【作者】 刘瑞新；

【导师】 杨占军；

【作者基本信息】 扬州大学 ， 化学， 2024， 硕士

【摘要】 快速且准确地检测肿瘤标志物可预测癌症或某些疾病的发生。电化学免疫分析作为一种定量检测肿瘤标志物的分析方法,因其灵敏度高、特异性好等优点而得到广泛应用。然而,传统的电化学免疫分析大多使用天然酶作为信号探针,其在长时间使用过程中容易失活且对环境要求严格。为解决上述问题,具有类酶活性的纳米酶展现出更高的稳定性和活性,逐渐作为天然酶的替代物开始被广泛应用。普鲁士蓝类似物(PBA)是一类由氰化物配体和过渡金属节点构成的纳米材料,具有成分可调、比表面积大、形貌多变等特点,在储能和催化等领域备受关注。此外,一些普鲁士蓝类似物材料还表现出类似于天然酶的催化特性,但由于其固有的元素组成和结构,其类酶活性受到限制,所以需要改变普鲁士蓝类似物的成分或结构以提高酶活性。本论文合成了NiFePBA@Pt纳米立方体、核壳开放笼状Ni Fe@Fe PBA和中空Ni Fe(Ⅱ)PBA(Ni Fe(Ⅱ)PBA HNCs)三种纳米酶。实验结果表明,这三种纳米酶均表现出优异的类过氧化物酶活性。将这三种纳米酶作为标记探针可用于构建新型电化学免疫传感器,从而实现对甲胎蛋白(AFP)高效、灵敏、准确的检测。主要研究内容如下:(1)本研究构建了一种基于NiFePBA@Pt纳米酶探针的新型电化学免疫传感器,用于高灵敏检测AFP。采用室温共沉淀法制备NiFePBA纳米酶,并通过原位生长铂纳米颗粒成功合成NiFePBA@Pt纳米酶。通过扫描电镜(SEM)、透射电镜(TEM)、X射线光电子能谱仪(XPS)及X射线衍射仪(XRD)证明NiFePBA@Pt纳米酶成功制备。镍和铁作为纳米酶的催化位点,协同作用使NiFePBA@Pt具有类过氧化物酶活性。紫外光谱、电子顺磁共振(EPR)、酶促动力学证明引入铂纳米颗粒后进一步提高了纳米酶的类酶活性。NiFePBA@Pt纳米酶探针在过氧化氢存在下催化邻苯二胺(o-PD)产生电化学信号(差分脉冲伏安信号),继而利用抗原浓度与电化学信号的线性关系实现对AFP定量检测。构建的基于NiFePBA@Pt纳米酶的新型电化学免疫传感器对AFP的线性范围为0.01-70 ng/m L,检测限低至1.1 pg/m L(S/N=3)。此外,制备的免疫传感器表现出良好的抗干扰性及储存稳定性,并成功应用于实际样品检测,具有广阔的临床应用前景。(2)为进一步提高NiFePBA纳米酶的活性,本研究设计一种开放笼状核壳NiFe@FePBA纳米酶。首先利用聚乙烯吡咯烷酮(PVP)的还原性将Fe PBA生长在NiFePBA表面合成了Ni Fe@Fe PBA纳米酶。通过SEM、TEM、XPS及XRD验证其独特的结构形态。紫外光谱、EPR和酶促动力学进一步证明该纳米酶相较于前体NiFePBA纳米酶展现出更优异的类过氧化物酶活性,这主要得益于其结构和元素分布变化。为构建电化学免疫传感器,将Ni Fe@Fe PBA纳米酶与二级抗体结合作为信号探针,并引入硬碳(HC)作为基底来提高电子传输速率。Ni Fe@Fe PBA纳米酶探针在过氧化氢存在下纳米酶探针能够催化o-PD产生电化学信号(差分脉冲伏安信号),利用抗原浓度与电化学信号的线性关系实现对AFP定量检测。基于Ni Fe@Fe PBA纳米酶构建的新型电化学免疫传感器的线性范围为0.005-100 ng/m L,检测限低至0.89 pg/m L(S/N=3)。此外制备的免疫传感器表现出良好的特异性和稳定性,并成功应用于实际样品检测,为临床应用提供了基础。(3)本研究以NiFePBA纳米酶为模板,利用PVP在高温下的还原性成功制得一种具有更高类过氧化物酶活性的中空Ni Fe(Ⅱ)PBA纳米酶(Ni Fe(Ⅱ)PBA HNCs)。SEM、TEM、XPS及XRD发现Ni Fe(Ⅱ)PBA HNCs纳米酶成功合成。紫外光谱、EPR和酶促动力学分析证明该纳米酶具有比前驱体NiFePBA纳米酶更优异的类过氧化物酶活性,这归因于其中空结构以及元素价态的变化。随后将Ni Fe(Ⅱ)PBA HNCs纳米酶与二级抗体结合作为信号探针,并引入HC作为基底以提高电子传输速率,构建一种新型电化学免疫传感器。在过氧化氢存在下纳米酶探针会催化o-PD产生电化学信号(差分脉冲伏安信号),利用抗原浓度与电化学信号的线性关系实现对AFP定量检测。基于Ni Fe(Ⅱ)PBA HNCs纳米酶的新型电化学免疫传感器在检测AFP方面展现出卓越的性能,其线性范围为0.001-150 ng/m L,检测限低至0.13 pg/m L(S/N=3)。此外所制备的免疫传感器展现出优异的特异性和稳定性,并成功应用于实际样品检测,为临床检测肿瘤标志物提供可能性。更多还原

【Abstract】 Rapid and accurate detection of tumor markers can predict the occurrence of cancer or certain diseases.As an analytical method for quantitative detection of tumour markers,electrochemical immunoassay has been widely used due to its advantages of high sensitivity and good specificity.However,most traditional electrochemical immunoassays use natural enzymes as signal probes,which are prone to deactivation and have strict environmental requirements during long-term use.In order to solve the above problems,nanozymes with enzyme-like activity exhibit higher stability and activity,gradually becoming widely used as substitutes for natural enzymes.Prussian blue analogues(PBAs),a class of nanomaterials consisting of cyanide ligands and excessive metal nodes with tunable compositions,large specific surface areas,and variable morphologies,have attracted much attention in areas such as energy storage and catalysis.In addition,some Prussian blue analogue materials exhibit catalytic properties like those of natural enzymes,but their enzyme-like activity is limited due to their intrinsic elemental composition and structure.Therefore,it is necessary to change the composition or structure of PBAs to improve the enzyme activity.Three nanozymes,NiFePBA@Pt nanocubes,core-shell open-cage Ni Fe@Fe PBA and hollow Ni Fe(II)PBA(Ni Fe(II)PBA HNCs),were synthesized in this thesis.The experimental results showed that all three nanozymes exhibited excellent peroxidase-like activity.These nanozymes were used as labelled probes for the construction of novel electrochemical immunosensors,which enabled efficient,sensitive,rapid and accurate detection of alpha-fetoprotein(AFP).The main research contents are as follows:(1)In this study,a new electrochemical immunosensor based on NiFePBA@Pt nanozyme probe was constructed for highly sensitive detection of AFP.NiFePBA nanozyme was prepared by room temperature co-precipitation method,and NiFePBA@Pt nanozyme was successfully synthesized by in-situ growth of platinum nanoparticles.It was demonstrated by scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray photoelectron spectroscopy(XPS),and X-ray diffraction(XRD)that NiFePBA@Pt nanozyme was successfully synthesized.Nickel and iron serve as the catalytic site of the nanozyme,and their synergistic effect makes NiFePBA@Pt have peroxidase-like activity.Ultraviolet‐visible(UV-vis)absorption spectra,electron paramagnetic resonance(EPR),and enzymatic kinetics prove that the introduction of platinum nanoparticles further improves the activity.The NiFePBA@Pt nanozyme probe catalyze o-PD generates electrochemical signal(differential pulse voltammetry signal)in the presence of hydrogen peroxide,and then utilizing the linear relationship between antigen concentration and electrochemical signal,thereby achieving quantitative detection of AFP.The constructed new electrochemical immunosensor based on NiFePBA@Pt nanozyme has a linear range for AFP of 0.01-70 ng/m L,and the detection limit is as low as 1.1 pg/m L(S/N=3).In addition,the prepared immunosensor showed well anti-interference and storage stability,and was successfully used in actual sample detection,showing broad clinical application prospects.(2)In order to further improve the activity of NiFePBA nanozyme,this study designed an open-cage core-shell Ni Fe@Fe PBA nanozyme.First,Ni Fe@Fe PBA nanozyme was synthesised by growing Fe PBA on the surface of NiFePBA using the reducing properties of polyvinylpyrrolidone(PVP).Its unique structural morphology was verified through SEM,TEM,XPS,and XRD.Further use of UV-vis absorption spectra,EPR and enzymatic kinetics proved that the nanozyme exhibits more superior peroxidase-like activity compared to the precursor NiFePBA nanozyme,which is mainly due to changes in its structure and element distribution.To construct an electrochemical immunosensor,Ni Fe@Fe PBA nanozyme was combined with secondary antibodies as signal probes,and hard carbon(HC)was introduced as a substrate to increase the electron transfer rate.When the probe reacts with the antigen to form an immune complex,the nanozyme probe can catalyze o-PD to generate an electrochemical signal(differential pulse voltammetry signal)in the presence of hydrogen peroxide.By utilizing the linear relationship between the antigen concentration and the electrochemical signal,quantitative detection of AFP was achieved.The constructed new electrochemical immunosensor based on Ni Fe@Fe PBA nanozyme detects AFP with a linear range of 0.005-100 ng/m L and a detection limit as low as 0.89 pg/m L(S/N=3).In addition,the prepared immunosensor showed well specificity and stability,and was successfully used in actual sample detection,providing a basis for clinical application.(3)In this study,using NiFePBA nanozyme as a template,a hollow NiFe(II)PBA nanozyme(Ni Fe(II)PBA HNCs)with higher peroxidase-like activity was successfully prepared by utilizing the reducibility of PVP at high temperatures.SEM,TME,XPS,and XRD confirmed the successful synthesis of Ni Fe(II)PBA HNCs nanozyme.UV-vis absorption spectra,EPR and enzymatic kinetics analysis proved that the nanozyme has better peroxidase-like activity than the precursor NiFePBA nanozyme,which is attributed to changes in its hollow structure and element valence state.Subsequently,Ni Fe(II)PBA HNCs nanozymes were combined with secondary antibodies as signal probes,and HC was introduced as a substrate to increase the electron transfer rate,and a new electrochemical immunosensor was constructed.The nanozyme probe will catalyze o-PD to generate an electrochemical signal(differential pulse voltammetry signal)in the presence of hydrogen peroxide.By utilizing the linear relationship between the antigen concentration and the electrochemical signal,quantitative detection of AFP was achieved.The constructed new electrochemical immunosensor based on Ni Fe(II)PBA HNCs nanozyme detects AFP with a linear range of 0.001-150 ng/m L and a detection limit as low as 0.13 pg/m L(S/N=3).In addition,the prepared immunosensor showed excellent specificity and stability,and has been successfully applied to actual sample detection,providing the possibility for clinical detection of tumor markers.更多还原