阅读说明：本技术 一种磷脂快速检测方法 (Rapid detection method for phospholipid ) 是由 沈清 于 2019-11-21 设计创作，主要内容包括：本发明公开了一种磷脂快速检测方法,包括以下步骤,a、对水产品中的磷脂进行粗提取,得磷脂粗提液,即A品,b、合成TiO<Sub>2</Sub>微孔阵列芯片,得B品,c、将A品置于B品,静置,使磷脂与TiO2反应完全,得C品,d、在C品上滴加super-DHB和9-AA基质溶液的混合液,得D品,e、干燥D品,使D品上的磷脂结晶,得E品,f、E品采用基质辅助激光解吸检测,得检测图。本发明具有检测效率高、检测结果精准和不容易对人体造成危害的优点。(The invention discloses a method for rapidly detecting phospholipid, which comprises the following steps of a, carrying out coarse extraction on phospholipid in aquatic products to obtain a phospholipid coarse extract, namely a product A, and b, synthesizing TiO 2 And (2) placing the product A on the product B to obtain a product B, standing to ensure that the phospholipid completely reacts with TiO2 to obtain a product C, dropwise adding a mixed solution of super-DHB and 9-AA matrix solution on the product C to obtain a product D, drying the product D to crystallize the phospholipid on the product D to obtain a product E, and carrying out matrix-assisted laser desorption detection on the product f and the product E to obtain a detection image. The invention has the advantages of high detection efficiency, accurate detection result and difficult harm to human body.)

1. A method for rapidly detecting phospholipid is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

a. the phosphatide in the aquatic product is roughly extracted to obtain phosphatide crude extract, namely A product,

b. synthesis of TiO₂Obtaining a product B by using the micropore array chip,

c. placing the product A on the product B, standing to make phospholipid and TiO2 completely react to obtain product C,

d. dripping mixture of super-DHB and 9-AA matrix solution on the product C to obtain product D,

e. drying product D to crystallize phospholipid on product D to obtain product E,

f. and detecting the product E by matrix-assisted laser desorption to obtain a detection image.

2. The method for rapidly detecting phospholipids according to claim 1, characterized in that: and a, performing coarse extraction on the phospholipid in the aquatic product by adopting a Bligh & Dyer method to obtain a phospholipid coarse extract, namely a product A.

3. The method for rapidly detecting phospholipids according to claim 2, characterized in that: step a, adopting an improved Bligh & Dyer method to carry out coarse extraction on phospholipid in aquatic products to obtain a phospholipid coarse extract, namely a product A; the modified Bligh & Dyer method, comprising the steps of,

a1, getting muscle tissue of aquatic product, getting A1 product,

a2, adding A1 into the mixture of chloroform and methanol, shaking and mixing uniformly to obtain A2,

extracting A3 and A2 products with ultrasonic ice bath for 5-15min to obtain A3 product,

a4, adding pure water into A3, shaking and mixing uniformly to obtain A4,

a5, centrifuging product A4 by a refrigerated centrifuge to separate the solution in product A4 into layers from top to bottom to obtain product A5,

a6, taking out the lower solution in the A5 product to obtain an A6 product,

a7, adding chloroform into A6 to obtain A7,

centrifuging with a8 and A7 freezing centrifuge to separate the solution in A7 into upper and lower layers to obtain A8,

a9, taking out the lower solution in the A8 product to obtain an A9 product,

a10, adding chloroform solution into A6 to obtain A10,

centrifuging with a11 and A10 freezing centrifuge to separate the solution in A10 into upper and lower layers to obtain A11,

a12, taking out the lower solution in A11,

a13, mixing the lower solution taken out in the step a6, the lower solution taken out in the step a9 and the lower solution taken out in the step a12 to obtain A13 product,

a14, drying the A13 product by nitrogen to obtain A14 product,

a15, dissolving A14 product with methanol water solution, wherein the concentration of methanol is 90 percent, and obtaining phospholipid crude extract, namely A product.

4. The method for rapidly detecting phospholipids according to claim 3, characterized in that: in the step a1, the muscle tissue of the aquatic product is homogenized;

in the step a2, 1 part of A1 and 1.75 parts of mixed solution are uniformly shaken and mixed according to the parts by weight, and the ratio of chloroform to methanol in the mixed solution is 1: 2;

in the step A3, the product A2 is extracted for 10min by adopting a probe type ultrasonic ice bath, the ultrasonic power is 25kHz, and the amplitude is 60 percent;

in the step a4, the adding amount of the pure water is 1.25 parts;

in the step a5, the rotation speed of the refrigerated centrifuge is 8000rpm, and the centrifugation time is 15 min;

in the step a6, the lower solution in the A5 product is taken out by a pipette gun;

in the step a7, the adding amount of the chloroform is 2 parts;

in the step a8, the rotation speed of the refrigerated centrifuge is 8000rpm, and the centrifugation time is 15 min;

in the step a9, the lower solution in the A8 product is taken out by a pipette gun;

in the step a10, the chloroform is added in an amount of 2 parts;

in the step a11, the rotation speed of the refrigerated centrifuge is 8000rpm, and the centrifugation time is 15 min;

in the step a12, the lower solution in the A11 sample is taken out by a pipette gun.

5. The method for rapidly detecting phospholipids according to claim 1, characterized in that: the TiO is₂The synthesis method of the micropore array chip comprises the following steps,

b1, immersing the gold-plated cover glass in 5-15mM MPA solution, standing for more than 5h to obtain B1 product,

b2, washing the B1 product with ethanol to obtain B2 product,

b3, washing the B2 product with deionized water to obtain B3 product,

b4, blowing the B3 product by nitrogen to obtain B4 product,

b5, spraying PAH solution on the surface of the B4 product, spraying TALH solution after the PAH solution is dried, forming a multilayer unit on the surface of the B4 after the TALH solution is dried, obtaining a B5 product,

b6, forming a multilayer unit on the surface of the B5 product again to obtain a B6 product,

products B7 and B6 are rinsed with deionized water to obtain product B7,

b8, drying the B7 product to obtain a B8 product,

b9, B8 products, aging at 550 ℃ for 4-6h to obtain TiO₂The cover glass plate, article B9,

b10 and B9 products are immersed in 5-15mM OTS toluene solution, a water conveying layer is synthesized on the surface of the products, B10 products are obtained,

b11, drying the B10 product to obtain a B11 product,

steaming products B12 and B11 in hot steam for 0.5-1.5min to obtain product B12,

b13, covering a mold with an array of micropores on the surface of the B12 product, irradiating for 0.8-1.2h under ultraviolet light to obtain a B13 product,

b14 rinsing the array spot of B13 with ethanol and 0.4-0.6mM sodium hydroxide solution to obtain B14,

b15, washing B14 product with deionized water to obtain TiO₂And (4) obtaining a finished product of the micropore array chip, namely a product B.

6. The method for rapidly detecting phospholipids according to claim 5, characterized in that: in step b1, the MPA solution is 10 mM;

in the step b5, the PAH solution is 1mg/mL and has pH of 7.5, and the TALH solution is 5 wt% and has pH of 7.5;

in the step B6, the number of the multiple layer units formed again on the surface of the B5 product is 1-19;

in the step B9, the B8 product is aged in a muffle furnace at the furnace temperature of 500 ℃ for 4 hours;

in the step b10, the concentration of the OTS toluene solution is 10 mM;

in the step B12, the fumigation time of the B11 product is 1 min;

in the step b13, the irradiation time of the ultraviolet light is 1h, and the wavelength of the ultraviolet light is 254 nm;

in step b14, the concentration of the sodium hydroxide solution is 0.5 mM.

7. The method for rapidly detecting phospholipids according to claim 1, characterized in that: and c, transferring the product A to the product B by using a liquid transfer gun for multiple times, wherein 10 mu L of the product A is transferred each time, and standing to ensure that the phospholipid and the TiO are mixed₂And (4) completely reacting to obtain a product C.

8. The method for rapidly detecting phospholipids according to claim 1, characterized in that: the step d comprises the following steps of,

d1, rinsing product C with 2% TFA solution to obtain product D1,

d2, rinsing product D1 with a solution containing 2% TFA and 40% methanol to obtain product D2,

d3, and D2 product, and adding dropwise mixture of super-DHB and 9-AA matrix solution at a ratio of 1:2, 1:1 or 2:1 to obtain D product.

9. The method for rapidly detecting phospholipids according to claim 1, characterized in that: and D, dropwise adding 1% phosphoric acid solution on the product C, and then dropwise adding a mixed solution of super-DHB and 9-AA matrix solution, wherein the ratio of the super-DHB to the 9-AA matrix solution is 1:2, 1:1 or 2:1, so as to obtain a product D.

Technical Field

The invention belongs to the field of phospholipid detection methods of aquatic products, and particularly relates to a rapid detection method of phospholipid of aquatic products.

Background

Through the research on the lipidomics of the aquatic products, the lipid nutrition knowledge of the aquatic products and the processing byproducts can be deepened; and a lipidomics fingerprint spectrum can be established, so that a technical means is provided for the identification and source tracing of aquatic products; and the method can also provide reference for preventing, diagnosing and treating aquatic animal diseases and provide scientific basis for water environment regulation and control and pollution monitoring.

Phospholipids are one of the main analysis objects of aquatic product lipidomics, the types of phospholipids are various, the phospholipids can be divided into multiple categories such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and the like according to the difference of polar groups of the phospholipids, and the detection of the content of the phospholipids of each category in the aquatic products is a necessary and few step in the whole analysis process. The whole detection process of the phospholipid comprises the extraction of the phospholipid in the aquatic product and the detection of the extract. Matrix-assisted laser desorption can be adopted for detecting the extract, so that a detection graph is obtained, each peak in the detection graph corresponds to one phospholipid, the concentration of the corresponding phospholipid is calculated according to the area of each peak, and subsequent analysis work can be carried out according to the detection graph. The extraction of the phospholipids in the aquatic products can adopt a Bligh & Dyer method, and although most of the phospholipids in the aquatic products can be extracted by the Bligh & Dyer method, the extraction speed is slow, the time consumption is long, the time consumption of the whole detection process is long, and the detection efficiency is low; impurities are easy to mix during extraction, interference is formed during matrix-assisted laser desorption, and an obtained detection image is inaccurate, namely a detection result is inaccurate; and because chloroform is used in a large amount in the Bligh & Dyer method, the chloroform reacts with oxygen in the air after being irradiated by light and is gradually decomposed to generate virulent phosgene (carbonyl chloride) and hydrogen chloride, which are easy to cause harm to human bodies.

Therefore, the existing phospholipid detection method has the defects of low detection efficiency, inaccurate detection result and easy harm to human bodies.

Disclosure of Invention

The invention aims to provide a method for rapidly detecting phospholipid. The invention has the advantages of high detection efficiency, accurate detection result and difficult harm to human body.

The technical scheme of the invention is as follows: a method for rapidly detecting phospholipid comprises the following steps,

a. the phosphatide in the aquatic product is roughly extracted to obtain phosphatide crude extract, namely A product,

b. synthesis of TiO₂Obtaining a product B by using the micropore array chip,

c. placing the product A on the product B, standing to make phospholipid and TiO2 completely react to obtain product C,

d. dripping mixture of super-DHB and 9-AA matrix solution on the product C to obtain product D,

e. drying product D to crystallize phospholipid on product D to obtain product E,

f. and detecting the product E by matrix-assisted laser desorption to obtain a detection image.

In the aforementioned method for rapidly detecting phospholipid, in step a, phospholipid in an aquatic product is subjected to crude extraction by a Bligh & Dyer method to obtain a phospholipid crude extract, i.e., product a.

In the method for rapidly detecting phospholipid, step a is to adopt an improved Bligh & Dyer method to carry out coarse extraction on phospholipid in aquatic products to obtain a phospholipid coarse extract, namely a product A; the modified Bligh & Dyer method, comprising the steps of,

a1, getting muscle tissue of aquatic product, getting A1 product,

a2, adding A1 into the mixture of chloroform and methanol, shaking and mixing uniformly to obtain A2,

extracting A3 and A2 products with ultrasonic ice bath for 5-15min to obtain A3 product,

a4, adding pure water into A3, shaking and mixing uniformly to obtain A4,

a5, centrifuging product A4 by a refrigerated centrifuge to separate the solution in product A4 into layers from top to bottom to obtain product A5,

a6, taking out the lower solution in the A5 product to obtain an A6 product,

a7, adding chloroform into A6 to obtain A7,

centrifuging with a8 and A7 freezing centrifuge to separate the solution in A7 into upper and lower layers to obtain A8,

a9, taking out the lower solution in the A8 product to obtain an A9 product,

a10, adding chloroform solution into A6 to obtain A10,

centrifuging with a11 and A10 freezing centrifuge to separate the solution in A10 into upper and lower layers to obtain A11,

a12, taking out the lower solution in A11,

a13, mixing the lower solution taken out in the step a6, the lower solution taken out in the step a9 and the lower solution taken out in the step a12 to obtain A13 product,

a14, drying the A13 product by nitrogen to obtain A14 product,

a15, dissolving A14 product with methanol water solution, wherein the concentration of methanol is 90 percent, and obtaining phospholipid crude extract, namely A product.

In the method for rapidly detecting phospholipid, in the step a1, the muscle tissue of the aquatic product is homogenized;

in the step a2, 1 part of A1 and 1.75 parts of mixed solution are uniformly shaken and mixed according to the parts by weight, and the ratio of chloroform to methanol in the mixed solution is 1: 2;

in the step A3, the product A2 is extracted for 10min by adopting a probe type ultrasonic ice bath, the ultrasonic power is 25kHz, and the amplitude is 60 percent;

in the step a4, the adding amount of the pure water is 1.25 parts;

in the step a5, the rotation speed of the refrigerated centrifuge is 8000rpm, and the centrifugation time is 15 min;

in the step a6, the lower solution in the A5 product is taken out by a pipette gun;

in the step a7, the adding amount of the chloroform is 2 parts;

in the step a8, the rotation speed of the refrigerated centrifuge is 8000rpm, and the centrifugation time is 15 min;

in the step a9, the lower solution in the A8 product is taken out by a pipette gun;

in the step a10, the chloroform is added in an amount of 2 parts;

in the step a11, the rotation speed of the refrigerated centrifuge is 8000rpm, and the centrifugation time is 15 min;

in the step a12, the lower solution in the A11 sample is taken out by a pipette gun.

In the above method for rapidly detecting phospholipid, the TiO is₂The synthesis method of the micropore array chip comprises the following steps,

b1, immersing the gold-plated cover glass in 5-15mM MPA solution, standing for more than 5h to obtain B1 product,

b2, washing the B1 product with ethanol to obtain B2 product,

b3, washing the B2 product with deionized water to obtain B3 product,

b4, blowing the B3 product by nitrogen to obtain B4 product,

b5, spraying PAH solution on the surface of the B4 product, spraying TALH solution after the PAH solution is dried, forming a multilayer unit on the surface of the B4 after the TALH solution is dried, obtaining a B5 product,

b6, forming a multilayer unit on the surface of the B5 product again to obtain a B6 product,

products B7 and B6 are rinsed with deionized water to obtain product B7,

b8, drying the B7 product to obtain a B8 product,

b9, B8 products, aging at 550 ℃ for 4-6h to obtain TiO₂The cover glass plate, article B9,

b10 and B9 products are immersed in 5-15mM OTS toluene solution, a water conveying layer is synthesized on the surface of the products, B10 products are obtained,

b11, drying the B10 product to obtain a B11 product,

steaming products B12 and B11 in hot steam for 0.5-1.5min to obtain product B12,

b13, covering a mold with an array of micropores on the surface of the B12 product, irradiating for 0.8-1.2h under ultraviolet light to obtain a B13 product,

b14 rinsing the array spot of B13 with ethanol and 0.4-0.6mM sodium hydroxide solution to obtain B14,

b15, washing B14 product with deionized water to obtain TiO₂And (4) obtaining a finished product of the micropore array chip, namely a product B.

In the aforementioned phospholipid rapid detection method, in step b1, the MPA solution is 10 mM;

in the step b5, the PAH solution is 1mg/mL and has pH of 7.5, and the TALH solution is 5 wt% and has pH of 7.5;

in the step B6, the number of the multiple layer units formed again on the surface of the B5 product is 1-19;

in the step B9, the B8 product is aged in a muffle furnace at the furnace temperature of 500 ℃ for 4 hours;

in the step b10, the concentration of the OTS toluene solution is 10 mM;

in the step B12, the fumigation time of the B11 product is 1 min;

in the step b13, the irradiation time of the ultraviolet light is 1h, and the wavelength of the ultraviolet light is 254 nm;

in step b14, the concentration of the sodium hydroxide solution is 0.5 mM.

In the aforementioned method for rapidly detecting phospholipid, in step c, the sample A is transferred to the sample B by a pipette in multiple times, 10 μ L of sample A is transferred each time, and the sample is allowed to stand to allow phospholipid and TiO to react₂And (4) completely reacting to obtain a product C.

In the aforementioned method for rapidly detecting phospholipids, the step d comprises the following steps,

d1, rinsing product C with 2% TFA solution to obtain product D1,

d2, rinsing product D1 with a solution containing 2% TFA and 40% methanol to obtain product D2,

d3, and D2 product, and adding dropwise mixture of super-DHB and 9-AA matrix solution at a ratio of 1:2, 1:1 or 2:1 to obtain D product.

In the aforementioned method for rapidly detecting phospholipid, in step D, 1% phosphoric acid solution is firstly dripped on the product C, and then mixed solution of super-DHB and 9-AA matrix solution is dripped, wherein the ratio of the super-DHB to the 9-AA matrix solution is 1:2, 1:1 or 2:1, so as to obtain the product D.

Compared with the prior art, the invention mainly improves the extraction method of phospholipid, and firstly utilizes the improved Bligh&The Dyer method is used for roughly extracting phospholipid in aquatic products, a centrifugal separation step is added, methanol is added, phospholipid is extracted for three times, and chloroform is added for several times, so that the extraction efficiency is improved, the using amount of chloroform can be reduced, and the detection is not easy to cause harm to human bodies; remanufacturing TiO₂Micro-porous array chip, TiO₂Has selective adsorption effect on phospholipids by bidentate bridging chelation, so that the phospholipids in the phospholipid crude extract can be enriched into TiO₂On the array point of the micropore array chip, the enriched phospholipid has high purity and low impurity content, the accuracy of the detection result can be improved, the enrichment speed is high, and the enrichment time can be completed within 5 minutes, so that the time consumed in the whole detection process is reduced, and the detection efficiency is improved. In addition, TiO is used according to the invention₂TiO obtained by synthesis method of micropore array chip₂Microwell array chip having array densityThe chip size can be reduced; and may be made TiO₂The growth form and the thickness of the probe are easy to control, and the result reproducibility of the probe used for matrix-assisted laser desorption is good, namely the probe is also beneficial to improving the accuracy of the detection result; the technology is simple, expensive equipment is not needed, and the popularization and the promotion are convenient.

Therefore, the invention has the advantages of high detection efficiency, accurate detection result and difficult harm to human body.

Drawings

FIG. 1 is a detection chart obtained by the present invention.

Fig. 2 is a diagram of a test according to the prior art.

Detailed Description

The invention is further illustrated by the following figures and examples, which are not to be construed as limiting the invention.