] The present invention aims to provide a mass spectrometry analysis application of bifunctional group-modified water-soluble melanin nanoparticles. The bifunctional group-modified water-soluble melanin nanoparticles are MNP-PEG, with a particle size of 4 nm to 5 nm, uniform particle size, good monodispersity in water, strong ultraviolet absorption at 355 nm, and an absorbance value of 0.35 to 0.56. As a new MALDI matrix for the detection of TBBPA and its derivatives in food and the analysis of dietary exposure, the melanin nanoparticles have good water solubility, dispersibility, and stability, and are safe, non-toxic, and biodegradable.

[0006] The MNP-PEG as a MALDI matrix can be prepared by the following preparation methods, including but not limited to:

[0007] S1. Preparation of water-soluble melanin nanoparticles: Weighing melanin raw materials to prepare small-sized water-soluble melanin nanoparticles MNP;

[0008] S2. Preparation of bifunctional group-modified water-soluble melanin nanoparticles: The water-soluble melanin nanoparticles prepared in S1 were PEGylated using PEG.

[0009] Preferably, the melanin raw material in S1 is melanin; 10 mg to 30 mg of melanin is added to 3 mL to 5 mL of 0.1 mol / L NaOH solution, and ultrasonically shaken until mixed and completely dissolved, and then slowly added to 0.1 mol / L HCl solution. After adjusting the pH value to 7 to 8, the solution is pipetted into a 30KD ultrafiltration centrifuge tube and centrifuged at 3000 rpm / min to 5000 rpm / min for 10 min. After the centrifugation, deionized water is continued to be added to the ultrafiltration centrifuge tube, and the washing and centrifugation are repeated 5 to 8 times. The liquid in the membrane is collected, frozen in a -80°C refrigerator for 40 min to 60 min, and then placed in a freeze dryer for 24 h to obtain water-soluble melanin nanoparticles MNP.

[0010] Preferably, the MNP prepared in S1 and 5 times the mass of PEG are placed in two sample bottles, 5 mL to 8 mL of deionized water are added to each of them, ultrasonically shaken until mixed and completely dissolved, and then 0.1 mol / L NaOH solution is added to adjust the pH value to 9 to 10; the solutions in the two sample bottles prepared above are thoroughly mixed, magnetically stirred for 24 hours, and then centrifuged at 3000 rpm / min to 5000 rpm / min for 15 minutes, and the washing and centrifugation are repeated 3 to 5 times. The liquid in the membrane is collected, frozen in a refrigerator at -80°C for 40 minutes to 60 minutes, and then placed in a freeze dryer for 24 hours to obtain water-soluble melanin nanoparticles MNP-PEG modified with two functional groups.

[0011] The present invention provides an application of MNP-PEG as a novel MALDI matrix for analyzing and detecting low molecular weight compounds in matrix-assisted laser desorption / ionization time-of-flight mass spectrometry.

[0012] Preferably, the MNP-PEG matrix is drop-coated on a matrix-assisted laser desorption ionization target plate, dried at room temperature to obtain a thin layer of matrix, and then the sample solution to be analyzed is spotted on the surface of the matrix layer to form a secondary recrystallization of the sample and the matrix, and ionization mass spectrometry detection is performed after natural air drying; or a mixed solution of the matrix and the analyte is drop-coated on a matrix-assisted laser ionization target plate, dried at room temperature, and then subjected to laser desorption ionization mass spectrometry analysis after drying.

[0013] Preferably, the low molecular weight compound refers to m / z < 1000.

[0014] Preferably, the low molecular weight compound is the environmental pollutant tetrabromobisphenol A (TBBPA) and its various derivatives.

[0015] Preferably, when the MNP-PEG is used as a MALDI matrix, the concentration of the MNP-PEG matrix solution is 0.1 mg / mL to 10 mg / mL.

[0016] Preferably, in matrix-assisted laser desorption / ionization time-of-flight mass spectrometry detection, the volume of TBBPA and its derivatives to be detected is about 0.5 μL to 2 μL, and the concentration is about 0.001 μg / mL to 100 μg / mL.

[0017] The present invention also applies for an MNP-PEG as a novel MALDI matrix for the detection and analysis of tetrabromobisphenol A and its various derivatives in food.

[0018] The present invention uses MNP-PEG as a novel MALDI matrix for mass spectrometry analysis, and usually uses a time-of-flight mass analyzer, but other mass analyzers can also be used instead.

[0019] The beneficial effects of the present invention are as follows:

[0020] The MNP-PEG provided by the present invention has the advantages of simple synthesis process, low preparation cost, safety, non-toxicity, and biodegradability.

[0021] The MNP-PEG provided by the present invention has a small particle size, good ultraviolet absorption ability, and strong electron donor performance, and can be used as a new matrix to replace the traditional matrix and is more conducive to MALDI mass spectrometry analysis.

[0022] The MNP-PEG provided by the present invention can be used as a novel MALDI matrix for the analysis and detection of low molecular weight substances, which can avoid the background noise and possible co-crystallization phenomenon existing in traditional organic acid matrices in the m / z range of <1000, thereby ensuring high-sensitivity MALDI mass spectrometry analysis.

[0023] When the MNP-PEG provided by the present invention is used as a novel MALDI matrix for analyzing and detecting low-molecular-weight TBBPA and its derivatives, it has low background signal and high ion intensity.

[0024] The MNP-PEG provided by the present invention serves as a novel MALDI matrix for the detection of TBBPA and its derivatives in food. It also serves as a novel matrix for matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), enabling efficient mass spectrometric analysis. The MNP-PEG exhibits advantages such as low background interference peaks, high ion intensity, and high sensitivity in the detection of tetrabromobisphenol A molecules. Furthermore, the MNP-PEG can be used for mass spectrometric analysis of small molecules of environmental pollutants in food, potentially providing insights into health risks associated with dietary intake of tetrabromobisphenol A and its derivatives.

[0025] The detection method of the present invention has the advantages of high throughput, rapidity, high sensitivity, etc., and provides a new efficient and accurate method for testing environmental pollution molecules.

[0026] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0028] Figure 1 1 is a transmission electron microscopy (TEM) characterization image of the MNP in Example 1 and the carboxyl-modified water-soluble melanin nanoparticles (COOH-MNP-COOH, MNPC) in Example 2 of the present invention;

[0029] Figure 2 1 is a Fourier transform infrared spectroscopy (FT-IR) characterization diagram of the MNP in Example 1 and the MNPC in Example 2 of the present invention;

[0030] Figure 3 1 is the mass spectra corresponding to the traditional organic acid α-cyano-4-hydroxycinnamic acid (CHCA), MNP and MNPC as MALDI matrices in negative ion mode in Example 3 of the present invention;

[0031] Figure 4 1 is the mass spectra of TBBPA and its derivatives analyzed in negative ion mode using traditional organic acids CHCA, MNP, and MNPC as MALDI matrices in Example 4 of the present invention;

[0032] Figure 5 The mass spectra of the traditional organic acids CHCA and MNPC used as MALDI matrices in the negative ion mode for analyzing small molecule environmental pollutants in meat food in Example 5 of the present invention are as follows;

[0033] Figure 6This is the mass spectra of analyzing small molecule environmental pollutants in milk samples using traditional organic acids CHCA and MNPC as MALDI matrices in negative ion mode in Example 6 of the present invention. DETAILED DESCRIPTION

[0034] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0035] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from the description. Therefore, the scope of protection of the present invention is not limited to the specific embodiments disclosed below.

[0036] Example 1

[0037] Preparation of water-soluble melanin nanoparticles (MNPs)

[0038] Weigh 20 mg of melanin, add 4 mL of 0.1 mol / L NaOH solution, and ultrasonically shake until mixed and completely dissolved.

[0039] Slowly add 0.1 mol / L HCl solution and shake with an ultrasonic oscillator to adjust the pH value to 7-8, mainly slightly alkaline.

[0040] The above solution was transferred to a 30KD ultrafiltration centrifuge tube and centrifuged at 3500rpm / min for 10min. After the centrifugation, deionized water was added to the ultrafiltration centrifuge tube and the washing and centrifugation were repeated 5-6 times until the filtered water was clear. The solution was then transferred to a 10mL centrifuge tube with a pipette and the centrifuge tube was placed in a -80℃ refrigerator and frozen for 40-60min. After being taken out, the tube was placed in a freeze dryer to obtain water-soluble melanin nanoparticles MNP after 24h.

[0041] Transmission electron microscopy (TEM) characterization of the prepared MNPs Figure 1 As shown. Figure 1 In the TEM of a, it can be observed that the particle size is only 3nm~4nm, the particle size is uniform, the aqueous solution has good monodispersity, and there is no obvious agglomeration phenomenon.

[0042] Example 2

[0043] Preparation of carboxyl-modified water-soluble melanin nanoparticles (MNPCs)

[0044] 12 mg of MNPs obtained in Example 1 were placed in a sample bottle, 6 mL of deionized water was added, and the mixture was dissolved by ultrasonic shaking in a water bath. Then 100 μL of 0.1 mol / L NaOH was added and the mixture was dissolved by ultrasonic shaking in a water bath again. The pH value was adjusted to 9-10 for later use.

[0045] Take 60 mg of COOH-PEG-COOH in another sample bottle, which is 5 times the mass of MNP, add 6 mL of deionized water, and dissolve it in an ultrasonic water bath. Then add 100 μL of 0.1 mol / L NaOH and dissolve it in an ultrasonic water bath again. Adjust the pH value to 9-10 for later use.

[0046] The MNPs and PEG solutions in the two sample bottles prepared above were thoroughly mixed and stirred on a magnetic stirrer at room temperature for 24 h.

[0047] After stirring, the above solution was transferred to a 30KD ultrafiltration centrifuge tube, centrifuged at 4500rpm / min for 15min, and washed and centrifuged three times. The liquid in the membrane was collected into a 10mL centrifuge tube, and the centrifuge tube was placed in a -80℃ refrigerator and frozen for 40min~60min. After being taken out, it was placed in a freeze dryer. After 24h, carboxyl-modified water-soluble melanin nanoparticles MNPC were obtained.

[0048] TEM characterization of the prepared MNPCs is shown in Figure 2. Figure 1 .from Figure 1 In the TEM image of b, it can be observed that the particle size is only 4nm~5nm, the particle size is uniform, the dispersion is good in aqueous solution, there is no obvious agglomeration, and the morphology has not changed compared with the unmodified MNP. In order to prove that the carboxyl group is successfully modified on the MNP material, the FT-IR characterization of the prepared MNPC is as follows Figure 2 As shown. Figure 2 It can be seen that the OH bond of -COOH is enhanced at the wavenumber of 1090 cm-1, indicating that the carboxyl group is successfully modified.

[0049] Example 3

[0050] Disperse 5 mg of the MNPs from Example 1 in 5 mL of deionized water and sonicate for 1 hour to obtain a 1 mg / mL MNP matrix solution. Similarly, disperse 5 mg of the MNPCs from Example 2 in 5 mL of deionized water and sonicate for 1 hour to obtain a 1 mg / mL MNPC matrix solution. Separately, dissolve 40 mg of traditional organic small molecule CHCA particles in 2 mL of a 7:3 mixture of acetonitrile and water containing 0.1% trifluoroacetic acid to obtain a 20 mg / mL CHCA matrix solution. Apply 1 μL of each of the three matrix solutions to a stainless steel MALDI target plate and air-dry at room temperature to form a thin matrix layer. Observe for matrix peaks within the m / z range of <1000.

[0051] from Figure 3 As can be seen in a, in the negative ion mode, in the m / z range of 100-1000, the CHCA traditional organic acid matrix has a large number of background noise peaks, which seriously affects the ionization intensity of small molecule analytes. Figure 3 b shows that MNP has a weak background signal when used as a new MALDI matrix. Figure 3 c) The presence of almost no background interference when MNPC was used as a novel matrix indicates that the carboxyl modification enhances the performance of MNP as a matrix. Therefore, MNP-PEG can be used as a novel MALDI matrix for the detection of low molecular weight compounds.

[0052] Example 4

[0053] 1 μL of the 1 mg / mL MNP matrix solution of Example 1, 1 μL of the 1 mg / mL MNPC matrix solution of Example 2, and 1 μL of a 20 mg / mL traditional organic acid matrix CHCA solution were drop-coated onto a MALDI stainless steel target plate. After air-drying at room temperature, 1 μL of a 50 mg / mL solution of TBBPA or its derivatives (TBBPA-BHEE, TBBPA-BAE, TBBPA-BDBPE) was spotted onto the matrix layer. After air-drying, the TBBPA and its derivative solutions were analyzed by mass spectrometry in negative ion mode.

[0054] from Figure 4 As can be seen in a, when TBBPA is detected in negative ion mode using the traditional CHCA matrix, the characteristic deprotonated [MH] peak of TBBPA is not observed. On the contrary, when MNP and MNPC are used as the new MALDI matrices, a clear TBBPA characteristic peak is observed at m / z 542.7434, and the peak intensity measured when MNPC is used as the matrix is higher. Figure 4 As shown in Figure b, the characteristic ionization peak [M-C2H5O] of TBBPA-BHEE is not observed in negative ion mode using the traditional CHCA matrix. However, when MNP and MNPC are used as novel MALDI matrices, a distinct characteristic peak at m / z 586.7752 is observed, with the peak intensity being higher when using MNPC as the matrix. Therefore, MNP-PEG can be used as a novel MALDI matrix for the highly sensitive mass spectrometric detection of the low-molecular-weight environmental pollutant TBBPA and its derivatives in negative ion mode.

[0055] Example 5

[0056] 1 μL of a 20 mg / mL conventional organic acid matrix CHCA solution and a 1 mg / mL MNPC matrix solution in Example 2 were drop-coated on a MALDI stainless steel target plate and air-dried at room temperature to form a thin matrix layer. Solid-phase extraction method was also used. 5 g of meat food sample was ground, and 10 mL of acetonitrile, 4 g of magnesium sulfate, and 1 g of sodium chloride were added. After thorough shaking, the sample was stored at a freezing temperature of -20°C for 30 min. The sample was then centrifuged at 4000 rpm / min at 4°C for 5 min. The resulting 6 mL of supernatant was mixed with 0.6 g of magnesium sulfate, 120 mg of N-propylethylenediamine solid phase adsorbent (PSA), 120 mg of C-18, and 30 mg of carbon black. The sample was centrifuged again at 4000 rpm / min at 4°C for 10 min. The supernatant obtained was the sample extract. 1 μL of the above extract was dropwise applied to the matrix layer and allowed to air-dry naturally at room temperature. After drying, it was used for MALDI-TOF MS analysis of small molecules of environmental pollutants in meat food samples in negative ion mode.

[0057] from Figure 5 As can be seen in a, when using the traditional CHCA matrix, the characteristic ion peaks of TBBPA and its derivatives in food are not observed. However, when using MNPC as the matrix, the [MH]-characteristic ionization peak corresponding to TBBPA at m / z 542.7434 can be observed. Figure 5 (b) Characteristic ionization peaks of other environmental pollutants may also be observed. Due to the lack of standards for other environmental pollutants, the composition of these characteristic ionization peaks of other possible small molecule environmental pollutants cannot be determined.

[0058] Example 6

[0059] 1 μL of a 20 mg / mL solution of the traditional organic acid matrix CHCA and a 1 mg / mL solution of the MNPC matrix from Example 2 were drop-coated onto a stainless steel MALDI target plate and air-dried at room temperature to form a thin matrix layer. Separately, using liquid phase extraction, 30 mL of milk sample was thoroughly mixed with 30 mL of acetonitrile and 800 μL of formic acid, stirred thoroughly for 5 minutes, and then 10 g of anhydrous magnesium sulfate and 3 g of sodium chloride were added and stirred again for 5 minutes. The mixture was then centrifuged at 12,000 rpm for 8 minutes at 4°C. The resulting supernatant was transferred to a new centrifuge tube containing 3 g of anhydrous magnesium sulfate and 300 mg of C-18 adsorbent. After shaking to mix thoroughly, the supernatant was centrifuged again at 12,000 rpm for 8 minutes at 4°C. The purified extract was evaporated to near dryness, and the residue was dissolved in 1 mL of methanol. 1 μL of the above extraction solution was applied to the matrix layer and allowed to air dry at room temperature. After drying, it was used for MALDI-TOF MS analysis of small molecules of environmental pollutants in milk samples in negative ion mode.

[0060] from Figure 6 As can be seen in a, when using the traditional CHCA matrix, the characteristic ion peaks of TBBPA and its derivatives in the milk sample are not observed. However, when using MNPC as the matrix, the [MH]-characteristic ionization peak corresponding to TBBPA at m / z 542.7434 can be observed. Figure 6 (b) Characteristic ionization peaks of other environmental pollutants may also be observed. Due to the lack of standards for other environmental pollutants, the composition of these characteristic ionization peaks of other possible small molecule environmental pollutants cannot be determined.

[0061] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An application of water-soluble melanin nanoparticles modified with bifunctional groups, characterized by: The bifunctional group-modified water-soluble melanin nanoparticles are MNP-PEG, with a particle size of 4 nm to 5 nm, uniform particle size, good monodispersity in water, strong ultraviolet absorption at 355 nm, and an absorbance value of 0.35 to 0.

56. They are used as MALDI matrices for the detection of TBBPA and its derivatives in food. PEG is COOH-PEG-COOH.

2. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 1, characterized in that: The specific steps of the MNP-PEG preparation method are as follows: S1. Preparation of water-soluble melanin nanoparticles: Weighing melanin raw materials to prepare small-sized water-soluble melanin nanoparticles MNP; S2. Preparation of bifunctional group-modified water-soluble melanin nanoparticles: The water-soluble melanin nanoparticles prepared in S1 were PEGylated using COOH-PEG-COOH.

3. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 2, characterized in that: The specific process of S1 includes the following: the melanin raw material is melanin, 10mg~30mg of melanin is taken, 3mL~5mL of 0.1mol / L NaOH solution is added, ultrasonic vibration is performed until the mixture is completely dissolved, 0.1mol / L HCl solution is slowly added, the pH value is adjusted to 7~8, and then the liquid is pipetted into a 30KD ultrafiltration centrifuge tube, and centrifuged at 3000rpm / min~5000rpm / min for 10min. After the centrifugation, deionized water is continued to be added to the ultrafiltration centrifuge tube, and the washing and centrifugation are repeated 5~8 times. The liquid in the membrane is collected, and it is frozen in a refrigerator at -80℃ for 40min~60min and then placed in a freeze dryer for drying for 24h to obtain water-soluble melanin nanoparticles MNP.

4. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 2, characterized in that: The specific process of S2 includes the following: taking the MNP prepared in S1 and 5 times the mass of COOH-PEG-COOH and placing them in two sample bottles, adding 5mL~8mL of deionized water respectively, ultrasonically shaking until mixed and completely dissolved, and then adding 0.1mol / L NaOH solution to adjust the pH value to 9~10; the solutions in the two sample bottles prepared above are fully mixed, magnetically stirred for 24 hours, and then centrifuged at 3000rpm / min~5000rpm / min for 15 minutes, repeated washing and centrifugation 3~5 times, collecting the liquid in the membrane, freezing it in a refrigerator at -80℃ for 40min~60min, and then drying it in a freeze dryer for 24 hours to obtain water-soluble melanin nanoparticles MNP-PEG modified with two functional groups.

5. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 1, characterized in that: The water-soluble melanin nanoparticles modified with double functional groups are used as MALDI matrix to analyze low molecular weight compounds in matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

6. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 5, characterized in that: The MNP-PEG matrix is drop-coated on a matrix-assisted laser desorption / ionization target plate, and dried at room temperature to obtain a thin layer of matrix. The sample solution to be analyzed is then spotted on the surface of the matrix layer to allow the sample and matrix to form a secondary recrystallization. After natural air drying, ionization mass spectrometry detection is performed; or a mixed solution of the matrix and the analyte is drop-coated on a matrix-assisted laser ionization target plate, naturally air dried at room temperature, and then subjected to laser desorption / ionization mass spectrometry analysis after drying.

7. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 5, characterized in that: The low molecular weight compound refers to m / z < 1000.

8. The use of a bifunctional group-modified water-soluble melanin nanoparticle according to claim 5, characterized in that: When the MNP-PEG is used as a MALDI matrix, the concentration of the MNP-PEG matrix solution is 0.1 mg / mL to 10 mg / mL.

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