CN113759108A - 一种基于血糖仪的生物传感器用于测定氯霉素含量的方法 - Google Patents
一种基于血糖仪的生物传感器用于测定氯霉素含量的方法 Download PDFInfo
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Abstract
本发明公开了一种基于血糖仪的生物传感器用于氯霉素含量测定的方法,通过生物素‑亲和素相互作用将特定互补链DNA序列连接到磁性颗粒表面,通过氯霉素与适配体特异性作用释放特定DNA序列,引发修饰有金纳米颗粒/淀粉酶的发夹结构循环自组装过程,并利用磁分离收集,与血糖仪相结合实现氯霉素含量的测定。具体步骤为:步骤S1:AuNPs的合成;步骤S2:氨基化磁性颗粒(MNPs)的合成;步骤S3:MNPs的修饰(MNPs/tDNA/Aptamer);步骤S4:DNA发夹预处理(H1和H2);步骤S5:AuNPs标记NDA和葡糖淀粉酶(AuNPs/H1/E,AuNPs/H2/E);步骤S6:基于血糖仪测定氯霉素;步骤S7:基于血糖仪测定氯霉素含量的选择性考察。本发明解决了使用血糖仪测定氯霉素含量的问题,实现了公众对于氯霉素的检测需求。
Description
技术领域
本发明涉及分子生物学技术领域,具体涉及一种基于血糖仪的生物传感器用于测定氯霉素含量的方法。
背景技术
氯霉素(2,2-二氯-N-[1,3-二羟基-1-(4硝基苯基)丙-2-基]乙酰胺)又名氯胺苯醇,是一种广谱抗生素药物。可有效抑制革兰氏阴性细菌、革兰氏阳性细菌等多种病原菌。具有非常明显的效果和极强的杀菌能力。但这种抗生素的过度使用也会损害人体健康,氯霉素经食物和药物吸收到人体内,很容易在肝脏中积累,从而对人类产生许多严重的副作用,如白血病、灰婴综合征和再生障碍性贫血。氯霉素对人类健康的危害已引起国际组织和多个国家或地区的高度重视,中国、美国、欧盟、韩国、日本等都已对该药物在食品中的残留做出了禁用和不得检出的规定。我国现行有效标准《动物源性食品中兽药最高残留限量》(农业部第235号公告)中也明确规定,氯霉素在动物源性食品中不得检出。欧洲国家为了食品安全控制,欧盟已经将氯霉素的“最低要求检出限值”设定为0.3×106克/千克,用于检测其在食品中的残留。因此,开发一种简单快速、选择性好的便携式氯霉素检测方法是必要且极其重要的。
目前,已经报道的氯霉素测定方法主要有:气相色谱-质谱法、高效液相色谱-质谱法、光电化学分析法、化学发光免疫分析法、酶联免疫吸附测定法、高效毛细管电泳、化学发光、包括荧光生物传感器、比色分析等。但大多数仪器分析方法通常表现出一些局限性,例如较差的灵敏度、准确性和稳定性,设备昂贵、仪器操作专业度高,前处理过程复杂耗时,生产的成本较高,难以推广到市场中进行现场检测。其中,基于抗原-抗体特异性识别反应建立起来的免疫分析方法,具有更高的特异性,但其原料抗体生产成本高、免疫活性易受离子强度、 pH等环境因素影响较大。荧光生物传感器因其简单、高特异性和高灵敏度的特性而日益受到关注。然而,大多数荧光生物传感器需要染料标记或修饰,操作复杂、标记效率低和成本昂贵。适配体通常是具有特定序列的短单链DNA或RNA分子,可用作特异性识别试剂,以高特异性和亲和力与目标物结合。由于适配体具有比抗体更容易修饰、成本更低和稳定性更好的优越性能,近年来基于适配体的传感器,逐渐开发并应用到抗生素的检测分析中。
发明内容
本发明所要解决的问题是:提供一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,解决了现有技术中存在的抗生素检测需要使用大型的仪器、进行复杂的样品前处理、无法实现现场实时检测等问题。
本发明为解决上述问题所提供的技术方案为:一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,所述方法包括以下步骤
步骤S1:AuNPs的合成
首先将所用到的玻璃器皿用王水浸泡,超纯水冲洗,干燥待用;然后得到将2mL1%(w/w, 34mM)的柠檬酸钠溶液加入到50ml煮沸的0.01%(w/w,0.24mM)HAuCl4溶液中;溶液由紫色逐渐变为酒红色;
步骤S2:氨基化磁性颗粒(MNPs)的合成
将[1,6-己二胺(6.5g)+无水醋酸钠(2g)+FeCl3.6H2O(1.0g)]加入到30mL乙二醇溶液中,搅拌得透明溶液,转移入高压釜,使用水和乙醇冲洗2-3次,烘干保存;
步骤S3:MNPs的修饰(MNPs/tDNA/Aptamer)
将100uL 100uM的生物素标记tDNA与100uL 100uM Aptamer混合,制得200uL 50uMtDNA/Aptamer,储存备用;
将2mg MNPs分散到2mL 10mM的PBS(pH 7.4)缓冲液中,超声15min;加入500uL25%戊二醛,室温条件下缓震2h,磁分离,使用PBS缓冲液洗三次;将上述MNPs再次超声分散到2mL 10mM PBS中,加入40uL 1.0mg/mL亲和素(Avidin)溶液中,室温条件下缓震12h,磁分离,PBS缓冲液洗三次,分散到1mL PBS缓冲液中;向上述溶液中加入100uL 50uM的tDNA/Aptamer,缓震12h,磁分离,使用2%的BSA溶液封闭,缓震6h后,磁分离洗涤,分散到1mL CAP结合缓冲液中备用;
步骤S4:DNA发夹预处理(H1和H2)
19uL 100uM H1(H2)+38uL 100mM Tris+26uL 1M NaCl+19uL H2O+88uL 100mMMgCl2,90℃处理5min后取出,置于37℃温育2h;
步骤S5:AuNPs标记NDA和葡糖淀粉酶(AuNPs/H1/E,AuNPs/H2/E)
取1mL 16mM的AuNPs,通过加入0.1M的Na2CO3溶液,调节pH到9.0,加入50uL 10uMDNA(H1/H2),缓震15min后,将混合溶液放置到4℃冰箱中,反应过夜;加入400uL 4.5mg/mL的葡糖淀粉酶,室温下反应6h;使用1%的BSA溶液封闭,缓震3h,离心(14000g),分散到1mL缓冲液中备用;
步骤S6:基于血糖仪对氯霉素进行测定
取30uL MNPs/tDNA/Aptamer放入离心管中,加入10uL待测氯霉素溶液,37℃温育1h;继续加入20uL AuNPs/H1/E和20uL AuNPs/H2/E,37℃温育3h,磁分离洗涤,将磁性颗粒复合物分散到50uL 5mg/mL淀粉溶液中,37℃温育1.5h,磁吸附,取5uL上清液,使用血糖仪进行测定;
步骤S7:基于血糖仪对氯霉素进行测定的选择性考察。
优选的,所述步骤S2中搅拌温度为50℃。
优选的,所述步骤S2中高压釜内反应温度为198℃,反应时间为6h。
优选的,所述步骤S2中烘干温度为50℃。
优选的,所述步骤S3中混合反应温度为37℃,反应时间为3h,储存温度为4℃。
优选的,所述步骤S7中选择性考察只需要将步骤“6”中氯霉素溶液分别换成TAP(甲砜霉素)、FF(氟苯尼考)、Kan(卡那霉素)、SS(硫酸链霉素)、GS(硫酸庆大霉素)进行检测,其他步骤不变。
与现有技术相比,本发明的优点是:
(1)检测设备简单、便携、易操作,检测时间短。
(2)实验所用原料易得,价钱便宜,可大规模工业生产。
(3)DNA适配体具有易制备、易储存的优点,在反应时,特异性识别能力强,生物相容性较好。
(4)目标物引发的DNA发夹自组装是一种循环进行的过程,具有优异的信号放大性能,可实现低含量氯霉素的高灵敏检测。
(5)合成的氨基化磁性颗粒(MNPs)能提供大的较大的比表面和更多的适配体结合位点。
(6)整个反应过程不产生任何有毒有害物质,本发明属于清洁生产。
本发明中使用到的,解决了使用便携式、易操作的血糖仪实现氯霉素含量测定的问题,因此本发明实现了公众对于待测氯霉素的检测需求。
附图说明
此处所说明的附图用来提供对本发明的进一步理解,构成本发明的一部分,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1是本发明用于氯霉素检测的工作原理图;
图2A是AuNPs的电镜图;
图2B是AuNPs、AuNPs/H1/E和AuNPs/H2/E的紫外吸收光谱图;
图3A是本发明用于氯霉素检测的可行性分析图;
图3B是血糖仪测量值随淀粉酶水解淀粉时间的变化图。
图3C是血糖仪测量值随淀粉浓度改变的变化图。
图4A是循环发夹自组装引发的信号放大对照图;
图4B是血糖仪测量信号重现性考察图。
图5A是血糖仪测量值与氯霉素浓度的相关性图;
图5B是基于血糖仪对氯霉素测定的选择性考察图。
具体实施方式
以下将配合附图及实施例来详细说明本发明的实施方式,借此对本发明如何应用技术手段来解决技术问题并达成技术功效的实现过程能充分理解并据以实施。
一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,具体按照以下步骤进行:
步骤S1:AuNPs的合成
首先将所用到的玻璃器皿用王水浸泡,超纯水冲洗,干燥待用。然后得到将2mL1%(w/w, 34mM)的柠檬酸钠溶液加入到50ml煮沸的0.01%(w/w,0.24mM)HAuCl4溶液中。溶液由紫色逐渐变为酒红色。
步骤S2:氨基化磁性颗粒(MNPs)的合成
将[1,6-己二胺(6.5g)+无水醋酸钠(2g)+FeCl3.6H2O(1.0g)]加入到30mL乙二醇溶液中, 50℃下搅拌得透明溶液,转移入高压釜,198℃反应6h,使用水和乙醇冲洗2-3次,50℃烘干保存。
步骤S3:MNPs的修饰(MNPs/tDNA/Aptamer)
将100uL 100uM的生物素标记tDNA与100uL 100uM Aptamer混合,37℃反应3h,制得 200uL 50uM tDNA/Aptamer,4℃储存备用。
将2mg MNPs分散到2mL 10mM的PBS(pH 7.4)缓冲液中,超声15min。加入500uL25%戊二醛,室温条件下缓震2h,磁分离,使用PBS缓冲液洗三次。将上述MNPs再次超声分散到2mL 10mM PBS中,加入40uL 1.0mg/mL亲和素(Avidin)溶液中,室温条件下缓震12h,磁分离,PBS缓冲液洗三次,分散到1mL PBS缓冲液中。向上述溶液中加入100uL 50uM的tDNA/Aptamer,缓震12h,磁分离,使用2%的BSA溶液封闭,缓震6h后,磁分离洗涤,分散到1mL CAP结合缓冲液中备用。
步骤S4:DNA发夹预处理(H1和H2)
19uL 100uM H1(H2)+38uL 100mM Tris+26uL 1M NaCl+19uL H2O+88uL 100mMMgCl2,90℃处理5min后取出,置于37℃温育2h。
步骤S5:AuNPs标记NDA和葡糖淀粉酶(AuNPs/H1/E,AuNPs/H2/E)
取1mL 16mM的AuNPs,通过加入0.1M的Na2CO3溶液,调节pH到9.0,加入50uL 10uMDNA(H1/H2),缓震15min后,将混合溶液放置到4℃冰箱中,反应过夜。加入400uL 4.5mg/mL的葡糖淀粉酶,室温下反应6h。使用1%的BSA溶液封闭,缓震3h,离心(14000g),分散到1mL缓冲液中备用。
步骤S6:基于血糖仪对氯霉素进行测定
取30uL MNPs/tDNA/Aptamer放入离心管中,加入10uL待测氯霉素溶液,37℃温育1h。继续加入20uL AuNPs/H1/E和20uL AuNPs/H2/E,37℃温育3h,磁分离洗涤,将磁性颗粒复合物分散到50uL 5mg/mL淀粉溶液中,37℃温育1.5h,磁吸附,取5uL上清液,使用血糖仪进行测定。
步骤S7:基于血糖仪对氯霉素进行测定的选择性考察
选择性考察只需要将步骤“6”中氯霉素溶液分别换成TAP(甲砜霉素)、FF(氟苯尼考)、 Kan(卡那霉素)、SS(硫酸链霉素)、GS(硫酸庆大霉素)进行检测,其他步骤不变。
图1为本发明用于氯霉素检测的工作原理图,如图中所示,本发明通过生物素-亲和素相互作用将与氯霉素适配体DNA序列杂交的互补cDNA序列修饰到氨基化磁性颗粒(MNPs) 表面,当氯霉素与适配体DNA特异性结合后,与发夹结构部分互补的cDNA链暴露,从而打开修饰有AuNPs/淀粉酶的DNA发夹结构H1和H2,引发H1和H2在磁性颗粒表面的发夹自组装过程,该过程中修饰到金颗粒表面的淀粉酶会不断组装到磁性颗粒表面。之后通过离心将其收集并分散到淀粉溶液中,基于淀粉酶水解淀粉产生葡萄糖的作用,使用血糖仪测定葡萄糖含量,即可实现对氯霉素含量的间接定量。制得的这种基于血糖仪的新型、高选择性的氯霉素含量测定方法,其检测灵敏度高、特异性强,且操作简单,价格低廉,避免使用大型仪器,且可进行现场检测。
图2A是AuNPs的电镜图;从图中可以看出合成的金纳米颗粒尺寸均一,具有良好的单分散性,表明金纳米颗粒制备成功。图2B是AuNPs、AuNPs/H1/E和AuNPs/H2/E的紫外吸收光谱图;对比AuNPs、AuNPs/H1/E和AuNPs/H2/E的紫外吸收曲线,发现金纳米颗粒在修饰H1/E或H2/E后,520nm的吸收峰基本保持不变,表明修饰过程并不会影响金纳米颗粒的分散性,且修饰后金纳米颗粒仍未发生团聚,保持稳定;对比AuNPs,AuNPs/H1/E和AuNPs/H2/E在260nm左右出现的吸收峰,表明H1/E或H2/E成功修饰到金纳米颗粒表面。
图3A是本发明用于氯霉素检测的可行性分析图;其中a为 MNPs/tDNA+AuNPs/H1/E+AuNPs/H2/E;b为MNPs/tDNA/Aptamer+ AuNPs/H1/E+AuNPs/H2/E;c为MNPs/tDNA+AuNPs/H1/E;d为MNPs/tDNA+AuNPs/H2/E; e为MNPs/tDNA/Aptamer+氯霉素+AuNPs/H1/E+AuNPs/H2/E;从图中a样品表示修饰磁性颗粒表面的tDNA未与Aptamer结合,当有AuNPs/H1/E+AuNPs/H2/E时,tDNA序列会引发 H1与H2的循环发夹自组装过程,使得金纳米颗粒表面结合的淀粉酶不断地修饰到磁性颗粒表面,经磁分离洗涤,磁性颗粒加入到淀粉溶液中,可水解淀粉产生葡萄糖,血糖仪显示信号较高。b样品表示修饰磁性颗粒表面的tDNA已经与Aptamer杂交结合,因此可引发H1与 H2的循环发夹自组装过程的tDNA序列未暴露,无法引发循环发夹自组装过程,金颗粒表面的淀粉酶无法结合到磁性颗粒表面,后续过程使用血糖仪检测信号非常低。c样品表示修饰磁性颗粒表面的tDNA未与Aptamer结合,当有AuNPs/H1/E时,H1与tDNA结合,金颗粒表面淀粉酶修饰到磁性颗粒表面,但因没有AuNPs/H2/E,无法进行循环自组装过程,因此后续血糖仪检测信号要远低于a样品。图中d样品表示修饰磁性颗粒表面的tDNA未与Aptamer结合,当有AuNPs/H2/E时,因tDNA无法打开H2发夹结构,无杂交过程,因此金颗粒表面淀粉酶无法结合到磁性颗粒表面,后续血糖仪检测信号非常低。图中样品e为在样品b的基础上加入氯霉素,此时,氯霉素与Aptamer特异性结合,磁性颗粒表面tDNA被释放出来,引发H1与H2的循环发夹自组装过程,金颗粒表面的淀粉酶循环组装到磁性颗粒表面,后续磁分离检测,血糖仪有明显信号。通过a、b、c、d、e五组样品对比发现,氯霉素与Aptamer结合引发的后续循环发夹自组装过程,后续可通过血糖仪检测的显著信号,因此表明本发明用于氯霉素检测是可行的。图3B是血糖仪测量值随淀粉酶水解淀粉时间的变化图;考察了淀粉酶水解淀粉反应的时间长短对测量结果的影响,结果表明最初血糖仪信号随着时间的增加而增大,1.5小时后信号增加缓慢。因此,选择1.5小时作为最佳时间。图3C是血糖仪测量值随淀粉浓度改变的变化图;考察了淀粉浓度对血糖仪测量值的影响,结果表明最初随淀粉浓度增加,血糖仪测量值逐渐增大,淀粉浓度到达5mg/mL后,血糖仪测量值随淀粉浓度增大缓慢。因此,选择5mg/mL为最佳淀粉浓度。
图4A是循环发夹自组装引发的信号放大对照图;其中a为MNPs/tDNA/Aptamer+AuNPs/H1/E+AuNPs/H2/E;b为MNPs/tDNA/Aptamer+氯霉素+AuNPs/H2/E;c为 MNPs/tDNA/Aptamer+氯霉素+AuNPs/H1/E;d为MNPs/tDNA/Aptamer+氯霉素+ AuNPs/H1/E+AuNPs/H2/E;通过图中c与d的信号对比,发现本发明中目标物氯霉素与Aptamer结合引发的循环发夹自组装过程,可有效的进行信号放大,提高了氯霉素检测灵敏度。图4B是血糖仪测量信号重现性考察图;其中1-6为六次平行测定实验,结果表明,六次平行测定实验,所得结果基本一致,表明本发明检测氯霉素含量具有良好的信号重现性。
图5A是血糖仪测量值与氯霉素浓度的相关性图;结果表明,随着氯霉素浓度上升,血糖仪检测信号显著增加。而且,血糖仪信号与5-2000pg/mL内的氯霉素浓度的对数呈现明显的正相关。此外,根据3σ规则估计检测限为0.16pg/mL,该结果表明本发明能够有效地灵敏检测待测物氯霉素。图5B是基于血糖仪对氯霉素测定的选择性考察图;其中TAP为甲砜霉素、FF为氟苯尼考、Kan为卡那霉素、SS为硫酸链霉素、GS为硫酸庆大霉素,CAP氯霉素。结果表明除了氯霉素(浓度为100pg/mL)以外,其他几种类似物(浓度均为1ng/mL),均不能产生明显信号,表明本发明对氯霉素检测具有良好的特异性,具有优异的抗干扰性能。
以上仅就本发明的最佳实施例作了说明,但不能理解为是对权利要求的限制。本发明不仅局限于以上实施例,其具体结构允许有变化。凡在本发明独立权利要求的保护范围内所作的各种变化均在本发明保护范围内。
Claims (6)
1.一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,其特征在于:所述方法包括以下步骤
步骤S1:AuNPs的合成
首先将所用到的玻璃器皿用王水浸泡,超纯水冲洗,干燥后使用;然后将2mL 1%(w/w,34mM)的柠檬酸钠溶液加入到50ml煮沸的0.01%(w/w,0.24mM)HAuCl4溶液中;溶液由紫色逐渐变为酒红色;继续煮沸2min,冷却装入深色玻璃瓶保持;
步骤S2:氨基化磁性颗粒(MNPs)的合成
将[1,6-己二胺(6.5g)+无水醋酸钠(2g)+FeCl3.6H2O(1.0g)]加入到30mL乙二醇溶液中,搅拌得透明溶液,将溶液转移入高压釜反应,冷却后使用水和乙醇冲洗2-3次,烘干保存;
步骤S3:MNPs的修饰(MNPs/tDNA/Aptamer)
将100uL 100uM的生物素标记tDNA与100uL 100uM Aptamer混合,制得200uL 50uMtDNA/Aptamer,储存备用;
将2mg MNPs分散到2mL 10mM的PBS(pH 7.4)缓冲液中,超声15min;加入500uL25%戊二醛,室温条件下缓震2h,磁分离,使用PBS缓冲液洗三次;将上述MNPs再次超声分散到2mL10mM PBS中,加入40uL 1.0mg/mL亲和素(Avidin)溶液中,室温条件下缓震12h,磁分离,PBS缓冲液洗三次,分散到1mL PBS缓冲液中;向上述溶液中加入100uL 50uM的tDNA/Aptamer,缓震12h,磁分离,使用2%的BSA溶液封闭,缓震6h后,磁分离洗涤,分散到1mL氯霉素结合缓冲液中备用;
步骤S4:DNA发夹预处理(H1和H2)
将19uL 100uM H1(H2)溶液、38uL 100mM Tris溶液、26uL 1M NaCl溶液、19uL水溶液和88uL 100mM MgCl2溶液混合,90℃处理5min后取出,置于37℃温育2h;
步骤S5:AuNPs标记NDA和葡糖淀粉酶(AuNPs/H1/E、AuNPs/H2/E)
取1mL 16mM的AuNPs,通过加入0.1M的Na2CO3溶液,调节pH到9.0,加入50uL 10uM DNA(H1/H2),缓震15min后,将混合溶液放置到4℃冰箱中,反应过夜;加入400uL 4.5mg/mL的葡糖淀粉酶,室温下反应6h;使用1%的BSA溶液封闭,缓震3h,离心(14000g),分散到1mL缓冲液中备用;
步骤S6:基于血糖仪测定氯霉素含量
取30uL MNPs/tDNA/Aptamer放入离心管中,加入10uL待测氯霉素溶液,37℃温育1h;继续加入20uL AuNPs/H1/E和20uL AuNPs/H2/E,37℃温育3h,磁分离洗涤,将磁性颗粒复合物分散到50uL 5mg/mL淀粉溶液中,37℃温育1.5h,磁吸附,取5uL上清液,使用血糖仪进行测定;
步骤S7:基于血糖仪对氯霉素进行测定的选择性考察。
2.根据权利要求1所述的一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,其特征在于:所述步骤S2中搅拌温度为50℃。
3.根据权利要求1所述的一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,其特征在于:所述步骤S2中高压釜内反应温度为198℃,反应时间为6h。
4.根据权利要求1所述的一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,其特征在于:所述步骤S2中烘干温度为50℃。
5.根据权利要求1所述的一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,其特征在于:所述步骤S3中混合反应温度为37℃,反应时间为3h,储存温度为4℃。
6.根据权利要求1所述的一种基于血糖仪的生物传感器用于测定氯霉素含量的方法,其特征在于:所述步骤S7中选择性考察只需要将步骤“6”中氯霉素溶液分别换成TAP(甲砜霉素)、FF(氟苯尼考)、Kan(卡那霉素)、SS(硫酸链霉素)、GS(硫酸庆大霉素)进行检测,其他步骤不变。
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