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2026 , Vol. 54 >Issue 1: 9 - 17

DOI: https://doi.org/10.15983/j.cnki.jsnu.2026002

A fluorescent aptamer probe for detection of microcystin-LR based on double-stranded DNA-silver nanoclusters/gold nanorods

  • LI Jun 1 ,
  • LUO Yan 1 ,
  • A Huaying 1 ,
  • ZHANG Yanli , 1, * ,
  • GAO Lianxun 1 ,
  • WANG Hongbin 1 ,
  • YANG Wenrong 2 ,
  • PANG Pengfei , 1, *
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  • 1 Functional Nanomaterial-based Chemical and Biological Sensing Technology Innovation Team of Department of Education of Yunnan Province, School of Chemistry and Environment, Yunnan Minzu University, Kunming 650504, Yunnan, China
  • 2 School of Life and Environmental Sciences, Deakin University, Geelong 3217, Victoria, Australia

Received date: 2025-09-23

  Online published: 2026-03-02

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Abstract

A fluorescent aptamer probe for highly sensitive sensing detection of microcystin-LR (MC-LR) was developed based on double-stranded DNA-silver nanoclusters (dsDNA-AgNCs) coupled with gold nanorods (AuNRs). Three DNA nucleotides were designed in this work, including one MC-LR aptamer chain and two C-riched single stranded DNA S1 and S2 complementary to aptamer. Single stranded DNA-AgNCs (ssDNA-AgNCs) with red fluorescence were synthesized by using single stranded DNA S1 and S2 as templates and reduction of silver ions (Ag+) with sodium borohydride (NaBH4). Two ssDNA-AgNCs hybridized with aptamer strands at its two ends to form negatively charged dsDNA-AgNCs with a maximum fluorescence emission at 624 nm. In the presence of positively charged AuNRs, fluorescent resonance energy transfer (FRET) occurred between dsDNA-AgNCs and AuNRs due to the electrostatic interaction, resulting in fluorescent quenching of dsDNA-AgNCs. After addition of the target MC-LR, MC-LR specifically bonded with aptamer of dsDNA-AgNCs, resulting in dissociation of dsDNA-AgNCs to ssDNA-AgNCs and restoring of fluorescence intensity. An “off-on” fluorescent aptamer probe was constructed for MC-LR quantitative detection. The proposed method exhibited a linear response for MC-LR detection in the concentration range of 5 ng/L to 500 μg/L with a detection limit of 1.7 ng/L. The proposed fluorescent aptamer probe possesses the advantages of simple preparation and high selectivity and sensitivity, and can be applied for the MC-LR detection in actual water samples.

Key words： microcystin-LR; double-stranded DNA-silver nanoclusters; gold nanorods; fluorescent aptamer probe

Cite this article

LI Jun , LUO Yan , A Huaying , ZHANG Yanli , GAO Lianxun , WANG Hongbin , YANG Wenrong , PANG Pengfei . A fluorescent aptamer probe for detection of microcystin-LR based on double-stranded DNA-silver nanoclusters/gold nanorods[J]. Journal of Shaanxi Normal University(Natural Science Edition), 2026 , 54(1) : 9 -17 . DOI: 10.15983/j.cnki.jsnu.2026002

近年来,荧光传感检测方法因具有快速响应、低成本、高灵敏度和高选择性等特点引起人们的关注和研究兴趣[1]。金属纳米团簇(MNCs)[2-3]、碳点(CDs)[4-5]和量子点(QDs)[6-7]等功能或复合纳米材料,已被应用于构建荧光传感探针。由于制备简单、易于表面功能化、 生物相容性好及优异的光致发光性能等因素,MNCs作为一种新兴的荧光纳米材料,正日益受到研究者们的关注[2,8]。MNCs是一种由数个至数百个原子构成的水溶性分子聚集体,其尺寸大小介于单一原子或分子与大颗粒之间,形成了一种特殊的尺寸过渡状态。这些MNCs粒子在尺寸接近电子费米波长时,呈现出与分子性质类似的特性,从而发挥出过渡的桥梁作用,弥补了单个金属原子与金属纳米颗粒之间的过渡形式[9-11]。由于金属纳米团簇(MNCs)能级离散性,其与较大尺寸的金属纳米颗粒在化学性质、光电子学等方面存在显著差异,如荧光和光致发光性能。不同类型的MNCs中,DNA-金属纳米团簇(DNA-MNCs)因其高量子产率、优良的生物相容性和稳定性、易于合成、荧光发射波长可调等特性而受到广泛关注。DNA-MNCs已被应用于化学生物传感、光电催化、生物医学及成像等多个领域[12-15]。
由于工农业不可持续发展引起生态系统中水污染物浓度增加,进而引发或加剧蓝藻水华形成,这对淡水生态系统及公众健康构成严重威胁[16-17]。蓝藻水华暴发期间,水体中会释放各种不同环境毒素,如微囊藻毒素(MC)、蚜虫毒素、结瘤菌素等细胞内毒素。其中,微囊藻毒素-LR(MC-LR)是最具代表性且具有最高浓度和最强毒性的微囊藻毒素。MC-LR是一种七肽单环肝毒素,具有极强的致癌性,并且不易被常规蛋白质或多肽水解酶降解[18]。近年的研究表明,MC-LR能够抑制肝细胞内蛋白磷酸酶的活性,进而诱导细胞角蛋白发生过度磷酸化,这一过程最终导致动物出现肝功能衰竭[19]。因此,世界卫生组织(WHO)设定生活饮用水中MC-LR的最高浓度限值为1 μg/L[20]。同时,由于MC-LR通常存在于各种湖泊和水体中,使得水体中的水生食物容易受到MC-LR的污染。因此,迫切需要建立一种选择性、灵敏度和准确度高的方法,以检测不同水体及水生食品中MC-LR的含量。近年来,研究人员已发展出多种检测方法,以实现对MC-LR的准确监测。这些方法包括高效液相色谱法(HPLC)[21-22]、酶联免疫吸附检验法(ELISA)[23-24]、蛋白磷酸酶抑制检验法(PPIA)[25],以及电化学、比色和荧光传感分析法[26-30]等。然而,这些检测方法大多需要昂贵的仪器设备和专业的操作人员,且分析过程耗时较长,一定程度上限制了这些方法的推广和普及。
本工作以DNA寡核苷酸链为模板,采用化学氧化还原法合成dsDNA-AgNCs,并结合AuNRs构建荧光适体探针,以实现对MC-LR的荧光传感分析。实验设计了3条DNA核苷酸链,以2条富含C碱基且与MC-LR适配体互补的ssDNA为模板,合成具有红色荧光特性的ssDNA-AgNCs。这些ssDNA-AgNCs分别与适配体杂交形成dsDNA-AgNCs,后者则通过静电相互作用力与AuNRs结合,从而产生荧光共振能量转移现象,导致dsDNA-AgNCs体系荧光淬灭。分析物MC-LR能够与适体特异性结合,导致dsDNA-AgNCs结构解体及探针荧光恢复。基于此,构建一种“off-on”型荧光适体探针,以实现对水体中MC-LR的灵敏和准确检测。

1 材料与方法

1.1 仪器与试剂

荧光分析仪(F-7000),日本日立;紫外-可见分光光度计(UV-2600i),日本岛津;透射电子显微镜(JEM-2100,transmission electron microscopy,TEM),日本电子;暗箱式三用紫外分析仪(WFH-203B),上海驰唐电子有限公司;恒温金属浴(HX-10F),上海沪析实业有限公司;高速离心机(TGL-16G),上海安亭科学仪器厂。
微囊藻毒素-LR(MC-LR)和微囊藻毒素-RR(MC-RR)购于北京伊普瑞斯科技有限公司;四水合氯金酸(HAuCl4·4H2O)、硝酸银(AgNO3)、硼氢化钠(NaBH4)和抗坏血酸(AA)购自上海国药集团化学试剂有限公司;十六烷基三甲基溴化铵(CTAB)、六水合硝酸镁(Mg(NO3)2·6H2O)、十二水合磷酸氢二纳(Na2HPO4·12H2O)和二水合磷酸二氢纳(NaH2PO4·2H2O)购于广东光华科技股份有限公司。实验所用的其他试剂均为分析纯级别,实验用水则采用高压灭菌去离子水。
本实验所使用的3条DNA核苷酸链由宝生物工程(大连)有限公司合成,并经高效液相色谱(HPLC)纯化和冻干处理。Aptamer碱基序列为5'-GGCGCCAAACAGGACCACCATGACAATT-ACCCATACCACCTCATTATGCCCCATCTCC-GC-3',ssDNA S1碱基序列为5'-ATTGTCATG-GTGGTCCTGTTTGGCCCCCCTAATTCCCCC-3',ssDNA S2碱基序列为5'-CCCCCTAATTCCCC-AGATGGGGCATAATGAGGTGGTA-3'。

1.2 实验方法

1.2.1 dsDNA-AgNCs的制备

采用DNA模板法改进后制备双链DNA-银纳米簇(dsDNA-AgNCs)[31-32]。分别移取5 μL aptamer、ssDNA S1和ssDNA S2溶液(物质的量浓度均为10 μmol/L)于3支离心管中,各加入5 μL 20 mmol/L PBS缓冲液(pH 7.4),振荡混合,95 ℃水浴退火处理5 min,自然冷却至室温。将上述3种溶液混合,漩涡振荡器振荡1 min,再依次加入AgNO3溶液(10 μL, 120 μmol/L)和新鲜配制的NaBH4溶液(10 μL, 120 μmol/L),混匀后4 ℃避光反应60 min,得到dsDNA-AgNCs。

1.2.2 AuNRs的制备

利用种子介导生长法合成金纳米棒(AuNRs)[33-34]。首先,称取0.35 g CTAB溶解于10 mL水中,加入250 μL HAuCl4溶液(10 mmol/L),超声混匀,再加入冰水浴中新鲜制备的NaBH4溶液(600 μL,10 mmol/L),混匀后室温静置2 h,得到金种子溶液。其次,配制生长液,称取0.40 g CTAB溶解于12 mL水中,加入500 μL HAuCl4溶液(10 mmol/L)、35 μL AgNO3溶液(10 mmol/L)、200 μL HCl溶液(1 mol/L)和80 μL新鲜配制的抗坏血酸溶液(0.1 mol/L),迅速加入1 mL金种子溶液,搅拌均匀后室温静置反应12 h。产物经12 000 r/min离心分离,去离子水洗涤2次,重新分散于水中,得到棕红色AuNRs溶液。

1.2.3 dsDNA-AgNCs/AuNRs荧光适体探针的制备

移取20 μL上述制备的dsDNA-AgNCs溶液,加入2 μL 2 mmol/L Mg(NO3)2溶液,振荡混匀,再加入2 μL制备的AuNRs溶液(1 mg/mL),用20 mmol/L PBS缓冲液(pH 7.4)稀释至50 μL。混合液在室温避光条件下孵育20 min,得到dsDNA-AgNCs/AuNRs荧光适体探针溶液。

1.2.4 MC-LR的分析检测

在合成的dsDNA-AgNCs/AuNRs荧光适体探针溶液(50 μL)中,加入5 μL不同浓度的MC-LR标准溶液,33 ℃培育70 min,采用荧光光谱仪对其荧光光谱及荧光强度进行测定。荧光激发波长和发射波长狭缝均设置为10 nm,光电倍增管电压为700 V,扫描速率为500 nm/min,在566 nm激发波长下记录dsDNA-AgNCs/AuNRs探针的荧光发射光谱。
河水和湖水2种水样中MC-LR的检测,水样利用高速离心机以10 000 r/min离心10 min后,使用0.22 μm×13 mm微孔滤膜过滤。测定MC-LR的步骤与之前一致,并开展加标回收实验以计算其加标回收率。

2 结果与讨论

2.1 dsDNA-AgNCs/AuNRs荧光探针检测原理

dsDNA-AgNCs/AuNRs荧光适体探针的制备过程及其对MC-LR的检测原理如图1所示。本研究设计了3条DNA核苷酸链,MC-LR适体链(aptamer)和2条富含C碱基且与aptamer互补的单链DNA S1和DNA S2。以单链DNA S1和S2为模板,利用硼氢化钠(NaBH4)还原Ag+,合成2条具有红色荧光的单链DNA-AgNCs(ssDNA-AgNCs)。2条ssDNA-AgNCs分别与aptamer两末端杂交形成带负电荷的dsDNA-AgNCs,其最大荧光发射波长为624 nm。当存在带正电的AuNRs时,通过静电相互作用力,dsDNA-AgNCs与AuNRs之间产生荧光共振能量转移,导致dsDNA-AgNCs荧光淬灭。分析物MC-LR存在时,MC-LR与dsDNA-AgNCs中aptamer之间高特异性结合作用下,导致双链结构解体,dsDNA-AgNCs转变为ssDNA-AgNCs,体系荧光恢复,根据dsDNA-AgNCs/AuNRs体系荧光恢复实现对MC-LR的定量检测。
图1 dsDNA-AgNCs/AuNRs荧光适体探针检测MC-LR原理示意图

注:网络版为彩图。

Fig.1 Schematic illustration of principle for MC-LR detection based on dsDNA-AgNCs/AuNRs fluorescent aptamer probe

2.2 dsDNA-AgNCs和AuNRs的光学特性及形貌表征

合成dsDNA-AgNCs的荧光激发(曲线i)和发射光谱(曲线ii)如图2a所示,其最大激发波长为566 nm,最大发射波长为624 nm,在可见光下呈现无色,而在365 nm紫外光照射下则显示出粉红色荧光(图2a插图)。图2b为dsDNA-AgNCs的TEM图,由图可见,合成的dsDNA-AgNCs呈球形均匀分散,粒径尺寸约为2.0 nm。图2c为合成AuNRs的紫外-可见吸收光谱,图中呈现AuNRs的2个特征吸收峰,分别为AuNRs的横向(峰i,528 nm)和纵向(峰ii,804 nm)局域表面等离子体共振吸收峰。图2d为AuNRs的TEM图,由图可见,合成的AuNRs呈粗细均匀的棒状,直径为8.2 nm,长度为28.0 nm,长径比为3.4。以上测试结果表明,成功合成了构建荧光适体探针的dsDNA-AgNCs和AuNRs。
图2 dsDNA-AgNCs和AuNRs的光学特性和形貌表征

注:网络版为彩图。

Fig.2 Optical properties and morphology characterization of dsDNA-AgNCs and AuNRs

2.3 dsDNA-AgNCs/AuNRs荧光探针可行性

为验证dsDNA-AgNCs和AuNRs荧光适体探针检测MC-LR的可行性,探究不同体系的荧光强度变化。图3a对比了ssDNA-AgNCs和dsDNA-AgNCs的荧光光谱,由图可见,ssDNA-AgNCs转变为dsDNA-AgNCs后,荧光强度和荧光发射波长基本不变,表明形成dsDNA结构后没有影响AgNCs的荧光性质。图3b为dsDNA-AgNCs依次与AuNRs、MC-LR作用后的荧光光谱,由图可观察到,dsDNA-AgNCs与AuNRs作用后,体系荧光强度发生显著淬灭,加入目标物MC-LR后,体系荧光恢复,据此可实现对MC-LR的定量分析检测。
图3 不同体系的荧光强度变化

Fig.3 The change of fluorescence intensity in different systems

此外,探究了dsDNA-AgNCs/AuNRs荧光体系的Zeta电位(图4)。dsDNA-AgNCs 的Zeta电位为-17.5 mV,AuNRs的Zeta电位为25.3 mV,dsDNA-AgNCs与AuNRs混合后的Zeta电位为15.4 mV,最后加入目标物MC-LR后,其电位值变为18.8 mV。以上结果表明,基于dsDNA-AgNCs/AuNRs荧光适体探针对MC-LR的检测是可行的。
图4 dsDNA-AgNCs/AuNRs荧光体系的Zeta电位

Fig.4 Zeta potentials of dsDNA-AgNCs/AuNRs fluorescent system

2.4 实验条件优化

为获得优良的荧光传感检测性能,对dsDNA-AgNCs和AuNRs荧光适体探针的合成条件和检测条件(nDNA∶nAgNO3∶nNaBH4、NaBH4反应时间、AuNRs体积、AuNRs孵育时间、MC-LR孵育温度和时间)进行优化(图5)。首先探究nDNA∶nAgNO3∶nNaBH4对合成dsDNA-AgNCs荧光强度的影响,当三者比例为1∶24∶24时,荧光强度达到最高(图5a)。加入NaBH4后,dsDNA-AgNCs荧光强度随着反应时间出现最高点,对应还原反应时间为60 min(图5b)。当dsDNA-AgNCs溶液中加入2 μL AuNRs溶液(1 mg/mL)后,体系荧光淬灭程度达到稳定(图5c)。当dsDNA-AgNCs与AuNRs孵育时间为20 min时,体系荧光淬灭程度达到最大(图5d)。最后对检测MC-LR时的孵育温度和时间进行优化,当孵育温度设定为33 ℃,孵育时间为70 min时,体系荧光强度恢复达到最大(图5e、图5f)。因此,选择nDNA∶nAgNO3∶nNaBH4=1∶24∶24、NaBH4反应时间60 min、AuNRs加入2 μL、孵育时间20 min、MC-LR孵育温度和时间分别为33 ℃和70 min,确定为dsDNA-AgNCs/AuNRs荧光适体探针的最佳制备条件和分析检测条件。
图5 实验条件优化

Fig.5 Optimization of experimental conditions

2.5 MC-LR的定量检测

在以上最优实验条件下,测定dsDNA-AgNCs/AuNRs探针对不同质量浓度MC-LR的荧光强度。如图6a所示,随着分析物MC-LR质量浓度的增加,dsDNA-AgNCs/AuNRs探针荧光强度逐渐恢复增强,探针荧光强度恢复与MC-LR质量浓度在0.005~500 μg/L范围内呈现出良好的线性关系(图6b),其线性回归方程为y=95.33lg x+469.18,计算得到检出限(LOD)为1.7 ng/L,决定系数(R2)为0.987 3。上述实验结果说明,构建的dsDNA-AgNCs/AuNRs荧光适体探针可用于MC-LR定量检测分析。
图6 MC-LR定量检测荧光光谱图

注:网络版为彩图。

Fig.6 Fluorescence spectra of MC-LR quantitative determination

2.6 荧光适体探针的特异性、重现性和稳定性

探索dsDNA-AgNCs/AuNRs荧光适体探针的特异性或选择性,考察MC-RR和一些可能共存物质对荧光探针的影响。如图7a所示,在MC-LR的质量浓度为20 μg/L、其他干扰物(MC-RR、葡萄糖、Al3+、Ca2+、NH4+、Cl-、SO42-、CO32-)的质量浓度为50 μg/L的情况下,dsDNA-AgNCs/AuNRs探针仅对MC-LR表现出明显的荧光响应,对其他干扰物及其混合物的荧光变化可忽略不计。
图7 dsDNA-AgNCs/AuNRs荧光适体探针的选择性(a)和重现性(b)

Fig.7 Selectivity and reproducibility of dsDNA-AgNCs/AuNRs fluorescent aptamer probe

图7b为5次单独实验考察dsDNA-AgNCs/AuNRs探针对20 μg/L MC-LR的响应重现性,其相对标准偏差为3.2%。此外,考察了荧光适体探针的时间稳定性,dsDNA-AgNCs/AuNRs探针溶液在室温下储存15 d后,其荧光响应值仍可保持初始值的90%以上。上述实验结果说明,构建的dsDNA-AgNCs/AuNRs荧光适体探针具有较好的选择性、重现性和稳定性,可应用于实际水样中MC-LR定量检测。

2.7 实际水样分析

验证dsDNA-AgNCs/AuNRs荧光适体探针对实际水样中MC-LR分析的可行性和有效性,选择湖水和河水2种水样作为实际样品,2种水样经离心分离和膜过滤处理后,采用标准加入法测定探针荧光强度并计算回收率,实验测定结果列于表1。构建的dsDNA-AgNCs/AuNRs荧光适体探针对2种水样中MC-LR测定的回收率范围为93.5%~101.1%,其相对标准偏差范围为2.2%~4.8%,且荧光法与MC-LR标准检测方法酶联免疫吸附法(ELISA)的测定结果基本一致。上述实际水样结果说明,dsDNA-AgNCs/AuNRs荧光适体探针可用于环境水体中MC-LR的检测。
表1 实际水样中MC-LR的测定结果

Tab.1 Determination results of MC-LR in real water samples

水样 加标量/
(μg·L-1)		 dsDNA-AgNCs/AuNRs荧光适体探针 ELISA测定结果
测得值/
(μg·L-1)		 相对标准
偏差/%		 加标回
收率/%		 测得值/
(μg·L-1)		 相对标准
偏差/%		 加标回
收率/%
河水 0 - - - - - -
10 9.35 2.9 93.5 9.64 3.2 96.4
50 49.11 3.6 98.2 49.55 3.8 99.1
100 101.05 4.4 101.1 101.42 4.1 101.4
湖水 0 - - - - - -
10 9.68 3.2 96.8 9.78 2.9 97.8
50 48.20 2.2 96.4 48.62 3.1 97.2
100 97.30 4.8 97.3 97.81 3.7 97.8

3 结论

本文采用DNA模板法合成双链DNA-银纳米簇耦合金纳米棒,以构建荧光适体探针,旨在实现对水体中微囊藻毒素-LR高灵敏传感检测。以2条富含C碱基且与MC-LR aptamer互补的ssDNA为模板,利用硼氢化钠还原Ag+,得到可见光下呈现红色荧光的ssDNA-AgNCs,其分别与aptamer杂交形成带负电荷的dsDNA-AgNCs。当存在带正电的AuNRs时,通过静电相互作用力,dsDNA-AgNCs与AuNRs之间产生荧光共振能量转移现象,导致dsDNA-AgNCs荧光淬灭。分析物MC-LR与aptamer特异性结合,导致dsDNA-AgNCs双链结构解体转变为ssDNA-AgNCs,体系荧光恢复,根据dsDNA-AgNCs/AuNRs探针荧光恢复实现对MC-LR的定量分析。构建的“off-on”型荧光适体探针具有制备简单、灵敏度高和选择性强等优点,为环境水体中微囊藻毒素-LR的快速、准确定量检测提供了新方法。

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