CN110438202A - 一种基于环金属Ir(III)配合物的凝血酶光电化学传感器的制备方法 - Google Patents
一种基于环金属Ir(III)配合物的凝血酶光电化学传感器的制备方法 Download PDFInfo
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Abstract
本发明公开了一种基于环金属Ir(III)配合物的凝血酶光电化学传感器的制备方法。该传感器以环金属配铱配合物作为光电活性材料,通过Au‑S键将其组装在纳米金上制备了纳米探针。凝血酶识别体系采用适体与凝血酶的结合产生临近效应而引发的DNA置换,当存在凝血酶时,含有凝血酶适体片段的单链DNA S1和S2的局部浓度增加,诱导预先杂交的S1/T与S2发生链置换反应使单链T被释放。释放的T与工作电极上固定的捕获发夹H1杂交,引发催化发夹自组装反应,在工作电极表面形成得到大量的H1/H2杂交链,其末端暴露的单链DNA用于捕获信号纳米金探针,实现光电流信号的响应,具有高的灵敏度及良好的选择性。
Description
技术领域
本发明涉及一种以环金属Ir(III)配合物作为光电材料,用于定量检测凝血酶的光电化学传感器的制备方法,属于光电化学定量分析技术领域。
背景技术
凝血酶是一种多功能的丝氨酸蛋白酶,它可促进血液凝固和调控凝血,其浓度和活性是衡量凝血机制的重要指标,在炎症、创伤愈合、心血管疾病以及肿瘤等生理及病理过程中发挥重要作用。因此,建立快速、高灵敏度检测凝血酶的方法对临床疾病诊断、病程发展、预后以及疗效监测和评估等都具有重要意义。到目前为止,用于检测凝血酶的技术方法主要包括比色、荧光、电化学、电化学发光、光电化学等,其中光学检测方法最为普遍,但背景荧光的干扰严重,且检测相对复杂,仪器成本高;电化学方法虽仪器简单,但灵敏度、特异性不够高,重现性差。相比较而言,光电化学方法由于光激发过程与电流检测分步进行,激发与检测信号属于不同的能量模式,可降低背景信号,灵敏度比传统的电化学方法高;相对于光学检测而言,采用电流作为检测信号,可以通过单波长或普通光源与电化学检测装置组装,因此具有设备简单、成本低廉、易于微型化和集成化等特点,在蛋白质检测方面具有明显的优势(Chem.Rev.,2014,114, 7421-7441;Chem.Soc.Rev.,2015,44,729-741)。
光电化学传感基于在光照下识别元件和目标分子之间的识别作用而产生相应电信号的改变来进行检测,其分析检测性能取决于两大因素:一是所采用光电活性材料的性质,二是所设计的识别元件。光电活性材料的性质决定了传感器的灵敏度及稳定性,而识别元件决定传感器的特异性,同时对灵敏度也有一定的影响。本发明一方面采用光电转换效率高、性质稳定的环金属Ir(III)配合物作为光电活性材料,提高传感器的灵敏度和稳定性;另一方面采用双适体识别产生邻近效应引发的链置换反应及催化发夹自组装的信号放大策略(J.Am.Chem.Soc.,2013,135,2443-2446),保证特异性的同时进一步提高灵敏度,实现对凝血酶的痕量检测。
发明内容:
本发明的目的是提供一种以可见光激发的环金属Ir(III)配合物作为光电活性材料的光电化学传感器,实现对凝血酶的高灵敏、高特异性检测。
基于上述目的,本发明所涉及的技术方案如下:
1、本发明所述的光电材料为环金属Ir(III)配合物,其结构式如下所示:
2、本发明同时提供一种基于以1所述环金属Ir(III)配合物作为光电活性材料,用于检测凝血酶的光电化学生物传感器的制备方法,包括以下步骤:
(1)环金属Ir(III)配合物纳米金探针的制备。所制备纳米探针的结构如附图1所示:
Cp-DNA序列为:AAAGACAAGTCGCTATG(5’端修饰有-SH)
环金属Ir(III)配合物通过Au-S键负载至纳米金上,步骤为:纳米金溶液离心洗涤后分散于1mL 0.02%十二烷基硫酸钠溶液中,加入经三(2-羧基乙基)膦盐酸盐(TCEP)活化的Ir(III)配合物溶液和信号DNA溶液,置于摇床震荡12h,环金属Ir(III)配合物和信号DNA组装完成,经离心洗涤后分散于缓冲溶液中。
(2)凝血酶的识别。凝血酶的识别过程如附图2所示,其中S1和S2为含有凝血酶适体的DNA单链, DNA单链T与S1部分杂交。当凝血酶不存在时,杂交的S1/T与S2不发生链置换反应,而当凝血酶存在时,凝血酶与适体的结合使S1和S2的局部浓度增加产生临近效应,诱导S1/T与S2发生链置换反应使单链DNAT被释放。
S1序列为:GGTTGGTGTGGTTGGTTTTTTTTGCTAGGTCTCGC;
S2序列为:GAGACCTAGCAATTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT;
T序列为:ATAGATCCTCATAGCGAGACCTAGCAA
(3)光电传感器的组装。包括ITO电极的修饰、催化发夹自组装信号放大过程及纳米金探针的捕获三个步骤,其中催化发夹自组装信号放大过程借助发夹DNAH1和H2进行。
ITO工作电极的制备:将清洗后的ITO电极浸入含有30%H2O2,NH3·H2O和H2O(体积比为1:1:5) 的混合溶液中15分钟,取出电极用超纯水冲洗,并在氮气流下干燥。将电极浸入5%的3-氨丙基三甲氧基硅氧烷乙醇溶液中过夜,使得在电极表面形成-NH2的自组装分子层。然后将其浸泡在纳米金溶液中孵化 12小时,得到纳米金修饰的ITO电极。然后与捕获发夹H1的溶液孵化后通过Au-S键将其固定在电极上得到工作电极。
H1序列为:AAAAAACTAGGTCTCGCTATGAGGATCTACCATCGTGTACTAGATCCTCA
TAGCGACTTGTCA(5’端修饰有-SH)。
催化发夹自组装:将修饰有捕获发夹H1的工作电极浸入含有发夹DNAH2的步骤(2)所述的凝血酶识别溶液中,释放的单链DNAT与发夹H1杂交,释放的一端与H2杂交发生链取代反应,释放单链DNA T,释放的T又与H1杂交,如此反复循环,在ITO工作电极表面得到大量的H1/H2杂交链,其末端暴露的单链DNA用于捕获信号纳米金探针。
H2序列为:AGGATCTAGTACACGATGGTAGATCCTCATAGCGACCATCGTGTAC。
纳米金探针的捕获:将上述修饰有H1/H2杂交链的ITO工作电极浸入(1)所述的环金属Ir(III)配合物金纳米探针溶液中孵化2小时,由于金纳米探针的信号DNA链与H1/H2杂交链的末端杂交而将信号材料捕获在工作电极上,完成光电传感器的组装。
(4)光电信号的检测。将步骤(3)组装好的光电传感器浸入0.01M PBS缓冲溶液中,以Pt丝为对电极,Ag/AgCl为参比电极,采用460nm可见光进行激发,每20s开关光源一次,偏置电压为-0.2–0.2V进行光电流检测,实现对不同浓度凝血酶的信号响应。
优选的,所述PBS缓冲溶液的pH=5.5。
优选的,所述偏置电压为-0.1V。
本发明的有益效果:
(1)本发明公开了一种可见光激发的环金属Ir(III)配合物作为光电材料用于制备凝血酶光电化学传感器,该材料具有稳定性好,光电转换效率高的特点。
(2)本发明公开的凝血酶光电化学传感器一方面采用双适体结合产生的邻近效应引发链置换反应识别凝血酶,另一方面采用催化发夹自组装的信号放大策略,既提高了检测的特异性,又提高了检测灵敏度。
附图说明
图1纳米探针的结构示意图;
图2凝血酶的识别过程示意图;
图3光电化学生物传感器制备示意图;
图4传感器对凝血酶浓度的光电流响应示意图,凝血酶的浓度分别为a)50fM,(b)100fM,(c)500fM, (d)1pM,(e)5pM,(f)10pM and(g)20pM;
图5传感器对凝血酶浓度的线性关系图;
具体实施方式
实施例1
Ir(III)配合物光电材料的合成
(1)取7-二乙氨基-3-醛基香豆素(127.9mg,0.68mmol),1,10-菲啰啉-5,6-二酮(104.1mg,0.5mmol),乙酸铵(805.4mg,11.3mmol),加入圆底烧瓶中,加入13mL冰乙酸溶解,氮气保护下加热回流6h,观察原料逐渐变成橙色后停止加热,自然冷却。在混合物中加浓氨水调节pH至7左右,得粉色沉淀物,过滤并用清水洗涤,样品真空干燥过夜,粗产物用柱色谱提纯(二氯甲烷:甲醇=8:1),得乳白色固体产物为环金属配体,60℃真空干燥,称重0.105g,产率:71%。1H NMR(500MHz,CDCl3):δ=9.16(d,J=4.2Hz,2H), 8.99(s,1H),8.78(d,J=5.8Hz,2H),7.70-7.72(m,2H),7.51(d,J=8.0Hz,1H),6.68(dd,J=2.0,7.0Hz,1H),6.55(d,J=2.0Hz,1H),3.47(q,J=7.5Hz,4H),1.27(t,J=7.5Hz,6H)。
(2)在2-乙氧基乙醇(15mL)和H2O(5mL)中的混合物中加入以上步骤制备的环金属配体(0.77g,2.2 mmol)和IrCl3·3H2O(0.34g,1.0mmol),在N2气氛120℃下搅拌24h。将混合物冷却至室温后,过滤得到的沉淀,依次用水、乙醇和丙酮洗涤,得到铱二氯桥联二聚体产物2。将二聚体(0.14g,0.08mmol)和4-(2- 吡啶基)苯甲醛(0.879g,4.8mmol)加入2-乙氧基乙醇(25mL)溶液中。搅拌溶解后,将过量的Na2CO3(5.0mmol) 加入到上述溶液中,将反应混合物搅拌加热,回流18h。然后,利用减压蒸馏除去溶剂,加入盐酸(1.0M,10 mL),搅拌10min。过滤产物,用水(2×15mL)洗涤,用甲醇萃取。加入饱和的六氟磷酸铵甲醇(5mL)溶液,将混合物搅拌30min。减压除去溶剂,粗产物用硅胶色谱纯化,用二氯甲烷/甲醇=10:1洗脱,得到深红色固体,50℃真空干燥,称重0.122g,产率:65%。1H NMR(500MHz,CDCl3)δ=9.79(s,2H),9.50(s,1H), 8.14(d,J=4.5Hz,2H),8.10(d,J=8.0Hz,2H),(d,J=8.0Hz,2H),7.98(d,J=8.0Hz,2H),7.88(t,J=8.0Hz, 2H),7.84(m,2H),7.69(d,J=9.0Hz,1H),7.62(d,J=8.0Hz,2H),7.45(d,J=5.5Hz,2H),7.05(t,J=6.0Hz, 2H),6.85(s,2H),6.65(d,J=8.9,1H),6.53(s,1H),3.47(q,J=7.5Hz,4H),1.24(t,J=7.5Hz,6H)。
(3)将以上得到的深红色固体(1.14g,1mmol)和2-胺基硫醇(0.15g,2mmol)溶于甲醇(25mL)中,回流6h。旋转蒸发除去溶剂,粗产物用硅胶色谱纯化,用二氯甲烷/甲醇=10:1洗脱,得到最终的环金属Ir(III)配合物光电材料。
实施例2
环金属Ir(III)配合物纳米金探针的制备
(1)纳米金的制备:将制备过程中所需的玻璃仪器、磁子及存放纳米金粒子的容器均用二次水洗净,用王水浸泡过夜,之后用大量超纯水冲洗至pH值为中性,烘干备用。将氯金酸HAuCl4(1.0mmol/L,100mL) 置于洗净的单口烧瓶中边搅拌加热至沸腾,然后快速把柠檬酸三钠(38.8mmol/L,10mL)加入到上述溶液,继续反应10min溶液由淡黄色慢慢变为深酒红色,继续回流15min,停止加热,边搅拌边自然冷却至室温,取500μL金胶溶液离心洗涤(10000rpm,10min),均匀分散于1mL 0.02%SDS溶液中备用。
(2)依次取1.5μL tris-HCl(0.5M,含0.1M NaCl,pH=7.4)、6μL 10.0mM TCEP、7.2μL 100.0μM的 Cp-DNA于4mL样品管中,室温活化1h。向上述溶液中加入步骤(1)制备的1mL纳米金SDS溶液混匀,置于摇床震荡6h(37℃,170rpm)。将制备的DNA-AuNPs中加入7.2μL1.0mM Ir(III)配合物溶液和 120μL 0.1M硼酸-硼砂缓冲溶液(pH=9.0),置于摇床上震荡12h(37℃,170rpm),,产物用0.025M的 Tris-HCl(含0.1M NaCl,pH=7.4)洗液洗3次,最后用200μL Tris-HCl(0.025M,含0.1M NaCl,pH=7.4) 分散,制得Ir(III)配合物纳米金探针溶液。
实施例3
基于实例2中所制备铱配合物金纳米探针,光电化学传感器的制备
(1)工作电极的制备:将ITO电极依次用丙酮、乙醇、超纯水进行超声清洗,然后在氮气氛围下干燥。将清洗后ITO电极浸入含有30%H2O2,NH4OH和H2O(体积比为1:1:5)的混合溶液中15分钟,取出用超纯水冲洗,并在氮气流下干燥。然后浸入5%APTMS乙醇溶液中过夜。用乙醇彻底清洗后并在110℃下干燥15min。浸入AuNPs溶液中孵育12h,以获得修饰金纳米粒子的电极表面。用超纯水清洗并在氮气流下干燥后,使用3M隐形胶带控制ITO电极面积0.5cm×0.5cm。将提前活化好的带有巯基的发夹DNA HP1溶液(1.0μM,20μL)滴加到用AuNPs修饰的ITO电极表面,37℃下反应12h。然后将其浸入2.0mM MCH溶液中1h,封板占位,获得AuNP/HP1工作电极。
(2)凝血酶的识别:S1/T制备:依次取4.8μL 5.0×10-6M T、4.0μL 5.0×10-6M O、11.2μL SPSC溶液于4mL样品管中,95℃下水浴加热10min,自然冷却,即得1.0μM S1/T。依次取不同浓度的凝血酶溶液、5.0×10-8M S1/T、5.0×10-8M S2加入样品管中,置于摇床上震荡2h(37℃,170rpm)。溶液用于接下来的电极修饰过程。
(3)光电化学生物传感器的组装:将20μL上述凝血酶反应液和DNAH2(2.0μM,20μL)滴在工作电极表面上,并在37℃下孵育4h,以进行催化发夹自组装信号放大反应。最后,将20μL上述金纳米探针滴到组装电极的表面,在37℃下进一步杂交2h。在每个组装步骤后,用0.01M PBS溶液冲洗ITO电极三次。
(4)光电信号检测:将制备好的传感器浸入0.01M PBS缓冲溶液中(pH=5.5),采用460nm可见光进行激发,测定光电流响应,实现对凝血酶的检测。光电流强度与凝血酶的浓度在50fM至20pM范围内成线性关系,线性方程为I=12.78lgc-2.91,相关系数为0.9935,其中I为光电流强度(nA),c为凝血酶浓度(fM)。
实施例4
光电化学生物传感器用于检测血清中的凝血酶
步骤(1)、(3)和(4)与实施例3相同。
步骤(2)中凝血酶的识别采用标准加入法,将不同浓度的凝血酶加入10倍稀释的人血清中,其它步骤与实施例3中步骤(2)相同。光电信号响应结果与实施例3一致,表明该传感器可以应用在实际生物血样中。
序列表
<110> 青岛科技大学
<120> 一种基于环金属Ir(III)配合物的凝血酶光电化学传感器的制备方法
<141> 2019-08-15
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Claims (2)
1.一种基于环金属Ir(III)配合物的凝血酶光电化学传感器的制备方法,其特征在于,包括以下步骤:
(1)环金属Ir(III)配合物纳米金探针的制备,其特征在于采用环金属Ir(III)配合物作为光电信号活性材料,其化学结构如下式所示:
将环金属Ir(III)配合物和信号DNA(Cp-DNA)按比例(摩尔比10:1)加入到含有0.02%十二烷基硫酸钠溶液的纳米金溶液中,通过Au-S键自组装为纳米金探针。
Cp-DNA序列为:5’-AAAGACAAGTCGCTATG-3’(5’端修饰有-SH)。
(2)凝血酶的识别,其特征在于采用凝血酶与双适体的结合产生临近效应引发链置换反应,此识别过程借助于含有凝血酶适体的单链DNA S1和S2以及与S1部分杂交单链T进行。当凝血酶不存在时,杂交的S1/T与S2不发生链置换反应,而当凝血酶存在时,凝血酶与适体的结合使S1和S2的局部浓度增加产生临近效应,诱导S1/T与S2发生链置换反应使单链DNAT被释放。
S1序列为:GGTTGGTGTGGTTGGTTTTTTTTGCTAGGTCTCGC
S2序列:GAGACCTAGCAATTTTTTAGTCCGTGGTAGGGCAGGTTGGGGTGACT
T序列为:ATAGATCCTCATAGCGAGACCTAGCAA
(3)工作电极的制备,将ITO电极浸入3-氨基丙基三乙氧基硅烷溶液中,在电极表面修饰氨基,然后将其浸泡在纳米金溶液中得到纳米金修饰的ITO电极,最后通过Au-S键将发夹DNA H1固定在电极上得到工作电极。
H1序列为:AAAAAACTAGGTCTCGCTATGAGGATCTACCATCGTGTACTAGATCCTCATAG
CGACTTGTCA(5’端修饰有-SH)。
(4)催化发夹自组装及传感器的制备,将步骤(2)中凝血酶识别后的溶液及发夹DNA H2溶液滴加到步骤(3)制备的工作电极上,经过催化发夹自组装反应后与步骤(1)制备的金纳米探针的Cp-DNA杂交,将探针捕获在工作电极上,制得光电化学传感器。
H2序列为:AGGATCTAGTACACGATGGTAGATCCTCATAGCGACCATCGTGTAC。
(5)光电信号检测:将步骤(4)制得的传感器浸入0.01M PBS(pH=5.5)的缓冲溶液中,以Pt丝为对电极,Ag/AgCl为参比电极,采用460nm可见光进行激发,每20s开关光源一次,记录光电流响应,实现对不同浓度凝血酶的检测。
2.根据权利要求1所述方法制备得到的光电化学传感器在检测凝血酶中的应用。
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CN115078490A (zh) * | 2022-06-02 | 2022-09-20 | 青岛科技大学 | 一种用于检测CN–的铱(III)配合物敏化NiO光阴极的制备方法 |
CN115078490B (zh) * | 2022-06-02 | 2024-04-30 | 青岛科技大学 | 一种用于检测CN–的铱(III)配合物敏化NiO光阴极的制备方法 |
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