# US 10,246,523 B2 · Fluorescent starch nanocrystal and preparation method and application thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,246,523 B2 - Document ID: US10246523B2 - Status: Granted - Kind: B2 - Application number: 15/314,861 - Filed: 2015-08-04 - Published: 2017-06-29 - Granted: 2019-04-02 - Earliest claimed: 2015-08-04 - Assignees: Jiangnan University - Applicants: Jiangnan University - Inventors: Yaoqi Tian; Canxin Cai; Benxi Wei; Zhengyu Jin; Jinpeng Wang; Xueming Xu; Na Yang; Xiuting Hu - Web page: https://patentgrep.com/patent/US10246523B2 ## CPC - C08B 31/006: {Crosslinking of derivatives of starch} - C08B 31/00: Preparation of derivatives of starch (derivatives of amylose C08B33/00; derivatives of amylopectin C08B35/00) - C09K 11/06: containing organic luminescent materials - C09K 2211/145: containing oxygen as the only heteroatom - C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21) - C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE ## Abstract The present disclosure discloses a fluorescent starch nanocrystal and a preparation method and application thereof. Mainly based on the character that starch nanocrystal has an active hydroxyl group on the surface, the present disclosure introduces fluorescein isothiocyanate into the surface of the starch nanocrystal in the form of stable covalent bonds through a two-step chemical modification method: silane coupling and electrophilic addition of fluorescein, so as to prepare the starch nanocrystal with fluorescent properties. The preparation process of the present disclosure is simple and easy to operate, uses cheap and readily available raw materials, involves low cost, and allows the non-fluorescent starch nanocrystal to produce significant fluorescent properties without affecting the properties of the starch nanocrystal itself, thereby expanding the application of starch. Moreover, the fluorescent starch nanocrystal thus obtained has significant fluorescent properties, good biocompatibility and biodegradability, has application prospects in the fields of biosensor preparation, bioimaging marking and fluorescent analysis and detection, and can be further prepared into a novel nano-fluorescent probe with organic nano-particles as the matrix. ## Claims ### Claim 1 A method for preparing a fluorescent starch nanocrystal, the method comprising: (1) an activating step consisting of adding 1 w/v % to 8 w/v % of starch nanocrystal to an alkaline solution having a pH of 8.0 to 11.0 and continuously stirring for 2 to 6 h to obtain an alkali-activated starch nanocrystal, and optionally separating and washing the alkali-activated starch nanocrystal with a mixed solution of ethanol and water; (2) adding 0.1 w/v % to 0.8 w/v % of benzoyl peroxide to a mixed solution of ethanol and water and continuously stirring at 100 to 400 rpm at room temperature until benzoyl peroxide is fully dissolved to form a benzoyl peroxide solution, then adding 1 v/v % to 5 v/v % γ-aminopropyl triethoxysilane to the benzoyl peroxide solution to carry out hydrolysis for 0.5 to 6 h to form a silane hydrolysate solution; (3) adding the activated starch nanocrystal of step (1) to the silane hydrolysate solution of step (2) and continuously stirring for 2 to 6 h followed by rotary evaporation and drying to obtain amino silane modified starch nanocrystal; and (4) adding 0.1 w/v % to 2.0 w/v % of the amino silane modified starch nanocrystal obtained from step (3) to a mixed solution of methanol and water containing 0.1 w/v % to 1.5 w/v % fluorescein isothiocyanate and keeping in dark place at room temperature for 12 to 36 h to obtain the fluorescent starch nanocrystal. ### Claim 2 (depends on 1) The method of claim 1, wherein the concentration of ethanol contained in the mixed solution of ethanol and water in step (2) is 60 v/v % to 95 v/v %. ### Claim 3 (depends on 1) The method of claim 1, wherein in step (3) the rotary evaporation is conducted under a vacuum degree of −0.1 to −0.06 Mpa and at a temperature of 50 to 55° C., followed by drying at 40 to 55° C. for 3 to 12 d, and the dried amino silane modified starch nanocrystal is re-dispersed and washed with water until pH value is constant before drying at 40 to 55° C. and then passing through a 60 to 100 mesh screen. ### Claim 4 (depends on 1) The method of claim 1, wherein the concentration of methanol contained in the mixed solution of methanol and water in step (4) is 55 v/v % to 90 v/v %. ### Claim 5 (depends on 1) The method of claim 1, wherein in step (4), after kept in dark place at room temperature for 12 to 36 h, the fluorescent starch nanocrystal is further subjected to centrifugal washing until the supernatant has no characteristic absorption peak in a scanning spectrum, and the washed fluorescent starch nanocrystal is then dried in the dark at 40° C. and passed through a 60 to 100 mesh screen. ### Claim 6 (depends on 1) The method of claim 1, wherein the alkaline is sodium hydroxide. ### Claim 7 (depends on 1) A fluorescent starch nanocrystal prepared using the method of claim 1, capable of sending out yellow-green fluorescence under ultraviolet light, having a characteristic absorption peak in the UV-visible spectrum at 490 nm, and having fluorescence emission at 547 nm with the excitation wavelength at 495 nm. ### Claim 8 (depends on 7) The fluorescent starch nanocrystal of claim 7, wherein the nanocrystal has a particle diameter of 30 to 120 nm. ### Claim 9 (depends on 7) A fluorescent detection reagent, comprising the fluorescent starch nanocrystal of claim 7. ### Claim 10 (depends on 7) A fluorescent detection device, comprising the fluorescent starch nanocrystal of claim 7. ### Claim 11 (depends on 7) The fluorescent starch nanocrystal of claim 7, wherein after continuous irradiation by a mercury lamp for 75 min, the fluorescent starch nanocrystal retains 62% to 85% fluorescence intensity. ### Claim 12 A method for preparing a fluorescent starch nanocrystal, the method comprising: (1) an activating step consisting of adding 1 w/v % to 8 w/v % of starch nanocrystal to a sodium hydroxide solution having a pH of 8.0 to 11.0 and continuously stirring for 2 to 6 h to obtain an alkali-activated starch nanocrystal, which is subsequently separated and then washed with aqueous ethanol; (2) adding 0.1 w/v % to 0.8 w/v % of benzoyl peroxide to 60 v/v % to 95 v/v % aqueous ethanol and continuously stirring at 100 to 400 rpm at room temperature until benzoyl peroxide is fully dissolved to form a benzoyl peroxide solution, then adding 1 v/v % to 5 v/v % γ-aminopropyl triethoxysilane to the benzoyl peroxide solution to carry out hydrolysis for 0.5 to 6 h to form a silane hydrolysate solution; (3) adding the activated starch nanocrystal of step (1) to the silane hydrolysate solution of step (2) and continuously stirring for 2 to 6 h to form a reaction mixture followed by conducting rotary evaporation of the reaction mixture under a vacuum degree of −0.1 to −0.06 Mpa and at a temperature of 50 to 55° C., followed by drying at 40 to 55° C. for 3 to 12 d, and the dried amino silane modified starch nanocrystal is re-dispersed and washed with water until pH value is constant before drying at 40 to 55° C. and then passing through a 60 to 100 mesh screen and (4) adding 0.1 w/v % to 2.0 w/v % of the amino silane modified starch nanocrystal obtained from step (3) to a 55 v/v % to 90 v/v % aqueous methanol containing 0.1 w/v % to 1.5 w/v % fluorescein isothiocyanate and keeping in dark place at room temperature for 12 to 36 h to obtain the fluorescent starch nanocrystal. ### Claim 13 (depends on 12) A fluorescent starch nanocrystal prepared using the method of claim 12, capable of sending out yellow-green fluorescence under ultraviolet light, having a characteristic absorption peak in the UV-visible spectrum at 490 nm, and having fluorescence emission at 547 nm with the excitation wavelength at 495 nm. ### Claim 14 (depends on 13) The fluorescent starch nanocrystal of claim 13, wherein the nanocrystal has a particle diameter of below 50 nm. ### Claim 15 (depends on 13) A fluorescent detection reagent, comprising the fluorescent starch nanocrystal of claim 13. ### Claim 16 (depends on 13) A fluorescent detection device, comprising the fluorescent starch nanocrystal of claim 13. ### Claim 17 (depends on 13) The fluorescent starch nanocrystal of claim 13, wherein after continuous irradiation by a mercury lamp for 75 min, the fluorescent starch nanocrystal retains 62% to 85% fluorescence intensity. ### Claim 18 A method for preparing a fluorescent starch nanocrystal, the method comprising: (1) an activating step consisting of adding 1 w/v % to 8 w/v % of starch nanocrystal to a sodium hydroxide solution having a pH of 8.0 to 11.0 and continuously stirring for 2 to 6 h to obtain an alkali-activated starch nanocrystal, which is subsequently separated and then washed with aqueous ethanol; (2) adding 0.1 w/v % to 0.8 w/v % of benzoyl peroxide to 60 v/v % to 95 v/v % aqueous ethanol and continuously stirring at 100 to 400 rpm at room temperature until benzoyl peroxide is fully dissolved to form a benzoyl peroxide solution, then adding 1 v/v % to 5 v/v % γ-aminopropyl triethoxysilane to the benzoyl peroxide solution to carry out hydrolysis for 0.5 to 6 h to form a silane hydrolysate solution; (3) adding the activated starch nanocrystal of step (1) to the silane hydrolysate solution of step (2) and continuously stirring for 2 to 6 h to form a reaction mixture followed by conducting rotary evaporation of the reaction mixture under a vacuum degree of −0.1 to −0.06 Mpa and at a temperature of 50 to 55° C., followed by drying at 40 to 55° C. for 3 to 12 d, and the dried amino silane modified starch nanocrystal is re-dispersed and washed with water until pH value is constant before drying at 40 to 55° C. and then passing through a 60 to 100 mesh screen and (4) adding 0.1 w/v % to 2.0 w/v % of the amino silane modified starch nanocrystal obtained from step (3) to a 55 v/v % to 90 v/v % aqueous methanol containing 0.1 w/v % to 1.5 w/v % fluorescein isothiocyanate and keeping in dark place at room temperature for 12 to 36 h to obtain the fluorescent starch nanocrystal, which is subjected to centrifugal washing until the supernatant has no characteristic absorption peak in a scanning spectrum, and the washed fluorescent starch nanocrystal is then dried in the dark at 40° C. and passed through a 60 to 100 mesh screen. ### Claim 19 (depends on 18) A fluorescent starch nanocrystal prepared using the method of claim 18, capable of sending out yellow-green fluorescence under ultraviolet light, having a characteristic absorption peak in the UV-visible spectrum at 490 nm, having fluorescence emission at 547 nm with the excitation wavelength at 495 nm, and a particle diameter of below 50 nm, and wherein after continuous irradiation by a mercury lamp for 75 min, the fluorescent starch nanocrystal retains 62% to 85% fluorescence intensity. ### Claim 20 (depends on 19) A fluorescent detection reagent, comprising the fluorescent starch nanocrystal of claim 19. ### Claim 21 (depends on 19) A fluorescent detection device, comprising the fluorescent starch nanocrystal of claim 19.