CN104198740A - 一种对葡萄糖和胆固醇同步检测的纳米生物传感器 - Google Patents
一种对葡萄糖和胆固醇同步检测的纳米生物传感器 Download PDFInfo
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Abstract
本发明提供一种纳米生物传感器,由已标记碳量子点的伴刀豆蛋白和胆固醇传感器通过自组装而成,所述的胆固醇传感器是由环糊精修饰的纳米金与罗丹明6G共孵育后除去多余反应物而得。并进一步提供了这种纳米生物传感器的制备方法以及应用。本发明的纳米生物传感器是基于荧光共振能量转移理论构建而成,可以同时检测葡萄糖和胆固醇,并且在检测过程中不会出现相互干扰,是一种新型、简便、低成本、快速、高灵敏度的双重物质定量检测传感器。
Description
技术领域
本发明属于生化分析领域,更具体地,涉及一种对葡萄糖和胆固醇同步检测的纳米生物传感器及其制备方法和应用。
背景技术
FRET即荧光共振能量转移是较早发展起来的一门技术,随着绿色荧光蛋白应用技术的发展,FRET已经成为检测活体中生物大分子纳米级距离和纳米级距离变化的有力工具,在生物大分子相互作用分析、细胞生理研究、免疫分析等方面有着广泛的应用。荧光共振能量转移是距离很近的两个荧光分子间产生的一种能量转移现象。当供体荧光分子的发射光谱与受体荧光分子的吸收光谱重叠,并且两个分子的距离在10 nm范围以内时,就会发生一种非放射性的能量转移,即FRET现象,使得供体的荧光强度比它单独存在时要低的多(荧光猝灭),而受体发射的荧光却大大增强(敏化荧光)。基于FRET理论构建的纳米生物传感器,在本领域的研究中一直占据着举足轻重的地位。
葡萄糖是最显著的能量源,在人体代谢过程中,血液中的葡萄糖的浓度被认为是对人体健康状况医疗诊断中的一项重要指标。截至目前,血糖测定主要是基于荧光和电化学方法。电化学方法是简单的和快速的,并已被广泛用于在葡萄糖传感器设计中,但该信号容易受血清中或者尿液中的还原性基质如抗坏血酸和尿素的影响,在实际测量中仍存在误差。作为一项高灵敏度和无损坏性的技术,荧光已被广泛应用于各种原理葡萄糖传感器的设计。虽然目前检测葡萄糖的报道层出不穷,但是基于复杂生物样品基质如血清里的生物分子(葡萄糖)的检测,仍有探索新技术的意义。迄今为止,现有技术中基于FRET原理的葡萄糖传感器尚不多见。
胆固醇是动物细胞膜的一个重要组成部分和合成不同生物分子如胆酸类、类固醇激素、维他命D的主要前体。但血清中过量的胆固醇会在血管通道中形成斑块,从而阻碍血液的循环和导致心血管疾病。现有技术中用于胆固醇检测的方法主要有荧光分析、电化学分析、分子印迹技术等,但是这些检测方法中的选择性大多依赖于对胆固醇特异性识别的酶或抗体,并具有损坏性和成本相当昂贵。因此,针对血清或者食物样品,设计出简单、高选择性、低成本、快速和高灵敏的胆固醇方法仍然极具临床意义。
构建低成本、多功能的生物传感检测技术以解决日常生活及临床诊断中出现的问题是当前的热点。由于糖尿病、心脑血管疾病这两类疾病的居高不下,使得开发葡萄糖和胆固醇同步检测的、低成本、快速和高灵敏的生物传感检测技术尤为重要。因此,本发明公开一种基于FRET原理的纳米生物传感器,用于血清中的葡萄糖和胆固醇的同步检测。
发明内容
本发明的目的在于提供一种用于同步检测血清样品中葡萄糖和胆固醇的简易、低成本、高灵敏度的荧光纳米生物传感器。
为了实现这一发明目的,本发明首先提供一种纳米生物传感器,由已标记碳量子点的伴刀豆蛋白和胆固醇传感器通过自组装构建而成,
所述的胆固醇的传感器是由环糊精修饰的纳米金与罗丹明6G共孵育后除去多余反应物而得。
所述的已标记碳量子点的伴刀豆蛋白是通过碳量子点表面的大量羧基与伴刀豆球蛋白上的氨基形成酰胺键连接而成。
所述的环糊精修饰的纳米金呈酒红色。
本发明更进一步提供一种上述的纳米生物传感器的制备方法,包括以下步骤
S1. 已标记碳量子点的伴刀豆蛋白的合成:所述的碳量子点是表面带大量羧基,通过将伴刀豆球蛋白的氨基进行活化,碳量子点以酰胺键的方式标记到伴刀豆球蛋白上;
S2. 环糊精修饰的纳米金的合成:所述的环糊精修饰的纳米金,通过氯金酸盐与巯基-β-环糊精混合溶液在激烈磁性搅拌中加入氢氧化钠一步法合成;
S3. 胆固醇传感器的合成:将步骤S2所得的环糊精修饰的纳米金与罗丹明6G混合,在摇床上共孵育;
S4 将步骤S1所得的已标记碳量子点的伴刀豆蛋白与步骤S3所得的胆固醇传感器在摇床上共孵育,即得。
步骤S3的共孵育的时间为10~20分钟;步骤S4所述的共孵育的时间为30~60分钟。
步骤S4所述的已标记碳量子点的伴刀豆蛋白与胆固醇传感器的重量比约为1~5:1。
步骤S3所述的环糊精修饰的纳米金与罗丹明6G的重量比约为1:1000~2000。
为了进一步说明本发明,发明人对本发明进行如下解释,但是不能理解为对本发明的限定。
本发明提供的是一种基于FRET荧光共振能量转移理论构建的生物传感器,是由碳量子点标记的伴刀豆球蛋白ConA与罗丹明6G共孵育后的环糊精修饰的纳米金自组装形成。巯基-β-环糊精修饰的纳米金作为所构建的生物传感器的荧光猝灭基团即能量受体,碳量子点标记的ConA和罗丹明6G作为能量的供体。罗丹明6G通过疏水作用进入环糊精的疏水空腔,而合成的碳量子点标记的ConA通过ConA上的糖的特异结合位点与环糊精上的D-吡喃葡萄糖单元自组装,简易高效得到目标纳米传感器。由于纳米金与ConA上的以及环糊精疏水空腔内的荧光基团距离很小,形成有效的荧光共振能量转移,使得碳量子点和罗丹明6G的荧光同时被纳米金猝灭。而当加入含葡萄糖的样本时,由于葡萄糖与ConA的结合力远大于环糊精与ConA的结合力,从而将纳米金从标记碳量子点的ConA上取代下来,伴随着纳米金与ConA距离的增加,FRET现象消失,碳量子点的荧光得以恢复。样本中葡萄糖含量与荧光的恢复情况呈线性关系,通过测定荧光发射强度从而可定量检测出样本的葡萄糖含量。而当加入含胆固醇的样本时,由于胆固醇与环糊精的疏水空腔的结合能力远强于罗丹明6G,从而取代出罗丹明6G,伴随着纳米金与罗丹明6G距离增加,FRET现象消失,使得其荧光恢复,荧光恢复情况与待测样本中的胆固醇含量有线性关系,通过测定荧光发射强度可定量检测出样本的胆固醇含量。由于选取的碳量子点与罗丹明6G这两种荧光染料具有不同波长的荧光发射,检测中不会发生相互干扰,从而实现多功能纳米生物传感器的构建。
本发明所述的纳米生物传感器的构建是将环糊精修饰的纳米金先与罗丹明6G共孵育后除去多余产物先构成检测胆固醇的传感器,再和已标记碳量子点的伴刀豆蛋白ConA通过自组装的简单过程来实现。
本发明中所述的碳量子点标记的伴刀豆球蛋白ConA,是碳量子点这种国际上公认的新型荧光染料在生物上的创新应用,碳量子点具有高量子产率、低成本、低毒性、低光漂白、高生物相容性等诸多优点,本发明公开的技术首次将碳量子点标记上伴刀豆球蛋白加以应用到生物传感器中,以提高检测的灵敏度及生物传感平台的稳定性。
所述的碳量子点是表面带大量羧基,通过将伴刀豆球蛋白ConA上的氨基进行活化,碳量子点以酰胺键的方式标记到ConA上,标记过程确保碳量子点的荧光特性及伴刀豆球蛋白ConA的蛋白特性不发生明显改变。
所述的ConA的标记以及涉及到具体样本中葡萄糖的检测环节需要用到含有Ca2+和Mn2+的pH为7.4的PBS缓冲溶液,ConA上的糖识别位点需要Ca2+和Mn2+的活化,并且pH为7.4的PBS溶液是生物医学上常用的缓冲溶液。
所述的环糊精修饰的纳米金采用新型的有别于传统柠檬酸钠法的一步合成方法进行制备,产物均一,分散性好,呈酒红色。
所述的纳米生物传感器核心步骤是碳量子点对ConA的成功修饰,以及纳米金表面充分修饰环糊精并且产物分散性很好。
本发明涉及的碳量子点、罗丹明6G染料的荧光激发和发射性质不同,检测所使用的荧光发射波形不发生重叠互相干扰,从而借助荧光分析手段可以实现简易、低成本、快速、高灵敏度的双重物质定量检测。
本发明中构建出新型双重检测的纳米生物传感器能同时检测血清中的葡萄糖与胆固醇,实现生物医学检测领域上的突破。所设计的传感器的构建过程只需45分钟,检测时间只需30分钟,成功研发后并无技术上的难度,从而对于发展中及不发达国家地区的推广应用具有很大的应用研发前景。
附图说明
图1 基于FRET理论构建的新型纳米生物传感器的原理示意图。
具体实施方式
下面结合附图和具体实施例进一步详细说明本发明。除非特别说明,本发明采用的试剂、设备和方法为本技术领域常规市购的试剂、设备和常规使用的方法。
实施例1 一种纳米生物传感器的制备,以及对葡萄糖和胆固醇的检测
(1)材料准备
Mili-Q超纯水,伴刀豆球蛋白ConA,1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC),N-羟基琥珀酰亚胺 (NHS),氯金酸(HAuCl4·3H2O),巯基-β-环糊精(SH-β-CDs),C量子点(CQDs,参考已有文献自行制备),柠檬酸钠,PBS缓冲液(10 mM, pH=7.4,含Ca2+和Mn2+各1 mM),六水合氯化镁,二水合氯化钙,罗丹明6G,葡萄糖,胆固醇,无水乙醇,牛/人血清,截留分子量为1000,3000、14000的透析袋,96孔酶标板,2、5、15、50mL离心管,锡纸。
(2)环糊精修饰的纳米金的制备:
室温下,首先取47.0 mL的Mili-Q超纯水,取10.0 mg的SH-β-CDs和2.0 mL 1.0%(w/w)HAuCl4加入100 mL圆底烧瓶中,超声溶解混合均匀,然后在激烈搅拌中将1.0 mL 1.0 M的NaOH溶液快速加入其中使得pH达到11,避光搅拌16小时后反应质得近酒红色的纳米金溶液,用截留分子量3500的透析袋纯化处理,做透射电镜、紫外吸收光谱(紫外最大吸收峰在520 nm)、粒径、和表面电位的各项性质检测后,待用。
(3)碳量子点的制备:
参考国际上已经发表的制备方法,一步简易合成制备出表面富含羧基的碳量子点水溶液,完成红外,高分辨电镜的各项测试后待用。
(4)碳量子点标记伴刀豆球蛋白ConA的制备:
室温下,ConA溶解在PBS缓冲溶液(10 mM,pH7.4)含CaCl2(1.0 mM)和MnCl2(1.0 mM)浓度为1.0 mg mL-1,存储在4℃冰箱中;取制备待用的碳量子点溶液1.00 mL,适量PBS缓冲液(10 mM,pH7.4)溶解0.5 mg EDC和0.25 mg NHS,在室温下摇床中共孵育30分钟;随后活化后的富含羧基的碳量子点溶液和0.5 mL的活化过后富含活化氨基的ConA溶液混合,于摇床中共孵育2小时,并在4℃冰箱中存储过夜;之后用YM-30K的超滤离心管,在12000 rpm下离心5分钟,重复纯化几次,得到碳量子点标记的ConA待用。
(5)罗丹明6G-环糊精修饰的纳米金的处理:
通过将0.01 mM罗丹明6G溶液与制备好的环糊精修饰的纳米金溶液做荧光猝灭实验,在得出的猝灭曲线上找到其最佳组装比例,最佳反应时间15分钟;将0.01 mM罗丹明6G水溶液与制备的纳米金按比例混合混合,于摇床共孵育15分钟后,用截留分子量3500的透析袋进行纯化透析出多余的罗丹明6G,待用。
(6)这种新型生物传感器的组装:
将制备好的罗丹明6G-环糊精修饰的纳米金溶液与碳量子点标记的ConA溶液做关于碳量子点的荧光猝灭实验,通过获得的荧光猝灭曲线找到两者组装的最佳比例和最佳猝灭时间。将制备好的罗丹明6G-环糊精修饰的纳米金溶液与碳量子点修饰的ConA溶液按2 : 1比例进行混匀并于室温下,在摇床上共孵育45分钟后,待用。
(7)胆固醇的检测:
先将胆固醇标准品配置成不同浓度待测的乙醇溶液作为待测样本,分别取800 μL的(6)中溶液,向其中分别加入100 μL不同浓度的待测样本,然后都用超纯水稀释至1 mL,于摇床上室温下,摇匀反应30分钟,然后各样本在设置为激发波长525 nm,发射波长565 nm的多功能酶标仪中进行荧光值的测定,找到胆固醇含量与体系荧光值的标准曲线,从而进行实际样本中的胆固醇含量的精确检测。
(8)葡萄糖的检测:
先将葡萄糖标准品配置成不同浓度的待测的水溶液作为待测样本,分别取800 μL的(6)中的溶液,向其中分别加入100 μL不同浓度的待测样本,然后都用超纯水稀释至1 mL,于摇床上室温下,摇匀反应45分钟,然后各样本在设置为激发波长370 nm,发射波长480 nm的多功能酶标仪中进行荧光值的测定,找到样本中的葡萄糖含量与体系荧光值的标准曲线,从而进行实际样本中的葡萄糖含量的检测。
(9)血清中的胆固醇和葡萄糖的检测:
取100 μL胎牛/人血清,适量乙醇,适量PBS缓冲溶液,适量的葡萄糖、胆固醇,600 μL组装完成的传感器溶液,稀释至1 mL配置成不同浓度的葡萄糖、胆固醇溶液样本,室温下于摇床上混匀,反应45分钟后,分别用设置为激发波长525 nm发射波长565 nm,和激发波长370 nm发射波长480 nm的多功能酶标仪进行荧光值测量,分别做出相关含量与荧光值的线性曲线图,从而获得适用于普遍样本的葡萄糖、胆固醇的定量测量。用本发明所述纳米生物传感器与国标法(WS/T350-2011血清葡萄糖测定参考方法;WS/T120-1999血清总胆固醇的酶法测定)测定同一血清样本中的胆固醇和葡萄糖,结果显示本发明所述的纳米生物传感器达到与国标法相同的准确度和精密度。
Claims (7)
1.一种纳米生物传感器,其特征在于,由已标记碳量子点的伴刀豆蛋白和胆固醇传感器通过自组装构建而成,
所述的胆固醇传感器是由环糊精修饰的纳米金与罗丹明6G共孵育后除去多余反应物而得。
2.根据权利要求1所述的纳米生物传感器,其特征在于,所述的已标记碳量子点的伴刀豆蛋白是通过碳量子点表面的大量羧基与伴刀豆球蛋白上的氨基形成酰胺键连接而成。
3.一种权利要求1所述的纳米生物传感器的制备方法,其特征在于,包括以下步骤:
S1. 已标记碳量子点的伴刀豆蛋白的合成:所述的碳量子点是表面带大量羧基,通过将伴刀豆球蛋白的氨基进行活化,碳量子点以酰胺键的方式标记到伴刀豆球蛋白上;
S2. 环糊精修饰的纳米金的合成:所述的环糊精修饰的纳米金,通过氯金酸盐与巯基-β-环糊精混合溶液在激烈搅拌中加入氢氧化钠一步法合成;
S3. 胆固醇传感器的合成:将步骤S2所得的环糊精修饰的纳米金与罗丹明6G混合,在摇床上共孵育,
S4 将步骤S1所得的已标记碳量子点的伴刀豆蛋白与步骤S3所得的胆固醇传感器在摇床上共孵育,即得。
4.根据权利要求3所述的制备方法,其特征在于,步骤S3的共孵育时间为10~20分钟;步骤S4所述的共孵育时间为30~60分钟。
5.根据权利要求3所述的制备方法,其特征在于,步骤S4所述的已标记碳量子点的伴刀豆蛋白与胆固醇传感器的重量比约为1~5 : 1。
6.根据权利要求3所述的制备方法,其特征在于,步骤S3所述的环糊精修饰的纳米金与罗丹明6G的重量比约为1 : 1000~2000。
7.一种根据权利要求1所述的纳米生物传感器在检测葡萄糖和/或胆固醇含量中的应用。
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