CN113831912B - 一种基于自身荧光增强的土霉素比率荧光探针及制备与应用 - Google Patents
一种基于自身荧光增强的土霉素比率荧光探针及制备与应用 Download PDFInfo
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Abstract
本发明提供了一种用于可视化检测土霉素的有机硅纳米点比率荧光探针(OSiNDs)及制备方法。有机硅纳米点能够在较广pH溶液中稳定存在,且在长时间紫外照射下释放稳定的荧光。本发明中比率荧光探针的检测机制包含两部分,有机硅纳米点(OSiNDs)基于自身的荧光猝灭机制用于检测四环素类抗生素,通过探针与土霉素(OTC)作用改变并增强土霉素自身荧光将土霉素与其他四环素类区分开来,能够通过比率荧光方法应用于OTC的痕量检测。本发明中比率荧光探针无需后处理加入昂贵的稀土金属或其他荧光材料作为参比,过程简单,条件温和、耗时短。
Description
技术领域
本发明涉及一种基于自身荧光增强的土霉素比率荧光探针的制备及应用,属于荧光分析检测技术领域。
背景技术
土霉素(Oxytetracyclin)是一种具有广谱抗菌活性和抑菌活性的四环素族抗生素,已广泛用于应用于畜禽肉制品生产,用于防治肠道感染和促进畜禽生长。然而,土霉素在人体内不断蓄积到一定浓度的剂量时,能导致人体各种内脏器官或组织的病变,其广泛使用会增加在食品中的残留风险,对人体健康具有潜在危害。因此,需要对食品和饮用水中的土霉素残留进行定性、定量评估。
目前,应用于抗生素残留检测的分析方法有很多,包括免疫分析,高效液相色谱(HPLC)和微生物检测等。然而,这些方法中的大多数通常需要复杂的仪器和复杂的提取程序来实现,既费时价格又昂贵。同时,重现性差也给大规模样品分析带来了负面影响。因此,构建一种高选择性、快速便捷检测实际水样中土霉素的传感器具有重要意义。荧光检测由于其高选择性、快速便捷性和更广泛的应用场景,是构建能在实际水样中检测土霉素的传感器的理想方法之一。
近年来,基于硅纳米点的荧光探针的荧光分析法受到了科研工作者的广泛欢迎。含硅纳米点制备简单、光学特性可调、稳定性高并且通常具有良好的生物相容性,已经在荧光分析领域受到了相当多的关注。然而大多数研究成果均是通过荧光猝灭来实现可视化检测抗生素,当遇到高浓度目标物时荧光微弱不易被肉眼观察,限制了其实际应用(Wang,J.;Cheng,R.;Wang,Y.;Sun,L.;Chen,L.;Dai,X.;Pan,J.;Pan, G.;Yan,Y.Surface-ImprintedFluorescence Microspheres as Ultrasensitive Sensor for Rapid and EffectiveDetection of Tetracycline in Real Biological Samples.Sensors Actuators,BChem.2018,263,533–542.),或者在检测体系中加入铕离子(Eu3+)构成比率荧光探针,通过建立内标,削弱其他因素的干扰(Li,X.;Ma,H.;Deng,M.;Iqbal, A.;Liu,X.;Li,B.;Liu,W.;Li,J.;Qin,W.Europium Functionalized Ratiometric Fluorescent TransducerSilicon Nanoparticles Based on FRET for the Highly Sensitive Detection ofTetracycline.J.Mater.Chem.C 2017,5(8),2149–2152.)。鲜有通过土霉素自身荧光增强构建比率荧光传感器的研究。
发明内容
本发明的目的在于提供一种用于可视化检测土霉素的比率荧光探针及制备方法,该探针为基于有机硅纳米点(OSiNDs)的比率荧光探针,所述有机硅纳米点荧光稳定性好,采用水热方法,制备方法简单,用时短。有机硅纳米点能够在较广pH 溶液中稳定存在,且在长时间紫外照射下释放稳定的荧光。本发明中比率荧光探针的检测机制包含两部分,有机硅纳米点(OSiNDs)基于自身的荧光猝灭机制用于检测四环素类抗生素,通过探针与土霉素(OTC)作用改变并增强土霉素自身荧光将土霉素与其他四环素类区分开来,能够通过比率荧光方法应用于OTC的痕量检测。本发明中比率荧光探针无需后处理加入昂贵的稀土金属或其他荧光材料作为参比,过程简单,条件温和、耗时短。
本发明提供一种新型有机硅纳米点比率荧光探针检测水溶液中土霉素含量的方法,可采用下述步骤:
(1)将4-硼酸三苯胺和3-氨丙基三乙氧基硅烷溶解在水中,超声分散后将混合物转移到内衬聚四氟乙烯的高压釜中,将其密封并在140~160℃的温度下保持2~4 h。
(2)等反应结束并冷却至室温后,将所得溶液进行透析,并通过旋转蒸发器浓缩收集。最终样品通过冷冻干燥获得有机硅纳米点(OSiNDs),并溶于水中配置成5 mg/mL储备液,在4℃下保存以备使用。取OSiNDs储备液加入到0.01M的PBS 缓冲液,制备成探针溶液。
(3)在测定不同的土霉素摩尔浓度下,通过获取探针溶液的荧光发射光谱(激发波长365nm),记录样品加入后在448nm和517nm处的荧光强度,计算荧光强度比值I517/I448,以该比值作为纵坐标,土霉素浓度为横坐标做工作曲线,得到对应方程式:F517/F448=-0.06378(C土霉素/μM)+2.7398(R2=0.9872)。
其中,步骤(1)所述4-硼酸三苯胺与3-氨丙基三乙氧基硅烷质量比优选1: 40~100,更优选1:40-60。
其中,步骤(2)中透析袋截留分子量为100~200Da,收集浓缩溶液为透析袋外溶液;最终配置的探针溶液中,所述OSiNDs质量浓度为0.4~0.6mg/mL,以0.5 mg/mL为最佳,此时既能满足OSiNDs自身荧光猝灭的可识别度又能保证土霉素自身荧光增强在一定范围,达到合适的比率荧光效果。
其中,步骤(3)中,优选的,土霉素自身在碱性环境下自身稳定性更差0.01M PBS缓冲液的pH值为6.5。检测土霉素的线性范围为1~35μM,检测限为0.085μM。
本发明所述有机硅纳米点(OSiNDs)探针基于自身的荧光猝灭机制用于检测四环素类抗生素,通过探针与土霉素(OTC)作用改变并加强土霉素自身荧光将土霉素与其他四环素类区分开来,猝灭波长在约448nm,荧光增强波长在约517nm,对OTC具有比率荧光特征。
与现有技术相比,本发明的有益效果:
(1)本发明提供了一种新型OSiNDs比率荧光探针对OTC进行比率荧光检测的应用,过程简单、易操作,条件温和、耗时短。基于所合成的OSiNDs比率荧光探针,表面含有羧基和羟基官能团能螯合OTC,导致其在448nm左右的荧光发射峰(F448) 猝灭;同时因为螯合作用导致OTC的构象由弱荧光的扭曲型转变为强荧光的扩展型,使得在517nm左右的荧光发射峰(F517)增强。说明OSiNDs比率荧光探针能够采用比率荧光方法即荧光强度比值(F448/F517)的变化可以实现对OTC的比率荧光检测。常见的金属离子及生物大分子对OSiNDs比率荧光探针检测OTC没有影响,说明该探针可实现对OTC的特异性识别与检测。
(2)与单一型的荧光检测方法相比,本发明利用比率型的荧光分析方法,将两个波长处的荧光强度比值作为响应信号,其不受光源强度和仪器灵敏度的影响,提高了检测的灵敏度和特异性。
附图说明
图1是OSiNDs、土霉素及OSiNDs/土霉素复合溶液的荧光发射光谱曲线;
图2是在不同pH值和和孵育时间下对应的荧光强度比值(F517/F448);
图3是探针对不同浓度的土霉素的荧光响应图与荧光强度比值(F448/F517)和土霉素浓度线性图谱;左图中,土霉素用量从0-35μM逐渐增大,内插图分别是OSiNDs 探针溶液和添加了土霉素后的OSiNDs探针溶液的荧光图片;
图4是OSiNDs比率荧光探针加入各种相同浓度四环素类抗生素荧光光谱图及对应的荧光强度比值(F517/F448);图中,*和**分别表示具有显著差异。
图5是在与线性检测相同的探针浓度和溶剂环境下,荧光探针对不同离子和常见生物大分子的荧光响应和分别与300μM离子和生物大分子共存情况下的荧光强度。其它物质的浓度为300μM;
图6半定量检测OTC的荧光检测图。
具体实施方式:
下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实例1
本发明提供了一种新型有机硅纳米点OSiNDs作为比率荧光探针对OTC进行比率荧光检测的应用,包括以下步骤:
(1)将0.02g 4-硼酸三苯胺和1mL3-氨丙基三乙氧基硅烷溶解在4mL水中,室温下超声10~20min使分散均匀,再将混合物转移到10mL内衬聚四氟乙烯的高压釜中,将其密封并在160℃的温度下保持2h。
(2)等反应结束并冷却至室温后,将所得溶液进行透析用100Da透析袋进行纯化分离,收集透析袋渗出液并通过旋转蒸发器浓缩样品,最终将浓缩样品通过冷冻干燥得到OSiNDs。将OSiNDs溶于水中配置成5mg/mL储备液,在4℃下保存以备使用。在本发明中,所述水优选为超纯水。
(3)比率型荧光探针的构建:取OSiNDs储备液加入到0.01M的PBS缓冲液,制备成探针溶液。在365nm紫外光照射下,0.5mg/mL OSiNDs探针溶液自身在448 nm左右有荧光,当向其中加入35μM土霉素时,原本探针溶液在448nm左右的蓝色荧光被淬灭,在517nm左右出现新的绿色荧光发射峰,而35μL土霉素溶液荧光非常微弱(如图1所示)。原本探针溶液在448nm处的发射峰峰强和加入35μL土霉素后517nm处发射峰峰强比例合适,蓝色至绿色的颜色变化可被肉眼观察,确定了探针溶液的浓度与土霉素浓度的大致范围。
实例2
将OSiNDs探针储备液用不同pH的1xPBS缓冲液稀释10倍,使其质量浓度为0.5mg/mL,再加入30μM土霉素测荧光光谱。图2是在不同pH值和和孵育时间下对应的荧光强度比值(F517/F448)的关系。在碱性条件下,荧光强度比值(F517/F448)更大,探针的灵敏度更高,但是更加不稳定,随着时间的增加,荧光强度比值(F517/F448) 逐渐降低。pH=6.5条件温和,荧光强度比值(F517/F448)稳定,且检测土霉素OTC无需孵育时间。
实例3
1xPBS缓冲液(pH为6.5)中加入所述OSiNDs储存液,得到OSiNDs比率荧光探针(其中OSiNDs浓度为0.5mg/mL)。然后取多组OSiNDs比率荧光探针溶液于石英比色皿中,分别加入10μL浓度不同的OTC,得到OTC浓度分别为0、1、2、 5、7、10、15、20、25、30、35μM的待测液体,在室温条件下进行记录荧光光谱 (如图3A所示,激发波长为365nm)。内插图分别是OSiNDs探针溶液和添加了 OTC后的OSiNDs探针溶液的荧光图片。随着OTC用量从0-35μM逐渐增大,以 448nm和517nm处荧光强度比值(F448/F517)为纵坐标,OTC的物质的量浓度(μM)为横坐标建立所述OSiNDs比率荧光探针对OTC检测的线性曲线(如图3B所示)。所述线曲线具体为F517/F448=-0.06378(C土霉素/μM)+2.7398。448nm和517nm处荧光强度比值(F448/F517)对于土霉素的物质的量浓度(C)的线性响应在1~35μM (R2=0.9872)之间,检测限为0.085μM。
实例3荧光比率探针的选择性
分别向多组OSiNDs比率型荧光探针的水溶液中加入相同浓度的四环素类抗生素(30μM):四环素(TC)、强力霉素(DOC)、金霉素(CTC)、二甲胺四环素(MOC),在与OTC相同的检测条件下进行荧光测定(如图4A所示)。结果表明,在最佳优化条件下,四环素类抗生素都能使OSiNDs比率型探针溶液在448nm左右的荧光猝灭,说明OSiNDs可检测四环素类抗生素。同时只有OTC能使OSiNDs比率型探针在517nm左右的荧光明显增强,且OTC在517nm和448nm处荧光强度比值 (F517/F448)显著地比其他四环素类抗生素高(如图4B),说明OSiNDs比率型荧光探针能区分OTC与其他四环素类抗生素。
为了证明OSiNDs比率型荧光探针能定性分析四环素类抗生素且对于OTC识别检测具有特异性,先选取一些比较常见的金属离子、抗生素和生物大分子进行干扰性测定。分别向多组比率型荧光探针溶液中加入相同体积的30μM干扰物质:K+, Na+,Ca2+,Mg2+,Co2+,Fe3 +,Al3+,谷胱甘肽(GSH),牛血清蛋白(BSA),抗坏血酸(Vc),组氨酸(His),葡萄糖(Glu),赖氨酸(Lys),在与OTC相同的检测条件下进行荧光测定。实验结果表明,除去OTC之外的其它干扰物质都不会引起 OSiNDs比率型荧光探针荧光强度比的变化,说明比率型荧光探针能够实现对OTC 的选择性识别(如图5A)。先向多组OSiNDs比率型荧光探针溶液中加入相同体积的300μM干扰物质:K+,Na+,Ca2+,Mg2+,Co2+,Fe3+,Al3+,GSH,BSA,Vc, His,Glu,Lys,再向每组中添加30μM OTC并进行荧光测定(如图5B)。实验结果表明,这些干扰物质即使浓度比OTC高出10倍,对OSiNDs比率型荧光探针特异性检测OTC干扰性很微弱。
实例4
制作试纸:将聚丙烯材料的纸片通过打孔器制成一张纸的圆纸片,然后浸入在浓度为0.5mg/mL的OSiNDs中,然后干燥得到检测OTC的荧光试纸。
取OTC母液浓度大约为1mM,再取1mL的OTC,稀释至10mL,此时浓度为0.1M。然后分别取0.5,1.5,2.5和3.5mL的OTC溶液定容至10mL。此时OTC 浓度依次为5,15,25,35μM。然后取8μL不同浓度的OTC溶液,滴加在试纸上面,在365nm的紫外灯下面拍照,可获得半定量检测OTC的荧光检测图。如图6。图中,A组和B组为对照组,分别是在日光灯下和365nm紫外灯下拍摄,各圆形试纸在日光灯下无颜色,在365nm紫外灯均显示蓝色荧光;C组为测试组,滴加OTC 溶液后,蓝色荧光猝灭,绿色荧光增强,随OTC溶液浓度升高,由蓝色变绿色越明显。
Claims (9)
1.一种基于自身荧光增强的土霉素有机硅纳米点比率荧光探针的制备方法,其特征在于,将4-硼酸三苯胺和3-氨丙基三乙氧基硅烷溶解在水中,超声分散后将混合物转移到内衬聚四氟乙烯的高压釜中,将其密封并在 140~160 oC 的温度下保持 2~4 h进行水热反应;所述4-硼酸三苯胺与3-氨丙基三乙氧基硅烷质量比1:40~100。
2. 如权利要求1所述制备方法,在所述水热反应之后,进行后处理纯化,所述后处理纯化是指用100~200 Da透析袋进行透析,将渗透液通过旋转蒸发器浓缩收集,并冷冻干燥得到。
3.由权利要求1或2所述制备方法得到的基于自身荧光增强的土霉素比率荧光探针。
4.权利要求3所述比率荧光探针在土霉素检测中的应用,所述应用不包括以有生命的人体或动物体为直接实施对象,用于诊断病因或治疗疾病的用途。
5.如权利要求4所述的应用,其特征在于,采用下述方法步骤:将荧光探针溶于水中,得到比率荧光探针储备液。
6. 如权利要求5所述的应用,其特征在于,取探针储备液加入到PBS缓冲液中,得到浓度为0.4~0.6 mg/mL的探针溶液,随后加入土霉素溶液,进行土霉素检测。
7.如权利要求6所述的应用,其特征在于,所述PBS缓冲液pH = 6.5。
8. 如权利要求5所述的应用,其特征在于,取探针储备液加入到PBS缓冲液中,得到浓度为0.4~0.6 mg/mL的探针溶液,取试纸浸入探针溶液,再干燥得到测试用荧光试纸,然后进行土霉素检测。
9.如权利要求8所述的应用,其特征在于,所述PBS缓冲液pH = 6.5。
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CN113429961B (zh) * | 2021-07-06 | 2023-08-25 | 西北农林科技大学 | 一种荧光探针、增强型比率荧光传感器、方法及应用 |
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