CN111766290B - 一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法 - Google Patents
一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法 Download PDFInfo
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Abstract
本发明涉及一种基于三维碳化钛‑二硫化钼复合物免疫传感器的制备方法及应用,属于新型纳米材料领域与生物传感技术领域;本发明首次通过混合‑干燥法制备三维多孔碳化钛‑二硫化钼(Ti3C2Tx‑MoS2)复合材料,并以鲁米诺作为还原剂制备铂功能化三维Ti3C2Tx/MoS2(Lum@Pt/Ti3C2Tx‑MoS2)作为传感基底,首次提出了一种用于非小细胞肺癌疾病标志物CYFRA 21‑1的高灵敏检测的免疫传感器的制备方法,Ti3C2Tx/MoS2具有高导电性、高电化学活性、大比表面积与生物相容性,可高效催化水中的溶解氧转化为超氧阴离子自由基O2 •−,从而增强鲁米诺与O2 •−之间的电致化学发光反应实现信号高效稳定输出,该传感器检出限低至18 pg/mL,线性范围50 pg/mL‑50 ng/mL,在非小细胞肺癌早期诊断中具有明显的潜在应用价值。
Description
技术领域
本发明属于新型纳米材料领域与生物传感技术领域。
背景技术
CYFRA 21-1是细胞角蛋白19的可溶性片段,被认为是一种主要用于检测肺癌的肿瘤标记物,尤其对非小细胞肺癌的诊断具有重要价值。如果肺部存在不清晰的环形阴影,同时血清CYFRA21-1浓度高于30 ng/mL,原发性支气管肺癌的可能性非常高,各类非小细胞肺癌阳性检出率为70%~85%,CYFRA21-1的血清浓度水平高低与肿瘤临床分期正相关,也可作为肺癌手术和放化疗后追踪早期复发的有效指标,CYFRA 21-1的血清高浓度水平提示疾病处于进展期和预后不良,质量成功的标志是CYFRA 21-1的血清浓度迅速下降,反之则表示病灶未完全清除,CYFRA 21-1对各型肺癌诊断的敏感性依次为:鳞癌>腺癌>大细胞癌>小细胞癌。到目前为止,只有荧光法、电化学分析、色谱分析等几种分析方法得到了发展。因此,发展一种简单、快速、准确的测定方法,对于CYFRA 21-1的即时检测具有重要意义。
作为一门由生物、化学、医学、电子技术等多种学科相互交叉而兴起的研究热点,电致化学发光免疫分析是电致化学发光技术与免疫分析方法的有机结合,制得的免疫传感器具有成本低、选择性好、灵敏度高、分析速度快,易于自动化、微型化与集成化等优点,已被广泛应用于疾病标志物分析、食品安全分析、环境污染分析等领域。
近年来,二硫化钼(MoS2)与金属碳化钛(Ti3C2Tx MXene)纳米材料凭借其优异的电催化活性与高导电性受到了广泛关注,在鲁米诺-过氧化氢体系中也有所应用。相比较于过氧化氢的生物毒性,无生物毒性、简单易得的溶解氧成为鲁米诺一种新兴的环保型共反应剂。为了进一步提高鲁米诺-溶解氧体系的发光效率,本发明通过一种混合-干燥方法首次制备了一种三维多孔的纳米复合材料Ti3C2Tx-MoS2,它集成了MoS2与Ti3C2Tx的性能优势,体现出更高的导电性与电化学活性、更大的比表面积与更加优秀的生物相容性,可高效催化溶解氧转变成超氧阴离子自由基O2 •−,从而大大增强鲁米诺-溶解氧体系的电致化学发光反应实现信号高效稳定输出。
发明内容
本发明的任务之一是为了拓宽鲁米诺-溶解氧体系在电致化学发光传感的应用,提出一种基于三维多孔的纳米复合材料Ti3C2Tx-MoS2增强鲁米诺与溶解氧之间电致化学发光反应的方法,通过原位还原法,制得Lum@Pt/Ti3C2Tx-MoS2纳米复合物作为传感基底,Ti3C2Tx-MoS2具有极好的导电性与电催化活性,可显著增强鲁米诺-溶解氧体系的发光效率;
本发明的技术任务之二是弥补现有CYFRA 21-1检测技术的不足,提供一种基于三维多孔Ti3C2Tx-MoS2电致化学发光免疫传感器的用途,该传感器能够快速检测CYFRA 21-1,具有灵敏度高、特异性强、重现性好的优点,检出限为18 pg/mL,线性范围50 pg/mL-50 ng/mL。
为实现上述目的,本发明采用的技术方案如下:
1. 一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法及应用,其特征在于,包括以下步骤:
(1)将直径为4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉抛光处理,用超纯水冲洗干净;
(2)在玻碳电极表面滴涂6 μL、浓度为2 ~ 4 mg/mL的Lum@Pt/Ti3C2Tx-MoS2溶液为传感基底,将其置于37 °C晾干;
(3)滴加6 μL、浓度为100 μg/mL的CYFRA 21-1抗体溶液,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(4)滴加3 μL、质量分数为1 ~ 3%的牛血清白蛋白溶液,以封闭电极表面的非特异性活性位点,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(5)滴加6 μL、一定浓度的 CYFRA 21-1标准溶液,37 °C下孵化0.5 ~ 2 h,用pH7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干,传感器构建完毕;
(6)在10 mL磷酸盐缓冲溶液中,对传感器进行电致化学发光测试,得到孵化不同浓度CYFRA 21-1时对应的电致化学发光信号强度,绘制工作曲线,其检出限为20 pg/mL,线性范围50 pg/mL-50 ng/mL。
2. 如权利要求1所述的一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法及应用,其特征在于,所述Lum@Pt/Ti3C2Tx-MoS2溶液,按以下步骤制备:
首先,配制质量浓度为1.5 ~ 3.5 mg/mL的Ti3C2Tx纳米片溶液,将其与1.5 ~ 3.5mg/mL的MoS2纳米片溶液,将上述两种溶液混合,在氩气保护条件下,超声处理0.5 ~ 2 h,形成了分散均匀的混合溶液,然后将混合溶液冷冻干燥12 ~ 36 h后得到最终产品Ti3C2Tx-MoS2复合物;
称取50 ~ 80 mg Ti3C2Tx-MoS2纳米材料溶于25 ~ 40 mL超纯水中,加入体积为3~ 6 mL、浓度为10 mmol/L的氯铂酸溶液超声处理5 ~ 20 min,随后加入5 ~ 10 mL、浓度为1 mmol/L的鲁米诺溶液,震荡0.5 ~ 2 h后,老化处理2 ~ 3 h,将溶液中的固体离心分离,70 ~ 90 °C下真空干燥12 h得到Lum@Pt/Ti3C2Tx-MoS2固体。
本发明的有益成果
(1)本发明首次提出一种基于Ti3C2Tx-MoS2促进鲁米诺-溶解氧体系电致化学发光性能的方法,Ti3C2Tx-MoS2具有更加优异的电化学活性,可高效催化电解液中的溶解氧O2转化为超氧阴离子自由基O2 •−,从而大大增强鲁米诺与O2 •−之间的电致化学发光反应,有助于鲁米诺-溶解氧体系在电致化学发光领域的进一步应用与研究;
(2)本发明基于Ti3C2Tx-MoS2促进鲁米诺-溶解氧电致化学发光原理提出了一种精准可靠的免疫传感技术。弥补了现有电化学检测技术操作复杂、灵敏度低、重现差的问题,将该技术应用于CYFRA 21-1的样品检测,检出限为18 pg/mL,线性范围50 pg/mL-50 ng/mL,具有响应速度快、灵敏度高、重现好、制备简单、成本低、绿色环保的优点。
具体实施方式
下面结合具体实施例,进一步阐述本发明,应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1.一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法及应用,其特征在于,包括以下步骤:
1. 一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法及应用,其特征在于,包括以下步骤:
(1)将直径为4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉抛光处理,用超纯水冲洗干净;
(2)在玻碳电极表面滴涂6 μL、浓度为2 mg/mL的Lum@Pt/Ti3C2Tx-MoS2溶液为传感基底,将其置于37 °C晾干;
(3)滴加6 μL、浓度为100 μg/mL的CYFRA 21-1抗体溶液,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(4)滴加3 μL、质量分数为1 %的牛血清白蛋白溶液,以封闭电极表面的非特异性活性位点,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(5)滴加6 μL、一定浓度的 CYFRA 21-1标准溶液,37 °C下孵化0.5 ~ 2 h,用pH7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干,传感器构建完毕;
(6)在10 mL磷酸盐缓冲溶液中,对传感器进行电致化学发光测试,得到孵化不同浓度CYFRA 21-1时对应的电致化学发光信号强度,绘制工作曲线,其检出限为20 pg/mL,线性范围50 pg/mL-50 ng/mL。
实施例2.一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法及应用,其特征在于,包括以下步骤:
(1)将直径为4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉抛光处理,用超纯水冲洗干净;
(2)在玻碳电极表面滴涂6 μL、浓度为3 mg/mL的Lum@Pt/Ti3C2Tx-MoS2溶液为传感基底,将其置于37 °C晾干;
(3)滴加6 μL、浓度为100 μg/mL的CYFRA 21-1抗体溶液,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(4)滴加3 μL、质量分数为2 %的牛血清白蛋白溶液,以封闭电极表面的非特异性活性位点,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(5)滴加6 μL、一定浓度的 CYFRA 21-1标准溶液,37 °C下孵化0.5 ~ 2 h,用pH7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干,传感器构建完毕;
(6)在10 mL磷酸盐缓冲溶液中,对传感器进行电致化学发光测试,得到孵化不同浓度CYFRA 21-1时对应的电致化学发光信号强度,绘制工作曲线,其检出限为20 pg/mL,线性范围50 pg/mL-50 ng/mL。
实施例3. 一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法及应用,其特征在于,包括以下步骤:
(1)将直径为4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉抛光处理,用超纯水冲洗干净;
(2)在玻碳电极表面滴涂6 μL、浓度为4 mg/mL的Lum@Pt/Ti3C2Tx-MoS2溶液为传感基底,将其置于37 °C晾干;
(3)滴加6 μL、浓度为100 μg/mL的CYFRA 21-1抗体溶液,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(4)滴加3 μL、质量分数为3 %的牛血清白蛋白溶液,以封闭电极表面的非特异性活性位点,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(5)滴加6 μL、一定浓度的 CYFRA 21-1标准溶液,37 °C下孵化0.5 ~ 2 h,用pH7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干,传感器构建完毕;
(6)在10 mL磷酸盐缓冲溶液中,对传感器进行电致化学发光测试,得到孵化不同浓度CYFRA 21-1时对应的电致化学发光信号强度,绘制工作曲线,其检出限为20 pg/mL,线性范围50 pg/mL-50 ng/mL。
实施例4.所述Lum@Pt/Ti3C2Tx-MoS2溶液,按以下步骤制备:
首先,配制质量浓度为1.5 mg/mL的Ti3C2Tx纳米片溶液,将其与1.5 mg/mL的MoS2纳米片溶液,将上述两种溶液混合,在氩气保护条件下,超声处理0.5 h,形成了分散均匀的混合溶液,然后将混合溶液冷冻干燥12 h后得到最终产品Ti3C2Tx-MoS2复合物;
称取50 mg Ti3C2Tx-MoS2纳米材料溶于25 mL超纯水中,加入体积为3 mL、浓度为10 mmol/L的氯铂酸溶液超声处理5 min,随后加入5 mL、浓度为1 mmol/L的鲁米诺溶液,震荡0.5 h后,老化处理2 h,将溶液中的固体离心分离,70 °C下真空干燥12 h得到Lum@Pt/Ti3C2Tx-MoS2固体。
实施例5. 所述Lum@Pt/Ti3C2Tx-MoS2溶液,按以下步骤制备:
首先,配制质量浓度为3.5 mg/mL的Ti3C2Tx纳米片溶液,将其与3.5 mg/mL的MoS2纳米片溶液,将上述两种溶液混合,在氩气保护条件下,超声处理2 h,形成了分散均匀的混合溶液,然后将混合溶液冷冻干燥36 h后得到最终产品Ti3C2Tx-MoS2复合物;
称取80 mg Ti3C2Tx-MoS2纳米材料溶于40 mL超纯水中,加入体积为6 mL、浓度为10 mmol/L的氯铂酸溶液超声处理20 min,随后加入10 mL、浓度为1 mmol/L的鲁米诺溶液,震荡2 h后,老化处理3 h,将溶液中的固体离心分离, 90 °C下真空干燥12 h得到Lum@Pt/Ti3C2Tx-MoS2固体。
实施例6.所述Lum@Pt/Ti3C2Tx-MoS2溶液,按以下步骤制备:
首先,配制质量浓度为2.5 mg/mL的Ti3C2Tx纳米片溶液,将其与2.5 mg/mL的MoS2纳米片溶液,将上述两种溶液混合,在氩气保护条件下,超声处理1.5 h,形成了分散均匀的混合溶液,然后将混合溶液冷冻干燥12 ~ 36 h后得到最终产品Ti3C2Tx-MoS2复合物;
称取60 mg Ti3C2Tx-MoS2纳米材料溶于30 mL超纯水中,加入体积为5 mL、浓度为10 mmol/L的氯铂酸溶液超声处理15 min,随后加入8 mL、浓度为1 mmol/L的鲁米诺溶液,震荡1.4 h后,老化处理2.5 h,将溶液中的固体离心分离,80 °C下真空干燥12 h得到Lum@Pt/Ti3C2Tx-MoS2固体。
Claims (1)
1. 一种基于三维碳化钛-二硫化钼复合物生物传感器的制备方法,其特征在于,按以下步骤制备:
(1)Lum@Pt/Ti3C2Tx-MoS2溶液的制备
首先,配制质量浓度为1.5 ~ 3.5 mg/mL的Ti3C2Tx纳米片溶液,将其与1.5 ~ 3.5 mg/mL的MoS2纳米片溶液,将上述两种溶液混合,在氩气保护条件下,超声处理0.5 ~ 2 h,形成了分散均匀的混合溶液,然后将混合溶液冷冻干燥12 ~ 36 h后得到最终产品Ti3C2Tx-MoS2复合物;
称取50 ~ 80 mg Ti3C2Tx-MoS2纳米材料溶于25 ~ 40 mL超纯水中,加入体积为3 ~ 6mL、浓度为10 mmol/L的氯铂酸溶液超声处理5 ~ 20 min,随后加入5 ~ 10 mL、浓度为1mmol/L的鲁米诺溶液,震荡0.5 ~ 2 h后,老化处理2 ~ 3 h,将溶液中的固体离心分离,70~ 90 °C下真空干燥12 h得到Lum@Pt/Ti3C2Tx-MoS2固体;
(2)将直径为4 mm的玻碳电极依次用1.0 μm、0.3 μm、0.05 μm氧化铝抛光粉抛光处理,用超纯水冲洗干净;
(3)在玻碳电极表面滴涂6 μL、浓度为2 ~ 4 mg/mL的Lum@Pt/Ti3C2Tx-MoS2溶液为传感基底,将其置于37 °C晾干;
(4)滴加6 μL、浓度为100 μg/mL的CYFRA 21-1抗体溶液,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(5)滴加3 μL、质量分数为1 ~ 3%的牛血清白蛋白溶液,以封闭电极表面的非特异性活性位点,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干;
(6)滴加6 μL、一定浓度的 CYFRA 21-1标准溶液,37 °C下孵化0.5 ~ 2 h,用pH 7.4的磷酸盐缓冲溶液PBS冲洗电极表面,将其置于4 °C晾干,传感器构建完毕;
(7)在10 mL磷酸盐缓冲溶液中,对传感器进行电致化学发光测试,得到孵化不同浓度CYFRA 21-1时对应的电致化学发光信号强度,绘制工作曲线,其检出限为20 pg/mL,线性范围50 pg/mL-50 ng/mL。
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