CN115201296B - 一种比率型电化学适配体传感器的制备方法 - Google Patents
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Abstract
本发明属于生物传感器技术领域，公开了一种可检测牛奶中卡那霉素（KAN）的比率型电化学适配体传感器制备及使用方法。本发明制备的比率型电化学适配体传感器基于二茂铁（Fc）标记引物和石墨炔‑亚甲蓝（GDY‑MB）纳米复合材料。GDY‑MB纳米复合材料修饰在电极上会产生MB电信号，设计5'端标记Fc的引物可以与适配体杂交形成互补双链，当KAN与适配体特异性结合后会释放出游离引物形成发夹结构，导致Fc接近电极表面引起Fc电信号增强。引入核酸外切酶I（Exo I）切割单链适配体以达到目标物循环扩增放大信号作用。引物3'端进行磷酸化修饰以保护发夹结构不被Exo I切割。进一步修饰壳聚糖‑氧化锡锑（CS‑ATO）和AuNPs起到双重信号放大作用，实现了卡那霉素的高灵敏检测。
Description
技术领域
本发明提供一种检测牛奶中卡那霉素残留的比率型电化学适配体传感器的制备方法，属于生物传感器技术领域。
背景技术
卡那霉素(KAN)是一种重要的氨基糖苷类抗生素，已用于治疗严重细菌感染和结核病，也被广泛用于畜牧业中治疗动物的各种疾病和作为动物生长促进剂，然而，卡那霉素的滥用会造成在食品中残留，通过食物链在人身体中积累，对人类健康构成严重威胁，包括耳毒性、肾毒性、过敏反应、甚至导致致病菌株产生抗生素耐药性等，为了消费者的健康，欧盟(EU)已经确定了牛奶中的卡那霉素最大残留限(MRLs)为150μg kg-1，目前，食品中卡那霉素残留的测定方法很多，包括毛细管电泳法(CE)，薄层色谱法(TLC)，高效液体色谱(HPLC)、气体色谱-质谱(LC-MS)、固相萃取(SPE)等，尽管这些分析方法对卡那霉素检测是成功的，但它们存在一些限制，涉及样品预处理繁琐，耗时和价格昂贵等。
近些年，一些传感器的报道，如适配体传感器和免疫传感器，为开发低成本、快速、简单、选择性好的检测卡那霉素方法提供了方向，适配体(Apt)是一种在体外通过指数富集的配体系统进化技术(SELEX)获得的单链核酸识别元件，能够识别和特异性结合细菌、金属离子、农兽药和蛋白质等多种目标，其具有体外合成，高稳定性、易于标记和可定制结构的独特优势。电化学适配体传感器通过Apt与换能器耦合产生电化学信号，由于其样品使用量低、简单、易于小型化等优点，已经开始成为越来越多的临床诊断和食品分析研究的热点，传统的电化学适配体传感器是基于在Apt修饰的电极上直接捕获靶标，由形成的Apt-靶标复合物造成电信号变化进行定量分析，然而这些方法往往灵敏度相对较低，为了获得高灵敏度，实现对低水平分析物的准确检测，会采用不同的信号放大技术，然而在放大信号的过程中，由于环境变化、电极表面修饰Apt及其他材料密度的变化等会造成干扰，目前，提高灵敏度及增强抗干扰能力是电化学传感器面临的挑战。
电极表面修饰导电性好的纳米材料对提高电化学适配体传感器的灵敏度起着至关重要的作用，纳米掺杂氧化物中的氧化锡锑(ATO)由于其较好的稳定性、低成本和良好的电导率，是一种很有前途的导电材料。纳米金颗粒(AuNPs)具有如易制备、成本低、稳定性高、优良的生物相容性和电子特性等优异特性，还可以作为固定探针分子的理想纳米载体，进一步提高传感器性能。通过共价结合与被修饰巯基的Apt形成金硫键连接，或以极高的亲和力与设计有连续腺嘌呤(polyA)的Apt吸附。polyA-Apt相较于硫化DNA，成本更低、能够可控地调整DNA分子的密度。基于酶辅助实现信号放大技术，也是一种很有前途的新策略，核酸外切酶Ⅰ(Exo I)可以将单链DNA(ssDNA)从3’端依次水解成单核苷酸，进而释放靶标实现目标物循环重复利用，实现信号放大，由于不需要特异性的识别序列，非常适合构建通用的生物传感平台。
使用单电信号输出的电化学传感器容易出现假阴性或假阳性，相比之下，具有比率双信号的电化学传感器具有内校准因子，可以强化干扰能力，提高检测精度，可以通过引入两个独立的电信号分子来构建比率方法，如亚甲基蓝(MB)、甲苯胺蓝(TB)、二茂铁(Fc)，其在适配体传感器中产生电信号的方式主要有把电信号分子直接标记在核酸链上或利用不同核酸(ssDNA/dsDNA/G-quadruplex)对＝电信号分子结合能力不同，目前，通过将电信号分子与纳米材料结合，制备具有电化学功能的纳米复合材料，具有更好的固定性及稳定性，在构建比率型电化学适配体传感器方面具有很大的潜力，因此选择一种新型的二维碳纳米材料-石墨炔(GDY)，由sp2和sp杂化碳原子组成平面网络结构，与其他碳纳米材料相比，GDY是一种富含π-电子的独特碳异素异形体，它具有均匀分布的空隙和优异的负载性，这些特性使GDY适合与MB电信号分子结合制备信号稳定的纳米复合材料。
综上所述，我们设计一种Fc标记的引物，制备了电化学功能的纳米复合材料GDY-MB，当KAN存在时引起Fc和MB两个信号变化，构建比率型电化学适配体传感器，并结合Exo I引起的靶标循环和CS-ATO/AuNPs纳米材料辅助的双信号放大策略检测卡那霉素。
发明内容
本发明的目的是提供一种检测卡那霉素残留的比率型电化学传感器的制备方法，实现卡那霉素高灵敏度的电化学检测。
本发明的第二个目的是放大比率型电化学传感器的信号，提高灵敏度。
为了实现上述目的，本发明所采用的技术方案为：一种比率型电化学适配体传感器的制备方法，包括以下步骤的方法制备而成。
(1)取等体积的Apt和其互补引物，使用TE缓冲液(含50mM氯化钠)制备得到2.5μM互补链溶液。
(2)在离心管中制备目标循环产物，当加入KAN时，KAN特异性结合互补链中的Apt，释放引物，引物中设计的自身互补部分通过碱基互补配对形成发夹结构，Exo I将游离出的Apt切割，释放目标物进行靶标循环，继续释放引物。
(3)制备GDY-MB，AuNPs和ATO，处理裸金电极，用氧化铝浆液仔细抛光，然后在乙醇和超纯水中超声5min，直到铁氰化钾溶液中循环伏安图的峰值电位差低于80mV。
(4)在电极表面逐层修饰ATO，GDY-MB和AuNPs，再滴加循环产物，等待polyA与AuNPs结合，从而使HP紧密的固定在电极表面，当目标物越多，生成的发夹结构越多，Fc信号越大，MB信号因为酶、Apt、纳米材料等因素影响也会发生减弱，计算Fc和MB的比值与KAN的对应关系，得到KAN的检测范围和检测限。
(5)对牛奶样品进行去脂去蛋白等预处理，取上清进行应用评估。
本发明的有益效果。
(1)基于GDY-MB复合材料和Fc标记引物制备了一种比率型电化学传感器，实现了卡那霉素高灵敏检测目的。
(2)Exo I诱导的目标再循环和AuNPs/ATO辅助信号放大实现了对电化学检测信号的双重放大。
(3)所制备的适配体传感器不仅适用于牛奶中卡那霉素的检测，还有望通过简单地改变Apt实现对其他抗生素的灵敏检测和大规模定量，在食品安全、环境监测、疾病诊断等方面具有巨大的应用潜力。
附图说明
图1是本发明制备的比率型电化学适配体传感器示意图。
图2是ATO透射电镜图像。
图3是AuNPs透射电镜图像。
图4是GDY透射电镜图像。
图5是GDY-MB透射电镜图像。
图6是GDY-MB的EDS图像。
图7是目标循环的琼脂糖凝胶电泳表征图。
图8是电化学传感器的循环伏安表征图。
图9是电化学传感器的电化学阻抗表征图。
图10是酶时间和浓度，GDY-MB稀释倍数，产物在电极上孵育时间的优化图。
其中，A为酶作用时间的优化图；B为酶浓度优化图；C为GDY-MB稀释倍数优化图；D为产物在电极上孵化时间优化图。
图11是测量不同浓度卡那霉素的差分脉冲伏安曲线，传感器的标准曲线和特异性图。其中，A为IFc与KAN浓度之间的标准曲线；B为IFc/IMB值与KAN浓度之间的标准曲线；C为不同浓度卡那霉素的差分脉冲伏安曲线；D为传感器的特异性图。
具体实施方式
实施例
实施例1：Exo I辅助目标循环：首先，取等体积的Apt及其互补引物，使用TE缓冲液(含50mM氯化钠)制备为10μM的互补链溶液，然后在95℃下退火5min，并冷却到室温下进行杂交，使用前稀释至2.5μM，然后，将6μL互补链、3μL KAN、1μL Exo I缓冲液和4U Exo I酶混合在离心管中，在37℃下反应105min，与目标KAN结合的Apt被Exo I裂解，然后释放KAN并进入循环反应，最后，在80℃下加热20min，以终止循环目标。
实施例2：通过3％琼脂糖凝胶电泳验证了Exo I消化循环的可行性，见附图7，第1、2和3道分别为Apt、Fc-HP-polyA以及Apt和KAN的混合物，第4、5和6号通道在上述三个样品中加入相同数量的ExoI酶，第4通道和第6通道的条带消失，说明KAN的存在并不影响ExoI酶解Apt，第5道的条带保留了下来，表明磷酸化的HP不受该酶的影响，第7道是由Apt和Fc-HP-polyA形成的互补链，第8道是互补链和ExoI酶的混合物，第9道是互补链和KAN的混合物，第10道是互补链和KAN和ExoI酶的混合物，结果表明，只有第10道的条带减弱，说明Apt可以被KAN成功竞争，并被ExoI消化。
实施例3：纳米材料的制备；
(1)制备具有电化学功能的GDY-MB纳米复合材料，首先制备石墨炔的良好分散液，取石墨炔分散于3:1的硫酸和硝酸混合液中，超声至完全溶解，将分散体多次离心并用超纯水洗涤至中性，用超纯水定容至2mg/mL，然后使亚甲基蓝负载于GDY，取1mM MB溶液和2mg/mL GDY溶液(2:1)进行超声混合处理，将复合物离心，并分别用乙醇和超纯水洗涤至溶液变成无色，最后定容至1mg/mL；
(2)制备AuNPs，首先将1mL 1％氯金酸加入100mL沸腾的超纯水中，然后快速加入2.7mL 1％柠檬酸三钠，继续煮沸10分钟，然后不停匀速搅拌冷却直至室温；
(3)制备分散性更好的1mg/mL ATO溶液，首先制备0.1％壳聚糖溶液，将0.1g的壳聚糖加入到1％的冰乙酸溶液中，搅拌至完全溶解。然后称取ATO分散于上述制备的壳聚糖溶液中，超声至完全溶解。
实施例4：对材料进行透射和EDS表征，结果见附图2-6。
实施例5：比率型电化学适配体传感器的制备，构建Au电极、参比电极和辅助电极三电极系统，测试前对裸金电极(AuE)进行处理：用氧化铝浆仔细抛光，然后在乙醇和超纯水中超声清洗5分钟，循环伏安(CV)峰值电位差低于80mV，最后，电极在氮气气氛中干燥；适配体传感器的具体制备过程如下：将6μL ATO，6μL GDY-MB和8μL AuNPs逐层滴在AuE上，取8μL循环产物滴加在修饰好的电极表面，等待polyA与AuNPs结合，从而使HP紧密的固定在电极表面，由此得到比率型电化学适配体传感器，为了验证所开发的电化学适应器传感器的组装过程，进行了CV和EIS表征，在含有5mM[Fe(CN)6]3-/4-和0.1M KCl的溶液中进行CV测试，K3Fe(CN)6溶液稀释4倍进行电化学阻抗(EIS)测试，结果见附图8-9。
实施例6：对实验条件进行优化，结果见附图10所示：为了达到最佳的KAN比率检测性能，优化了Exo I的剂量和反应时间，GDY-MB的浓度以及在电极上和纳米金的孵育时间等实验参数，在滴加ATO及纳米金后，孵育不同剂量Exo I酶产生的循环样品，结果如附图10A所示，在Exo I酶浓度为4U时，出现了Fc电流峰值，在酶浓度为4U时，对反应时间进行优化，根据附图10B所示，随着时间的增加，Fc电流峰值逐渐增加然后趋于平行，根据5nM和500nMKAN产生的Fc电流峰差值(ΔFc)，选取折线图峰值105min为Exo I酶的最佳反应时间；为了达到比率型的最佳效果，保证两种电信号值相近，因此对制备的GDY-MB溶液的稀释倍数进行优化，结果如附图10C所示，当稀释倍数较小时，由于IMB较大，IFc由于太小，峰无法测出，稀释量过大又会影响IMB出峰，因此选择最佳效果，将稀释GDY-MB溶液60倍；采用IFc和比值峰值电流(IFc/IMB)研究了最佳循环产物的孵育时间，结果如附图10D所示，IFc和IFc/IMB均在120min出现峰值，因此选择最佳孵育时间为120min。
实施例7：分析传感器性能，结果见附图11，在最佳实验条件下，使用所构建的比率型电化学适配体传感器检测不同浓度的KAN，在PBS缓冲液(pH 7.0)中进行DPV测试，KAN浓度与IFc/IMB的比值呈线性关系，回归方程为IFc/IMB＝6.07268×10-4CKAN+0.17298，相关系数为0.9967，其中I和C代表峰值电流强度和浓度，对比KAN浓度与IFc的线性关系，其回归方程为IFc＝2.31415×10-4CKAN+0.09954，相关系数为0.9599，根据相关系数表明，制备的比率传感器性能优于单独使用Fc作为检测信号的性能，计算比率型策略的检测限(LOD)为6.044nM(S/N＝3)。
实施例8：为了评价该适配体传感器的特异性，用10μM其他不同的氨基糖苷类抗生素庆大霉素(GEN)、大观霉素(SPE)、链霉素(STR)、新霉素(NEO)和1μM KAN进行测试，该适配体传感器具有良好的特异性。
实施例9：制备四种不同浓度的KAN残余乳进行电化学检测：0.1、0.4、0.6和0.8μM，根据实验结果得到，所制备比率型电化学适配体传感器的回收率在92.6％-109.25％之间，RSD为0.36％～4.11％，在实际牛奶样品测试中表现出良好的效果，说明在实际牛奶样品检测中具有良好的灵敏性。
Claims (6)
1.一种比率型电化学适配体传感器的制备方法，其特征在于：设计一种5'端二茂铁(Fc)标记的引物，制备电化学功能的纳米复合材料石墨炔-亚甲基蓝(GDY-MB)，当卡那霉素(KAN)存在时会引起Fc和MB两电信号变化，构建比率型电化学适配体传感器，并结合核酸外切酶I(Exo I)引起的靶标循环和氧化锡锑和纳米金(ATO/AuNps)纳米材料导电辅助的双信号放大策略检测牛奶中KAN。
2.根据权利要求1所述的比率型电化学适配体传感器的制备方法，其特征在于：设计一种5'端二茂铁Fc标记引物的方法是：设计部分序列能够与适配体(Apt)部分序列杂交形成双链的引物，在引物的5’端标记二茂铁电信号分子，在3’端增加polyA短核酸链并在末端腺嘌呤上进行磷酸化修饰防止被Exo I剪切，由于双链的刚性结构Fc远离电极表面，Fc电信号弱，当KAN存在时，特异性结合Apt释放引物，引物中设计的自身互补部分通过碱基互补配对形成发夹结构，Fc接近电极表面，引起Fc电信号增加。
3.根据权利要求1所述的比率型电化学适配体传感器的制备方法，其特征在于：结合Exo I引起的靶标循环的方法是：取等体积的Apt和其互补引物，使用TE缓冲液配置成互补链溶液，然后取互补链，KAN，Exo I酶缓冲液和Exo I酶于离心管中混合孵育，被目标物竞争下来的游离Apt被Exo I剪切，然后重新释放循环，最后，将混合物加热以终止循环靶标。
4.根据权利要求1所述的比率型电化学适配体传感器的制备方法，其特征在于：制备具有电化学功能的GDY-MB纳米复合材料的方法是：首先制备石墨炔的良好分散液，取石墨炔分散于硫酸和硝酸混合液中，超声至完全溶解，将分散体多次离心并用超纯水洗涤至中性，用超纯水定容，然后取MB溶液和GDY溶液进行超声混合处理，将复合物离心，并分别用乙醇和超纯水洗涤至溶液变成无色，最后定容，MB负载于GDY的GDY-MB纳米复合材料制备完成。
5.根据权利要求1所述的比率型电化学适配体传感器的制备方法，其特征在于：氧化锡锑的处理方法是：首先制备壳聚糖溶液作为分散剂和稳定剂，然后称取ATO分散于上述制备的壳聚糖溶液中，超声至完全溶解。
6.根据权利要求3所述的比率型电化学适配体传感器的制备方法，其特征在于：构建比率型电化学Apt传感器的方法是：将ATO，GDY-MB和AuNPs逐层滴在金电极上，最后取循环产物滴加在修饰好的电极表面，等待polyA与AuNPs结合，从而使发夹结构(HP)紧密固定在电极表面，由此，得到比率型电化学适配体传感器。
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