Gold nanobipyramid functionalized electrospun fiber membrane, preparation method and visual analysis application thereof

Abstract

The application discloses a kind of gold nanometer double-cone functionalized electrospun fiber membrane and its preparation method and visual analysis application, the application is by gold nanometer double-cone load in the surface of electrospun fiber membrane and mobile phone chroma identification technology two combinations, finally develops a kind of acetylcholinesterase and its inhibitor visual POCT technology;The functionalized electrospun membrane overcomes the defect that the quantitative effect of vision is not good in the single color of visual effect, the resolution is not high in traditional visual equipment, shows higher level in the quantitative and qualitative analysis detection of acetylcholinesterase, with good visual signal effect, high sensitivity, good reproducibility, multicolor high efficiency and the like advantages.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a gold nanobicone functionalized electrospun fiber membrane, its preparation method, and its visualization analysis application. Background Technology

[0002] Visualization analysis technology is favored due to its convenience, speed, ease of direct observation with the naked eye, and direct readings via UV-Vis spectrophotometers or fluorescence spectrophotometers. With the rapid development of analytical science and point-of-care testing (POCT) technology, mounting visual signal probes on solid-phase carriers to form visual solid-phase sensing platforms is a promising option. Compared to traditional liquid-phase-based analytical methods, solid-phase sensing platforms offer superior analytical performance, including ease of functionalization, effective separation from samples, and convenient storage and transportation. To date, researchers have explored and developed several sensors for visual analysis of disease biomarkers. However, some existing visualization sensing devices suffer from limited color options and low resolution, resulting in poor quantitative results. Therefore, further development and utilization of multi-color visualization probes for colorimetric POCT sensing devices suitable for target visualization remains of significant application value.

[0003] Patent document CN 107761366 A discloses a method for preparing electrospun fibers supported on gold nanorods. The method includes the following steps: (1) preparation of gold nanorods; (2) preparation of PVA / PAA electrospun fibers; (3) self-assembly of gold nanorods on the fiber surface.

[0004] Patent document CN104422679B discloses a method for assembling gold nanoparticles into a SERS active substrate using electrospun polymer nanofiber membranes. This method is based on assembling gold nanoparticles using electrospun polycaprolactone nanofiber membranes. First, ultraviolet light is used to induce acrylic acid to graft and modify the hydrophobic surface of the polycaprolactone nanofiber membrane. Then, the polycaprolactone nanofiber membrane is immersed in a gold nanoparticle solution to promote the assembly of gold nanoparticles onto the polycaprolactone nanofibers to form a SERS active substrate.

[0005] Acetylcholinesterase (AChE) is a key enzyme in biological neurotransmission, catalyzing the rapid hydrolysis of acetylcholine (ATCh) into choline and acetate, thereby terminating the excitatory effect of neurotransmitters on the postsynaptic membrane. Many neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, and Huntington's disease) may be closely related to AChE dysfunction. Currently, there are no reports on the solid-phase visualization analysis of cholinesterase using AuNBP-functionalized electrospun fiber membranes achieved through electrostatic assembly of gold nanobipyramidal nanoparticles (AuNBPs) with electrospun fiber membranes. Therefore, this invention is proposed. Summary of the Invention

[0006] This invention overcomes the shortcomings of the prior art and provides a gold nanobiconical functionalized electrospun fiber membrane, its preparation method, and its application.

[0007] A first aspect of the present invention provides a method for preparing a gold nanoparticle-functionalized electrospun fiber membrane, the method comprising the following steps:

[0008] (1) Preparation of gold nanobipyramidal structures;

[0009] (2) Preparation of PEI / PVA electrospun fiber membrane;

[0010] (3) The electrospun fiber membrane obtained from the modification treatment step (2);

[0011] (4) Self-assembly of gold nanobicones on the surface of electrospun fiber membranes.

[0012] Furthermore, step (1) includes the synthesis of a seed solution and the synthesis of a growth solution.

[0013] Furthermore, the synthesis of the seed solution involves mixing tetrachloroauric acid (HAuCl4), hexadecyltrimethylammonium chloride (CTAC), sodium borohydride (NaBH4), and citric acid, and reacting them.

[0014] Further, the reaction molar ratio of tetrachloroauric acid (HAuCl4), hexadecyltrimethylammonium chloride (CTAC), sodium borohydride (NaBH4) and citric acid is 1-5:700-900:1-5:70-100, preferably 1-5:750-850:1-5:75-85, more preferably 1-5:800:1-5:80, and even more preferably 1-3:800:1-3:80.

[0015] In one embodiment of the present invention, the molar ratio of tetrachloroauric acid (HAuCl4), hexadecyltrimethylammonium chloride (CTAC), sodium borohydride (NaBH4) and citric acid is 1:800:1:80.

[0016] Furthermore, in the seed solution synthesis step, the reaction time is 1 to 5 minutes (e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes), preferably 2 minutes.

[0017] Furthermore, in the synthesis step of the seed solution, the reaction temperature is room temperature.

[0018] Furthermore, the synthesis of the seed solution also includes a heating and reflux step.

[0019] Furthermore, the temperature of the heating reflux is 80°C.

[0020] Furthermore, the heating reflux time is 90 minutes.

[0021] Furthermore, the synthesis of the growth solution involves mixing hexadecyltrimethylammonium bromide (CTAB), tetrachloroauric acid (HAuCl4), silver nitrate (AgNO3), hydrochloric acid (HCl), ascorbic acid (AA) with 1.09 mL of seed solution and reacting the mixture.

[0022] Furthermore, the concentration of the hexadecyltrimethylammonium bromide (CTAB) is 80–120 mM, preferably 100 mM.

[0023] Furthermore, the concentration of the tetrachloroauric acid (HAuCl4) is 8–12 mM, preferably 10 mM.

[0024] Furthermore, the concentration of silver nitrate (AgNO3) is 8–12 mM, preferably 10 mM.

[0025] Furthermore, the concentration of the hydrochloric acid (HCl) is 0.1–2 mM, preferably 1 mM.

[0026] Furthermore, the concentration of ascorbic acid (AA) is 80-120 mM, preferably 100 mM.

[0027] Furthermore, the concentration of the seed solution is 0.01–1 mM, preferably 0.125 mM.

[0028] Furthermore, in the synthesis step of the growth solution, the reaction time is 2 hours.

[0029] Furthermore, in the synthesis step of the growth solution, the reaction temperature is 30°C.

[0030] Furthermore, step (1) also includes a purification step of gold nanobipyramidal nanoparticles.

[0031] Furthermore, the purification of the gold nanobipyramidal nanoparticles includes a centrifugation step.

[0032] Furthermore, the purification of the gold nanobipyramidal nanoparticles includes a washing step.

[0033] Furthermore, the purification of the gold nanobipyramidal nanoparticles includes an ultrasonic step.

[0034] Furthermore, the purification temperature is 30°C.

[0035] Furthermore, in the centrifugation step, the rotation speed is 8000-12000 rpm.

[0036] Furthermore, in the centrifugation step, the centrifugation time is 5 to 10 minutes.

[0037] Furthermore, in the ultrasound step, the ultrasound time is 0.5 to 2 minutes.

[0038] Further, step (2) includes the following steps:

[0039] (a) Mix PVA with water, heat, and cool to room temperature;

[0040] (b) Add PEI to the material obtained in step (a), mix, and obtain electrospinning precursor solution;

[0041] (c) The obtained electrospinning precursor solution is electrospinned to obtain PEI / PVA electrospun fiber membrane.

[0042] Furthermore, the mixing time in step (a) is 2 to 5 hours.

[0043] Furthermore, the mixing temperature in step (a) is 85–95°C.

[0044] Furthermore, the mixing time in step (b) is 6 to 8 hours.

[0045] Furthermore, the electrospinning time in step (c) is 1 to 5 hours.

[0046] In one embodiment of the present invention, the electrospinning time in step (c) is 2 hours.

[0047] Furthermore, the voltage for electrospinning in step (c) is 20–30 kV.

[0048] In one embodiment of the present invention, the voltage of electrospinning in step (c) is 25kV.

[0049] Further, the feed rate of the electrospinning in step (c) is 0.005 to 0.2 mm / min.

[0050] In one embodiment of the present invention, the feed rate of the electrospinning in step (c) is 0.1 mm / min.

[0051] Furthermore, the receiving distance for electrospinning in step (c) is 10–20 cm.

[0052] In one embodiment of the present invention, the receiving distance of the electrospinning in step (c) is 15 cm.

[0053] Furthermore, step (2) also includes a post-treatment step of the PEI / PVA electrospun fiber membrane.

[0054] Furthermore, the post-processing includes the step of crosslinking the PEI / PVA electrospun fiber membrane with GA vapor.

[0055] Furthermore, the crosslinking time is 5–15 h.

[0056] Furthermore, the crosslinking temperature is 55–65°C.

[0057] In one embodiment of the invention, the post-treatment includes crosslinking the PEI / PVA electrospun fiber membrane with GA vapor at 60°C for 10 hours.

[0058] Furthermore, the post-processing also includes washing and drying the prepared PEI / PVA electrospun fiber membrane.

[0059] Further, step (3) includes immersing the PEI / PVA electrospun fiber membrane obtained in step (2) in a PSS solution.

[0060] Further, the concentration of the PSS solution is 1 to 5 mg / ml (e.g., 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml), preferably, the concentration of the PSS solution is 1.5 to 2.5 mg / ml.

[0061] Further, the soaking temperature in step (3) is 30-45℃ (e.g., 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃), preferably, the soaking temperature in step (3) is 35-40℃.

[0062] Further, the soaking time in step (3) is 1 to 5 hours (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours), preferably, the soaking time in step (3) is 1 to 3 hours.

[0063] In one embodiment of the present invention, step (3) includes immersing the PEI / PVA electrospun nanofiber membrane prepared in step (2) in a 2 mg / ml PSS solution at 37°C for 2 h.

[0064] Furthermore, step (3) also includes washing the modified PEI / PVA electrospun fiber membrane.

[0065] Further, step (4) includes immersing the modified PEI / PVA electrospun fiber membrane obtained in step (3) in the gold nanobipyramidal (AuNBPs) solution obtained in step (1).

[0066] Further, the concentration of the gold nanoparticle bipyramidal (AuNBPs) solution in step (4) is 0.1–2 mM (e.g., 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM). M, 1.9mM, 2mM), preferably, the concentration of the gold nanoparticle bipyramidal solution in step (4) is 0.1-1.5mM, more preferably, the concentration of the gold nanoparticle bipyramidal solution in step (4) is 0.1-1mM, even more preferably, the concentration of the gold nanoparticle bipyramidal solution in step (4) is 0.2-0.8mM, particularly preferably, the concentration of the gold nanoparticle bipyramidal solution in step (4) is 0.5mM.

[0067] Further, the soaking temperature in step (4) is 30-45℃ (e.g., 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃), preferably, the soaking temperature in step (4) is 35-40℃, more preferably, the soaking temperature in step (4) is 37℃.

[0068] Further, the soaking time in step (4) is 5 to 20 hours (e.g., 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours), preferably, the soaking time in step (4) is 8 to 15 hours, more preferably, the soaking time in step (4) is 12 hours.

[0069] In one embodiment of the present invention, step (4) includes immersing the modified PEI / PVA electrospun nanofiber membrane prepared in step (3) in a 0.5 mM AuNBPs solution for 12 h at 37 °C.

[0070] Furthermore, step (4) also includes washing the gold nanobicone functionalized PEI / PVA electrospun fiber membrane.

[0071] A second aspect of the present invention provides a gold nanobipyramidal functionalized electrospun fiber membrane obtained by the preparation method described in the first aspect.

[0072] A third aspect of the present invention provides a method for detecting acetylcholinesterase, the method comprising detecting acetylcholinesterase using a gold nanobipyramidal functionalized electrospun fiber membrane as described in the second aspect.

[0073] Furthermore, the detection can be qualitative or quantitative.

[0074] In one embodiment of the present invention, the detection is a quantitative detection.

[0075] In one embodiment of the present invention, the detection is the detection of the concentration of acetylcholinesterase.

[0076] In one embodiment of the present invention, the detection is a visual detection of acetylcholinesterase concentration.

[0077] In one embodiment of the present invention, the detection is a qualitative detection.

[0078] In one embodiment of the present invention, the detection is the detection of acetylcholinesterase activity.

[0079] In one embodiment of the present invention, the detection is a visual detection of acetylcholinesterase activity.

[0080] Furthermore, the detection method involves determining the activity of acetylcholinesterase by observing the color of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction.

[0081] Furthermore, the visualization detection is as follows:

[0082] As the concentration of acetylcholinesterase (AChE) increases, the color of the gold nanobipyramidal nanoparticles in solution changes from orange to green to cyan to purple to purplish-red.

[0083] As the concentration of acetylcholinesterase (AChE) increases, the color of the gold nanobipyramidal functionalized electrospun fiber membrane changes from orange to green to cyan to purple to purplish-red.

[0084] Furthermore, the detection is performed by obtaining the concentration of acetylcholinesterase through the RGB values ​​of the gold nanobipyramidal functionalized electrospun fiber membrane after the reaction.

[0085] Furthermore, the relationship between the concentration of acetylcholinesterase (AChE) and the R / B value of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction is as follows:

[0086] When the concentration of AChE is 0.01–50 U / L, R / B = -0.26lgc + 1.13;

[0087] When the concentration of AChE is 50-500 U / L, R / B = 0.75lgc - 0.53.

[0088] Furthermore, the detection includes the following steps:

[0089] (1) Mix acetylcholinesterase with thioacetylcholine;

[0090] (2) Add ferric hydroxide and acidic solution to the material obtained in step (1) and react;

[0091] (3) Add hydrogen peroxide and gold nanoparticle-functionalized electrospun fiber membrane to the material obtained in step (2) and react;

[0092] (4) Obtain the RGB values ​​of the gold nanobicone functionalized electrospun fiber membrane after the reaction in step (3).

[0093] Furthermore, the RGB values ​​in step (4) can be obtained by any known method existing in the prior art.

[0094] In one embodiment of the present invention, step (4) involves taking a picture of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction (color development) with a mobile phone, importing the picture into the "color recognizer" mobile phone software, and obtaining the RGB value of the gold nanoparticle-functionalized electrospun fiber membrane.

[0095] Further, the mixing time in step (1) is 1 to 5 hours (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours), preferably 1 to 3 hours, and more preferably 2 hours.

[0096] Further, the mixing temperature in step (1) is 25-45°C (e.g., 25°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C), preferably 30-40°C, and more preferably 37°C.

[0097] Further, the thioacetylcholine in step (1) can be iodinated acetylthiocholine or acetylthiocholine chloride.

[0098] In one embodiment of the present invention, the thioacetylcholine is iodinated acetylthiocholine.

[0099] Further, the concentration of thioacetylcholine in step (1) is 0.2 to 1.0 mM (e.g., 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM), preferably 0.7 to 0.9 mM, and even more preferably, the concentration of thioacetylcholine is 0.8 mM.

[0100] Further, the hydrogen peroxide concentration in step (1) is 2.5–5 mM (e.g., 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM). Preferably, the hydrogen peroxide concentration in step (1) is 2.5–4.5 mM. More preferably, the hydrogen peroxide concentration in step (1) is 2.8–4 mM. Even more preferably, the hydrogen peroxide concentration in step (1) is 2.8–3.5 mM.

[0101] In one embodiment of the present invention, the hydrogen peroxide concentration in step (1) is 3 mM.

[0102] Further, the concentration of iron hydroxyoxide in step (2) is 0.05 to 0.07 mg / mL (e.g., 0.05 mg / mL, 0.051 mg / mL, 0.052 mg / mL, 0.053 mg / mL, 0.054 mg / mL, 0.055 mg / mL, 0.056 mg / mL, 0.057 mg / mL, 0.058 mg / mL, 0.059 mg / mL, 0.061 mg / mL, 0.062 mg / mL, 0.063 mg / mL, 0.064 mg / mL, 0.065 mg / mL, 0.066 mg / mL, 0.067 mg / mL, 0.068 mg / mL, 0.069 mg / mL, 0.07 mg / mL), preferably, the concentration of iron hydroxyoxide is 0.055 to 0.065 mg / mL.

[0103] In one embodiment of the present invention, the concentration of iron hydroxyoxide in step (2) is 0.06 mg / mL.

[0104] Further, the acidic solution in step (2) can be selected from one or more of the following: hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, formic acid, valeric acid, oxalic acid and hydrobromic acid. Preferably, the acidic solution in step (2) is hydrochloric acid.

[0105] Further, the concentration of the acidic solution in step (2) is 1 to 5M (e.g., 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.5M, 4M, 4.5M, 5M), preferably 1 to 3M, more preferably 2M.

[0106] Furthermore, the reaction time in step (2) is 10 min.

[0107] Further, the reaction time in step (3) is 9 to 11 min (e.g., 9 min, 9.1 min, 9.2 min, 9.3 min, 9.4 min, 9.5 min, 9.6 min, 9.7 min, 9.8 min, 9.9 min, 10 min, 10.1 min, 10.2 min, 10.3 min, 10.4 min, 10.5 min, 10.6 min, 10.7 min, 10.8 min, 10.9 min, 11 min), preferably, the reaction time in step (3) is 9.5 to 10.5 min.

[0108] In one embodiment of the present invention, the reaction time in step (3) is 10 min.

[0109] Furthermore, the detection is performed by obtaining the concentration of acetylcholinesterase from the RGB values ​​of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction.

[0110] A fourth aspect of the present invention provides a method for detecting acetylcholinesterase inhibitors, the method comprising detecting acetylcholinesterase inhibitors using a gold nanobipyramidal functionalized electrospun fiber membrane as described in the second aspect.

[0111] Furthermore, the detection can be qualitative or quantitative.

[0112] In one embodiment of the present invention, the detection is a qualitative detection.

[0113] In one embodiment of the present invention, the detection is a visual detection of acetylcholinesterase inhibitors and non-acetylcholinesterase inhibitors.

[0114] Furthermore, the detection method involves determining whether the inhibitor is an acetylcholinesterase inhibitor by observing the color of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction.

[0115] Furthermore, the detection method involves determining whether the inhibitor is an acetylcholinesterase inhibitor by analyzing the RGB values ​​of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction.

[0116] Furthermore, the detection is as follows:

[0117] When the inhibitor is an acetylcholinesterase inhibitor, the color of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction is orange.

[0118] When the inhibitor is an acetylcholinesterase inhibitor, the RGB value of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction is 1.5 to 1.7.

[0119] When the inhibitor is a non-acetylcholinesterase inhibitor, the color of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction is cyan.

[0120] When the inhibitor is a non-acetylcholinesterase inhibitor, the RGB value of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction is 0.4 to 0.6.

[0121] Furthermore, the acetylcholinesterase inhibitor can be a reversible acetylcholinesterase inhibitor and / or an irreversible acetylcholinesterase inhibitor.

[0122] Furthermore, the reversible acetylcholinesterase inhibitor is selected from one or more of the following: alkaloid acetylcholinesterase inhibitors, quaternary ammonium acetylcholinesterase inhibitors, and tertiary amine acetylcholinesterase inhibitors.

[0123] Furthermore, the irreversible acetylcholinesterase inhibitor can be an organophosphorus pesticide.

[0124] Furthermore, the acetylcholinesterase inhibitor may be selected from one of donepezil, rivastigmine, berberine, galantamine, rivastigmine, memantine, and takrine. Preferably, the acetylcholinesterase inhibitor may be selected from one or a combination of two or more of donepezil, rivastigmine, and berberine.

[0125] Furthermore, donepezil is a free base of donepezil or a pharmaceutically acceptable salt thereof, wherein the salt is selected from one or more combinations of hydrochloride, oxalate, hydrobromide, sulfate, nitrate, phosphate, valerate, fumarate, methanesulfonate, benzenesulfonate and toluenesulfonate, preferably, donepezil is donepezil hydrochloride.

[0126] Further, the rivastigmine is a free rivastigmine base or a pharmaceutically acceptable salt thereof, wherein the salt is selected from one or more combinations of tartrate, benzoate, isobutylphenylpropionate, maleate, fumarate, caprylate, salicylate, butyrate and laurate, preferably, the rivastigmine is rivastigmine tartrate.

[0127] Further, the berberine is berberine or berberine salt, wherein the berberine salt is selected from one or more combinations of berberine hydrochloride, berberine sulfate and berberine hydrogen sulfate, preferably, the berberine is berberine hydrochloride.

[0128] In one embodiment of the present invention, the acetylcholinesterase inhibitor may be selected from: donepezil hydrochloride, rivastigmine tartrate, and berberine hydrochloride.

[0129] Furthermore, the non-acetylcholinesterase inhibitor may be selected from one or more combinations of camptothecin, caffeine, and evodiamine.

[0130] Furthermore, the detection steps are as follows:

[0131] (1) Mix acetylcholinesterase with an inhibitor;

[0132] (2) Add thioacetylcholine to the material obtained in step (1) and react;

[0133] (3) Add ferric hydroxide and acidic solution to the material obtained in step (2) and react;

[0134] (4) Add hydrogen peroxide and gold nanoparticle-functionalized electrospun fiber membrane to the material obtained in step (3) and react;

[0135] (5) Observe the color of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction.

[0136] Further, the mixing time in step (1) is 10 to 20 minutes (e.g., 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes), preferably 12 to 18 minutes, and more preferably 15 minutes.

[0137] Further, the mixing temperature in step (1) is 25-45°C (e.g., 25°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C), preferably 30-40°C, and more preferably 37°C.

[0138] Further, the concentration of the inhibitor in step (1) is 40-60 μM (e.g., 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, 60 μM), preferably 45-55 μM, more preferably 50 μM.

[0139] Further, the concentration of acetylcholinesterase in step (1) is 100-300 U / L (e.g., 100 U / L, 150 U / L, 160 U / L, 170 U / L, 180 U / L, 190 U / L, 200 U / L, 210 U / L, 220 U / L, 230 U / L, 240 U / L, 250 U / L, 300 U / L), preferably 200 U / L.

[0140] Further, the concentration of hydrogen peroxide in step (2) is 2.5–5 mM (e.g., 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM). Preferably, the concentration of hydrogen peroxide in step (1) is 2.5–4.5 mM. More preferably, the concentration of hydrogen peroxide in step (1) is 2.8–4 mM. Even more preferably, the concentration of hydrogen peroxide in step (1) is 2.8–3.5 mM.

[0141] Furthermore, the thioacetylcholine in step (2) can be iodinated acetylthiocholine or acetylthiocholine chloride.

[0142] In one embodiment of the present invention, the thioacetylcholine in step (2) is iodinated acetylthiocholine.

[0143] Further, the concentration of thioacetylcholine in step (2) is 2-5 mM (e.g., 2 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.5 mM, 5 mM), preferably 2.5-3.5 mM, more preferably 3.2 mM.

[0144] Further, the reaction time in step (2) is 1 to 5 hours (e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours), preferably 1.5 to 2.5 hours, and more preferably 2 hours.

[0145] Further, the concentration of iron hydroxyoxide in step (3) is 0.1 to 5 mg / mL (e.g., 0.1 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL), preferably 0.5 to 2 mg / mL, more preferably 1.2 mg / mL.

[0146] Further, the acidic solution in step (3) can be selected from one or more of the following: hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, formic acid, valeric acid, oxalic acid and hydrobromic acid. Preferably, the acidic solution in step (3) is hydrochloric acid.

[0147] Further, the concentration of the acidic solution in step (3) is 0.1 to 5M (e.g., 0.1M, 0.5M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, 2.1M, 2.2M, 2.3M, 2.4M, 2.5M, 2.6M, 2.7M, 2.8M, 2.9M, 3M, 3.5M, 4M, 4.5M, 5M), preferably 1.5 to 2.5M, more preferably 2M.

[0148] Further, the reaction time in step (3) is 5 to 35 min (e.g., 5 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 25 min, 30 min, 35 min), preferably 10 to 20 min, and more preferably 15 min.

[0149] Further, the reaction time in step (4) is 5 to 30 min (e.g., 5 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 25 min, 30 min), preferably 5 to 15 min, more preferably 10 min.

[0150] Furthermore, the detection also includes the following step: obtaining the RGB value of the gold nanobipyramidal functionalized electrospun fiber membrane after the reaction in step (4).

[0151] Furthermore, the RGB values ​​of the gold nanobicone functionalized electrospun fiber membrane after the reaction in step (4) can be obtained by any known method existing in the prior art.

[0152] In one embodiment of the present invention, step (4) involves taking a picture of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction (color development) with a mobile phone, importing the picture into the "color recognizer" mobile phone software, and obtaining the RGB value of the gold nanoparticle-functionalized electrospun fiber membrane.

[0153] Furthermore, the detection method involves determining whether the inhibitor is an acetylcholinesterase inhibitor by analyzing the RGB values ​​of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction.

[0154] The basic principle of this invention is as follows:

[0155] (1) First, a two-dimensional material FeOOH sheet structure containing a large amount of Fe was synthesized by hydrothermal method. 2+ .

[0156] (2) Thiocholine is produced by the hydrolysis of thioacetylcholine by acetylcholinesterase. The strong reducing property of the sulfhydryl group in thiocholine decomposes FeOOH, releasing a large amount of Fe. 2+ Fe 2+ The reaction with hydrogen peroxide produces ·OH, which etches the gold nanobipyramids, causing a blue shift in the local surface plasmon resonance (LSPR) absorption peak of AuNBPs. This results in significant color changes in the gold nanobipyramid solution and the electrospun fiber membrane modified with gold nanobipyramids. The presence of acetylcholinesterase inhibitors suppresses acetylcholinesterase activity, reducing the content of thiocholine produced during decomposition and decreasing its ability to decompose FeOOH, leading to Fe… 2+The amount of reactive oxygen species (·OH) that catalyzes the formation of corrosion precipitates from hydrogen peroxide in AuNBPs also decreased, resulting in a negligible blue shift in the LSPR of the AuNBPs colloidal solution. Therefore, under LED light, the electrospun fiber membrane after color development was photographed with a mobile phone, and the image was imported into a "color recognition" mobile app to read the RGB values ​​of the electrospun fiber membrane. This led to the construction of a multicolor visualization sensing membrane for monitoring acetylcholinesterase activity and visually screening its inhibitors (donepezil hydrochloride, rivastigmine tartrate, berberine hydrochloride, etc.).

[0157] The gold nanoparticle bipyramidal functionalized electrospun fiber membrane of this invention utilizes acetylcholinesterase to catalyze the production of thiocholine from thioacetylcholine, and thiocholine catalyzes the production of Fe from iron hydroxyl oxide. 2+ Fe 2+ Catalytic hydrogen peroxide is used to generate reactive oxygen species, which then etch gold nanobipyramidal nanoparticles under catalytic conditions. By observing the color changes of the electrospun membrane functionalized with gold nanobipyramidal nanoparticles after the reaction, a visual analysis of acetylcholinesterase is achieved. A visual screening method for acetylcholinesterase inhibitors and non-acetylcholinesterase inhibitors is constructed. The functionalized electrospun membrane overcomes the shortcomings of traditional visualization equipment, such as single color and low resolution, which lead to poor quantitative results. It shows a high level of performance in the quantitative (concentration) and qualitative (enzyme inhibitor screening) analysis of acetylcholinesterase, with good visual signal effects, high sensitivity, good reproducibility, and high efficiency of multicolor analysis.

[0158] The term AuNBPs used in this invention refers to gold nanobipyramidal nanoparticles.

[0159] In this invention, the term RGB value refers to chromaticity value.

[0160] The term FeOOH used in this invention refers to iron hydroxyoxide.

[0161] The term H2O2 used in this invention refers to hydrogen peroxide.

[0162] In this invention, the term PEI refers to polyethyleneimine.

[0163] In this invention, the term PVA refers to polyvinyl alcohol.

[0164] In this invention, the term PSS refers to sodium poly(4-styrene sulfonate).

[0165] The term AChE used in this invention refers to acetylcholinesterase.

[0166] The term ACTh used in this invention refers to acetylthiocholine iodide.

[0167] The term CTAB used in this invention refers to hexadecyltrimethylammonium bromide.

[0168] The term C6H8O7·H2O used in this invention refers to citric acid monohydrate.

[0169] The term HAuCl4 used in this invention refers to tetrachloroauric acid.

[0170] The term AgNO3 used in this invention refers to silver nitrate.

[0171] In this invention, the term HCl refers to hydrochloric acid.

[0172] In this invention, the term AA refers to ascorbic acid.

[0173] In this invention, the term PSA refers to prostate antigen.

[0174] In this invention, the term ALP refers to alkaline phosphatase.

[0175] In this invention, the term PPase refers to pyrophosphatase.

[0176] In this invention, the term α-GAA refers to α-glucosidase.

[0177] The term Thr as used in this invention refers to threonine. Attached Figure Description

[0178] Figure 1 The images show actual photographs of an electrospun fiber membrane receiving substrate with a coverslip (where A is a TEM image of the electrospun fiber membrane; B is a TEM image of an electrospun fiber membrane loaded with AuNBPs).

[0179] Figure 2 The effect of different reaction parameters on Δλ (longitudinal wavelength difference) during the detection of AChE by electrospun fiber membranes modified with AuNBPs (Figure A shows the Δλ trend corresponding to the addition of different concentrations of H2O2; Figure B shows the Δλ trend corresponding to the addition of different concentrations of ATCh; Figure C shows the Δλ trend corresponding to the addition of different concentrations of FeOOH; Figure D shows the Δλ trend corresponding to different reaction times after the addition of AuNBPs).

[0180] Figure 3 Electrospun fiber membranes modified with AuNBPs were used for AChE detection (where A is the UV-Vis absorption curve of AuNBPs after corrosion by using different concentrations of AChE; B is the calibration curve obtained by plotting Δλ against the logarithm of AChE concentration; C is the color change of AuNBPs in solution; D is the color change of the electrospun fiber membrane modified with AuNBPs; and E is the calibration curve obtained by plotting the RGB of the electrospun fiber membrane modified with AuNBPs against the logarithm of AChE concentration).

[0181] Figure 4 The images show SEM images of AuNBPs / PEI / PVA NFs reacted with different analytical systems containing different concentrations of AChE (where A is the SEM image corresponding to an AChE concentration of 0 U / L; B is the SEM image corresponding to an AChE concentration of 1.0 U / L; C is the SEM image corresponding to an AChE concentration of 50.0 U / L; and D is the SEM image corresponding to an AChE concentration of 100.0 U / L).

[0182] Figure 5 A mobile page for detecting the RGB values ​​of electrospun fiber membranes.

[0183] Figure 6 Selectivity experiment of PEI / PVA electrospun fiber membrane modified with AuNBPs for AChE.

[0184] Figure 7 Electrospun fiber membranes modified with AuNBPs were used for the detection of AChE inhibitors (Figure A shows the relationship between inhibition rate and donepezil hydrochloride concentration; in the inset, a is blank, b is AChE with 50 U / L added, and c is donepezil hydrochloride with 50 nM added; Figure B shows the R / B inhibition curve of the electrospun fiber membrane versus donepezil hydrochloride concentration; Figure C shows the relationship between inhibition rate and rivastigmine tartrate concentration; in the inset, a is blank). Figure 1 shows the relationship between the R / B inhibition of the electrospun fiber membrane and the concentration of rivastigmine tartrate; Figure 2 shows the relationship between the inhibition rate and the concentration of berberine hydrochloride. In the inset, a is blank, b is AChE with 50 U / L added, and c is berberine hydrochloride with 50 μM added; Figure 3 shows the relationship between the R / B inhibition of the electrospun fiber membrane and the concentration of berberine hydrochloride.

[0185] Figure 8 The screening of acetylcholinesterase inhibitors under different conditions is shown in Figure A (screening of acetylcholinesterase inhibitors in a liquid phase environment; and screening of acetylcholinesterase inhibitors using POCT technology based on solid-phase electrospun membranes and mobile phones). Detailed Implementation

[0186] In order to better understand the technical content of the present invention, the following embodiments are provided in detail. The purpose of these embodiments is only to better understand the content of the present invention and not to limit the scope of protection of the present invention.

[0187] Example 1

[0188] 1. Materials and reagents

[0189] Polyvinyl alcohol powder (PVA, degree of hydrolysis 99%, average relative molecular mass 89,000-98,000) was provided by Shanghai Sigma-Aldrich. Polyethylene imine polymer (PEI, average relative molecular mass = 70,000), acetylcholinesterase (AChE, derived from fly heads), acetylthiocholine iodide (ACTh), donepezil hydrochloride, rivastigmine tartrate, and berberine hydrochloride were purchased from Shanghai Aladdin Chemical Co., Ltd. Sodium poly(4-styrenesulfonate) (PSS) was purchased from Ron Reagent Co., Ltd. Cetyltrimethylammonium bromide (CTAB), HCl, citric acid monohydrate (C6H8O7·H2O), HAuCl4·3H2O, and AgNO3 were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0190] 2. Instruments

[0191] Commercial electrospinning equipment (YFSP-T, Tianjin Yunfan Technology Co., Ltd., China) was used to prepare electrospun nanofiber films. UV-Vis spectral measurements were performed using a UV-2700 spectrophotometer (Shimadzu Corporation, Japan). The morphologies of AuNBPs, PEI / PVANFS, and AuNBPs / PEI / PVA NFS were characterized using transmission electron microscopy (TEM, JEM2100, Tokyo, Japan) and scanning electron microscopy (SEM, S-4800, Hitachi, Japan). A smartphone app called "Color Recognizer" (developer: Lin Zhimou) was used to read the RGB values ​​of the electrospun fiber films.

[0192] 3. Synthesis of gold nanobipyramidal structures

[0193] 3.1 Synthesis of Seed Solution

[0194] At room temperature, in a round-bottom flask (10 mL, 0.25 mM) of HAuCl4 and 5 mL of 200 mM hexadecyltrimethylammonium chloride (CTAC), the solution was rapidly reduced by freshly prepared NaBH4 (25 mM, 0.25 mL) and 5 mL of citric acid (20 mM). The mixed solution changed from pale yellow to brown. After capping and vigorous stirring for 2 min, the solution was gently heated under reflux in an oil bath at 80 °C for 90 min with stirring. The color gradually changed from brown to red. Finally, the gold seed solution was obtained and stored at room temperature.

[0195] 3.2 Synthesis of growth solution

[0196] In a 250 mL Erlenmeyer flask, CTAB (100 mL, 100 mM), HAuCl4 (5 mL, 10 mM), AgNO3 (1 mL, 10 mM), HCl (2 mL, 1 M), and AA (0.8 mL, 100 mM) were added sequentially and mixed thoroughly. The solution changed from yellow to colorless. 1.09 mL of the prepared seed solution was added to the above solution, mixed thoroughly, and the mixture was reacted at 30 °C for 2 h. The ultraviolet absorption spectrum was then measured.

[0197] 3.3 Purification of gold nanobipyramidal particles

[0198] The colloid was centrifuged at 10,000 rpm for 8 minutes at 30°C and washed twice with 100 mL of 1 mM CTAB. After removing the supernatant, the precipitate was redispersed in 30 mL of 1 mM CTAB solution for further purification. A certain volume of BDAC (62 mL 0.5 mol / L) solution and ultrapure water were added to 30 mL of crude bipyramidal solution, followed by 8 mL of the solution to obtain 100 mL of solution with the desired BDAC concentration. The mixture was then incubated at 30°C for 11 hours. The pink supernatant was carefully removed, and 30 mL of 1 mM CTAB was added to the conical flask to redisperse the precipitate. The solution was sonicated for 1 minute. The resulting purified solution (brown) was centrifuged at 8,000 rpm for 8 minutes and washed twice with 30 mL of 1 mM CTAB to remove excess BDAC. Finally, the purified bipyramidal solution was redispersed in 15 mL of 1 mM CTAB solution for subsequent use.

[0199] 4. Preparation of α-FeOOH

[0200] 1.08 g (4 mmol) of FeCl3·6H2O was added to a solution containing 5 mL (0.075 mol) of ethylenediamine and 40 mL of H2O, and the mixture was stirred vigorously for 10 minutes. The solution was then sealed in a 100 mL PTFE-coated stainless steel autoclave and placed in an oven maintained at 85 °C for 12 hours. The precipitate in the reaction mixture was collected by centrifugation at 8000 rpm for 5 minutes and washed three times with water. The precipitate was then freeze-dried to give a yellow solid product.

[0201] 5. Preparation of gold nanoparticle-functionalized electrospun fiber membranes

[0202] (1) Preparation of electrospinning solution: 1.36g of PVA powder was placed in a round bottom flask and dissolved in an oil bath at 90°C for 3 hours. After cooling to room temperature, 0.45g of PEI was added and mixed thoroughly for 6-8 hours to obtain the electrospinning precursor solution (PEI / PVA electrospinning solution).

[0203] (2) Preparation of electrospinning receiving device: The width of the aluminum foil is the same as the width of the original electrospinning roller receiving device, and the length of the aluminum foil is the same as the circumference of the roller. Use a piece of conductive adhesive or double-sided tape (the length is the same as the circumference of the roller) to stick in the middle of the aluminum foil. Then, cross-attach a 6mm diameter circular cover glass to the conductive adhesive or double-sided tape. 40 covers can be attached in one experiment. Finally, 40 electrospinning fiber sensing films are obtained by receiving the film through the roller of the electrospinning equipment.

[0204] (3) Electrospinning process: The electrospinning precursor solution prepared in (1) is placed in a 10mL syringe (medical disposable syringe with a needle tip diameter of 0.8mm), and then placed in an electrospinning device to prepare an electrospinning fiber sensing membrane.

[0205] Electrospinning equipment conditions: Voltage: 25.0kV, distance from spinneret (syringe needle) to roller receiving device: 15cm, injection pump feed speed: 0.1mm / min, roller receiving device speed: 5rpm. Under preferred conditions, after 2 hours of electrospinning, electrospinning is stopped. The obtained PEI / PVA electrospinned nanofiber membrane is crosslinked with GA vapor for 10 hours in a vacuum drying oven at 60℃ to form water-stable electrospinned fibers. Afterwards, it is rinsed three times with distilled water and dried for later use.

[0206] The prepared PEI / PVA electrospun fiber membrane had a positively charged surface. It was immersed in 500 μL of 2 mg / mL PSS solution and reacted in a mixer at 37°C for 2 h. Afterwards, it was rinsed three times with distilled water to modify the positively charged PEI / PVA electrospun fiber membrane into a negatively charged interface. Next, it was immersed in 500 μL of AuNBPs solution and reacted in a mixer at 37°C for 12 h. Since the AuNBPs were dispersed in CTAB, their surface was positively charged. Through electrostatic interaction, the AuNBPs were modified onto the electrospun fiber membrane. It was then washed three times with distilled water, placed in a 1.5 mL centrifuge tube, and 1 mL of distilled water was added. The membrane was then stored at 4°C for later use.

[0207] like Figure 1 As shown in Figure A, the PEI / PVA electrospun fiber membrane exhibits a three-dimensional network structure, such as... Figure 1 As shown in Figure B, gold nanobipyramids can be uniformly distributed on the surface of the PEI / PVA electrospun fiber membrane, indicating that the electrospun fiber membrane has been successfully modified with gold nanobipyramids.

[0208] 6. AChE Visualized POCT Analysis Technology

[0209] (1) Prepare a series of AChE standard solutions

[0210] Prepare 1 mL of AChE solutions with concentrations of 0, 0.04, 0.2, 0.4, 2, 4, 20, 40, 200, 400, 1200, 2000, 2400, and 2800 U / L in a PBS buffer (pH 7.5) environment.

[0211] (2) Detection of AChE

[0212] First, four key parameters in the analytical system were optimized: the concentration of H₂O₂, the concentration of ATCh, the concentration of FeOOH, and the reaction time after the addition of gold nanoparticles. Each experimental group included a blank experiment, in which the system solution was not supplemented with AChE, but with the same volume of buffered deionized water. In the experimental group, the concentration of AChE used was 1 U / L. The concentrations of added H₂O₂ were: 0.5, 1, 2, 3, 4, 5 mM; the concentrations of added ATCh were: 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mM; the concentrations of added FeOOH were: 0.02, 0.04, 0.06, 0.08, 0.10 mg / mL; and the reaction times after adding AuNBPs were: 2, 4, 6, 8, 10, 12, 14 min.

[0213] like Figure 2 As shown in (A~D), the optimal experimental conditions are as follows: the optimal concentration of H2O2 is 3mM, the optimal concentration of ATCh is 0.8mM, the optimal concentration of FeOOH is 0.06mg / mL, and the optimal reaction time for adding AuNBPs is 10min.

[0214] Afterwards, 50 μL of each AChE solution in (1) was taken out with a pipette and transferred to a centrifuge tube. 3.2 mMATCh was added and the mixture was incubated at 37 °C for 2 h. FeOOH (1.2 mg / mL, 10 μL) and HCl (2 M, 10 μL) were added and reacted on a constant temperature mixer for 10 min. 10 μL of 60 mM H2O2 was added and the electrospun fiber membrane loaded with the above solution was immersed in the above solution for 30 min. The color change of the electrospun fiber membrane was observed. The electrospun fiber membrane after the change was displayed was photographed under an LED light. The photograph was imported into the "Color Recognizer" mobile phone software and the RGB values ​​were read. The visualization analysis of acetylcholinesterase was achieved through R / B.

[0215] like Figure 3 As shown in Figure A, with increasing AChE concentration, the longitudinal wavelength of AuNBPs gradually changed from a longer wavelength to a shorter wavelength, ranging from 0 (blank) to 700.0 U / L. The longitudinal wavelength difference (Δλ) showed a good linear relationship with the logarithm of AChE concentrations from 0.01 to 500.0 U / L. Figure 3 B). The limit of detection (LOD) is 0.0074 U / L, at 3σ (control) Estimation of signal-to-noise ratio (where σ)(control) (This is the standard deviation of the blank sample, n=11). The regression equation is: Δλ=27.83lgc+60.45(R) 2 =0.9988). With increasing AChE concentration, AuNBPs in solution exhibited a significant color change from orange, green, cyan, purple, and purplish-red. Figure 3 C). Similarly, with increasing AChE concentration, the color of the electrospun fiber membrane assembled with AuNBPs ( Figure 3 D) Gradually, changes to orange, green, cyan, purple, and magenta appear. After photographing the electrospun fiber membrane under LED light, the photo is imported into the "Color Recognizer" mobile app to read the RGB values ​​(e.g., ...). Figure 5 As shown), the calibration curve obtained by plotting R / B against the logarithm of AChE concentration (as shown) Figure 3 E) It was found that the R / B value had a good linear relationship with the logarithm of AChE concentration (0.01-50 U / L, R / B = -0.26lgc + 1.13, 50-500 U / L, R / B = 0.75lgc - 0.53).

[0216] SEM images of AuNBPs / PEI / PVA NFs reacting with different analytical systems containing different AChE concentrations are shown below. Figure 4 A through D (photographs of the AuNBPs / PEI / PVA nanofiber films above the corresponding analysis system), where, Figure 4 The AChE concentration corresponding to A is 0 U / L (control); Figure 4 The AChE concentration corresponding to B is 1.0 U / L; Figure 4 The AChE concentration corresponding to C is 50.0 U / L; Figure 4 The concentration of AChE corresponding to D is 100.0 U / L. This is based on the microstructure of AuNBPs ( Figure 4 As can be seen from the TEM images in A to D, AuNBPs shorten from bipyramidal to quasi-nanospheres as the concentration of AChE increases.

[0217] 7. Selectivity test of AChE

[0218] Following the same experimental steps as in 6(2) above, the detection system was reacted with AChE (1 U / L), alkaline phosphatase (ALP, 100 U / L), pyrophosphatase (PPase, 100 U / L), α-glucosidase (α-GAA, 100 U / L), and threonine (Thr, 100 mM). FeOOH (1.2 mg / mL, 10 μL) and HCl (2 M, 10 μL) were then added for further reaction. Finally, a blank experiment was used as a control. The specificity of this system for AChE detection was investigated by comparing the change in the longitudinal localized surface plasmon resonance peak shift of AuNBPs with the recognition of different targets. All tests were performed using three groups, and the average value was taken.

[0219] like Figure 6 As shown, under all identical experimental conditions, when this system was used to identify ALP (100 U / L), PPase (100 U / L), α-GAA (100 U / L), and Thr (100 mM), the longitudinal plasmonic peak of AuNBPs showed almost no blue shift. However, when used to detect AChE (1 U / L), the longitudinal plasmonic peak of AuNBPs showed a significant blue shift, demonstrating that this system has a clear selectivity for AChE. When coexisting AChE (1 U / L) was added to each of the four solutions, the longitudinal peak of AuNBPs showed a significant blue shift, demonstrating that this system has good anti-interference ability against AChE.

[0220] 8. POCT analysis technology for visualizing AChE inhibitors

[0221] 10 μL of inhibitors (donepezil hydrochloride: 2,4,10,20,40,100,200,400,1000,2000,4000 nM, neostigmine bromide: 2,4,10,20,40,100,200,400,1000,2000,4000 nM, berberine hydrochloride: 2,4,10,20,40,100,200,400,1000,1600,2000 μM) were incubated with 50 μL of 200 U / L acetylcholinesterase at 37 °C for 15 min, followed by the addition of 50 μL of 3.2 mM ATCh and a further reaction time of 2 h. FeOOH (1.2 mg / mL, 10 μL) and HCl (2 M, 10 μL) were added, and the mixture was reacted for 15 min on a constant temperature mixer. Then, 60 mM, 10 μL H2O2 and 50 μL gold nanoparticles were added, and the mixture was reacted for 10 min. The longitudinal plasmon absorption peak λ of AuNBPs was measured. i As described above, a parallel experiment was conducted without the addition of inhibitors, and the longitudinal plasmon absorption peak of its AuNBPs was measured to be λ. Simultaneously, another experiment was conducted without the addition of inhibitors and AChE, and the longitudinal plasmon absorption peak of its AuNBPs was λ0. The inhibition rate was calculated using Equation 1:

[0222] Inhibition rate = (λ) i -λ) / (λ0-λ)×100%

[0223] like Figure 7 A. As the concentration of donepezil hydrochloride increases, the inhibition rate of acetylcholinesterase also increases, ranging from 0.1 to 200 nM. In the illustration, a represents the longitudinal plasma absorption peak of AuNBPs at ~720 nm in a solution without acetylcholinesterase; b represents the longitudinal plasma absorption peak of AuNBPs with the addition of 50 U / L acetylcholinesterase, which blue-shifts to ~615 nm; c represents the longitudinal plasma absorption peak of AuNBPs with the addition of 50 U / L acetylcholinesterase and the inhibitor donepezil hydrochloride at 50 nM, which blue-shifts to ~715 nm. This indicates that donepezil hydrochloride inhibits AChE activity. Figure 7 B is the curve showing the R / B value of the electrospun fiber membrane versus the concentration of donepezil hydrochloride. It was found that the R / B value of the electrospun fiber membrane has a good linear relationship with the logarithm of the donepezil hydrochloride concentration in the range of 0.1–50 nM (R / B = 0.34lgc + 1.06, R...). 2 =0.9963). For example... Figure 7 C. As the concentration of rivastigmine tartrate increases, the inhibition rate of acetylcholinesterase also increases, ranging from 0.1 to 200 nM. In illustration a, the longitudinal plasma absorption peak of AuNBPs in the solution without acetylcholinesterase is at ~720 nm; in illustration b, the longitudinal plasma absorption peak of AuNBPs with the addition of 50 U / L acetylcholinesterase is blue-shifted to ~615 nm; and in illustration c, the longitudinal plasma absorption peak of AuNBPs with the addition of 50 U / L acetylcholinesterase and the inhibitor rivastigmine tartrate is blue-shifted to ~711 nm, indicating that rivastigmine tartrate inhibits the activity of AChE. Figure 7 D represents the R / B value of the electrospun fiber membrane, which shows a good linear relationship with the logarithm of the concentration of 0.1–50 nM rivastigmine tartrate (R / B = 0.45lgc + 0.90, R). 2 =0.9962). For example... Figure 7 E. As the concentration of berberine hydrochloride increases, the inhibition rate of acetylcholinesterase also increases, ranging from 0.1 to 100 μM. In illustration a, in the solution without acetylcholinesterase, the longitudinal plasma absorption peak of AuNBPs is at ~720 nm; in illustration b, with the addition of 50 U / L acetylcholinesterase, the longitudinal plasma absorption peak of AuNBPs blue-shifts to ~615 nm; and in illustration c, with the addition of 50 U / L acetylcholinesterase and the inhibitor 50 μM berberine hydrochloride, the longitudinal plasma absorption peak of AuNBPs blue-shifts to ~704 nm. This indicates that berberine hydrochloride inhibits the activity of AChE. Figure 7F is the curve showing the R / B value of the electrospun fiber membrane versus the concentration of rivastigmine tartrate. It was found that the R / B value of the electrospun fiber membrane had a good linear relationship with the logarithm of the concentration of donepezil hydrochloride (0.1-50 μM) (R / B = 0.38lgc + 0.90, R...). 2 =0.9962).

[0224] Figure 8 As shown in Figure A, under the same conditions described above, when 50 μM donepezil hydrochloride, rivastigmine tartrate, berberine hydrochloride, caffeine, camptothecin, and evodiamine were used as inhibitors of acetylcholinesterase, reacted with acetylcholinesterase (50 U / L) and then with acetylcholine (0.8 mM), the inhibitory efficiencies of donepezil hydrochloride, rivastigmine tartrate, berberine hydrochloride, camptothecin, caffeine, and evodiamine as inhibitors of acetylcholinesterase were 94.35%, 92.58%, 85.71%, 27.03%, 9.20%, and 32.21%, respectively. Among them, donepezil hydrochloride, rivastigmine tartrate, and berberine hydrochloride showed better inhibitory efficiencies. Furthermore, as... Figure 8 As shown in B, under the same experimental conditions, screening for acetylcholinesterase inhibitors using electrospun membranes assembled with AuNBPs can be achieved through obvious visualization of both acetylcholinesterase inhibitors and non-acetylcholine inhibitors.

[0225] The electrospun membranes turned orange under the action of acetylcholinesterase inhibitors donepezil hydrochloride, rivastigmine tartrate, and berberine hydrochloride, respectively; and turned cyan under the action of non-acetylcholine inhibitors camptothecin, caffeine, and evodiamine, respectively.

Claims

1. A method for detecting acetylcholinesterase, comprising the following steps: (a1) Mix acetylcholinesterase with thioacetylcholine; (a2) Add ferric hydroxide and an acidic solution to the material obtained in step (a1) and react; (a3) Add hydrogen peroxide and gold nanoparticle-functionalized electrospun fiber membrane to the material obtained in step (a2) and react; (a4) Obtain the RGB values ​​of the gold nanoparticle-functionalized electrospun fiber membrane after the reaction in step (a3). The concentration of acetylcholinesterase is obtained from the RGB values ​​of the reacted gold nanoparticle-functionalized electrospun fiber membrane. The relationship between the concentration of acetylcholinesterase and the R / B value of the reacted gold nanoparticle-functionalized electrospun fiber membrane is as follows: When the concentration of AChE is 0.01~50 U / L, R / B = -0.26 lgc + 1.13; When the concentration of AChE is 50–500 U / L, R / B = 0.75 lgc - 0.53; The preparation method of the gold nanoparticle biconical functionalized electrospun fiber membrane includes the following steps: (b1) Preparation of gold nanobipyramidal structures; (b2) Preparation of PEI / PVA electrospun fiber membrane; (b3) The electrospun fiber membrane obtained from the modification treatment step (b2); (b4) Immerse the modified PEI / PVA electrospun fiber membrane obtained in step (b3) in the gold nanobicone solution obtained in step (b1).

2. The detection method according to claim 1, characterized in that, The thioacetylcholine mentioned in step (a1) is iodinated acetylthiocholine or acetylthiocholine chloride.

3. The detection method according to claim 2, characterized in that, The thioacetylcholine in question is iodinated acetylthiocholine.

4. The detection method according to claim 1, characterized in that, The hydrogen peroxide concentration mentioned in step (a3) ​​is 2.5~5mM.

5. The detection method according to claim 1, characterized in that, The concentration of thioacetylcholine in step (a1) is 0.2~1.0 mM.

6. The detection method according to claim 1, characterized in that, The concentration of iron hydroxyoxide in step (a2) is 0.05~0.07 mg / mL.

7. The detection method according to claim 1, characterized in that, The reaction time in step (a3) ​​is 9 to 11 minutes.

8. The detection method according to claim 1, characterized in that, The concentration of the gold nanoparticle bipyramidal solution in step (b4) is 0.1~2 mM.

9. The detection method according to claim 8, characterized in that, The concentration of the gold nanoparticle bipyramidal solution in step (b4) is 0.1~1.5 mM.

10. The detection method according to claim 9, characterized in that, The concentration of the gold nanoparticle bipyramidal solution in step (b4) is 0.1~1 mM.

11. The detection method according to claim 10, characterized in that, The concentration of the gold nanoparticle bipyramidal solution in step (b4) is 0.2~0.8 mM.

12. The detection method according to claim 11, characterized in that, The concentration of the gold nanoparticle bipyramidal solution in step (b4) is 0.5 mM.

13. The detection method according to claim 1, characterized in that, The soaking temperature in step (b4) is 30~45℃.

14. The detection method according to claim 13, characterized in that, The soaking time in step (b4) is 5 to 20 hours.

15. A method for detecting acetylcholinesterase inhibitors, comprising the following steps: (c1) Mix acetylcholinesterase with an inhibitor; said inhibitor is one of donepezil hydrochloride, rivastigmine tartrate, and berberine hydrochloride; (c2) Add thioacetylcholine to the material obtained in step (c1) and react; (c3) Add ferric hydroxide and an acidic solution to the material obtained in step (c2) and react; (c4) Add hydrogen peroxide and gold nanoparticle-functionalized electrospun fiber membrane to the material obtained in step (c3) and react; (c5) Observe the color of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction, and obtain the RGB value of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction in step (c4). Use the color and RGB value of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction to determine whether the inhibitor is an acetylcholinesterase inhibitor. When the inhibitor is an acetylcholinesterase inhibitor, the color of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction is orange, and the RGB value of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction is 1.5~1.

7. When the inhibitor is a non-acetylcholinesterase inhibitor, the color of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction is cyan, and the RGB value of the electrospun fiber membrane with gold nanoparticles functionalized after the reaction is 0.4~0.

6.

16. The detection method according to claim 15, characterized in that, The concentration of acetylcholinesterase in step (c1) is 100~300 U / L.

17. The detection method according to claim 15, characterized in that, The concentration of hydrogen peroxide in step (c4) is 2.8~3.5mM.

18. The detection method according to claim 15, characterized in that, The thioacetylcholine mentioned in step (c2) is iodinated acetylthiocholine or acetylthiocholine chloride.

19. The detection method according to claim 15, characterized in that, The concentration of thioacetylcholine in step (c2) is 2-5 mM.

20. The detection method according to claim 15, characterized in that, The concentration of iron hydroxyoxide in step (c3) is 0.1~5 mg / mL.

21. The detection method according to claim 15, characterized in that, The reaction time in step (c4) is 5 to 30 minutes.

Images