基于纳米材料新型电化学传感器的制备及其在生物样品分析中的应用研究
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摘要
电化学生物传感器结合了信息技术与生物技术,涉及化学、生物学、物理学以及电子学等学科。由于其具有体积小、分辨率高、响应时间短、所需样品少、对活细胞损伤小等特点,电化学生物传感器在医药工业、食品检测和环境保护等诸多领域有着广阔的应用前景。近年来,随着材料科学、化学、物理学等学科的发展,纳米材料因具有特殊的结构效应,如小尺寸效应、表面界面效应、量子尺寸效应、宏观量子隧道效应及介电限域效应等,使其在许多领域得到了广泛应用。目前,采用纳米材料构建新型的电化学生物传感器日益成为研究热点。纳米材料应用于电化学生物传感器领域后,不仅提高了传感器的检测性能,而且使传感器的化学和物理性质以及它对生物分子或者细胞的检测灵敏度大幅提高,检测时间也得以缩短,并且可实现高通量的实时分析检测。
本论文的工作主要集中在将纳米技术和电化学传感技术相结合,开发了基于纳米材料的新型电化学生物传感器并将其用于检测水体中大肠杆菌和细胞表面的多聚糖。该传感技术为水体中大肠杆菌的快速检测提供了新方法,同时也为肿瘤疾病的早期诊断及治疗提供了新途径。具体研究内容如下：
第一部分：Cu@Au复合纳米粒子标记抗体的电化学免疫方法用于水体中大肠杆菌的快速检测
本文制备了Cu@Au复合纳米粒子,并将其用于标记大肠杆菌抗体,利用电化学免疫技术实现了对水体中大肠杆菌的快速检测。Cu@Au复合纳米粒子具有优良的生物相容性、电化学活性和稳定性。与单独的金纳米颗粒相比,Cu@Au复合纳米粒子作为抗体标记物大幅提高了电化学检测的灵敏度。在实验过程中,首先将大肠杆菌吸附在聚苯乙烯修饰的ITO导电玻璃表面,利用抗体和大肠杆菌之间的免疫反应把Cu@Au复合纳米粒子标记的抗体结合在ITO导电玻璃表面。将Cu@Au复合纳米粒子在溴氢酸中氧化为离子形式,然后用阳极溶出伏安法定量检测溶液中的Cu2+。为了提高检测灵敏度,采用Nafion/汞膜修饰的玻碳电极(GCE/Nafion/Hg)作为工作电极,Cu2+的检测限可达9.0×10-12 M。结果表明,在50 cfu/mL～5.0×104 cfu/mL浓度范围内,铜的响应电流与大肠杆菌浓度的对数呈线性关系,检测限为30 cfu/mL,总的分析时间为2 h。将研究的电化学免疫方法用于地表水中大肠杆菌的测定,通过对实际水样进行预富集,能够检测到大肠杆菌的浓度为3 cfu/10 mL。
第二部分：基于磁性高分子微球的电化学DNA生物传感器用于水体中大肠杆菌的检测
本文研制了一种新型的基于磁性高分子微球的电化学DNA生物传感器,并将其用于水体中大肠杆菌的检测。以海藻酸包裹钴的磁性高分子微球作为DNA探针的固体基质,根据大肠杆菌细胞体内uid A基因合成了特异性的DNA序列,制备了用于大肠杆菌检测的DNA探针。利用透射电镜技术对制备的磁性高分子微球进行了表征,并通过红外光谱法证实了特定DNA序列与磁性高分子微球的成功连接。在DNA杂交前后,分别对嵌入式杂交指示剂柔红霉素进行电化学测定,根据电化学信号的变化对目标DNA进行检测。采用非互补DNA序列、三个碱基错配的DNA序列及完全互补DNA序列验证了DNA探针的选择性。实验过程中利用聚合酶链反应(PCR)技术提取了大肠杆菌细胞体内uid A基因片断,并用电化学DNA生物传感器对经过热处理后的PCR产物和水体中大肠杆菌进行了测定。结果表明,本文研制的电化学DNA生物传感器可以检测到0.30 nM完全互补DNA序列和0.50 ng/μL的PCR产物,对大肠杆菌的检出限为50 cfu/mL。
第三部分：基于二茂铁修饰氧化锌纳米棒的信号放大策略用于水体中大肠杆菌的电化学免疫检测
本文将大肠杆菌检测抗体(dAb)和二茂铁(Fc)共同修饰于氧化锌纳米棒(ZnO NRs)表面制备了{dAb-ZnO-Fc}生物复合物,并将其用于水体中大肠杆菌的电化学免疫检测。采用BCA蛋白测定法(BCA protein assay)和电感耦合等离子体原子发射光谱法(ICP-AES)分别对检测抗体和二茂铁在氧化锌纳米棒上的最优配比进行了研究。该生物复合物利用二茂铁作为电活性物质产生电信号,检测抗体用于免疫结合大肠杆菌。采用“三明治”夹心结构,首先将捕获抗体固定在巯基乙酸修饰的金电极表面,然后通过免疫反应结合大肠杆菌,进而吸附{dAb-ZnO-Fc}生物复合物,最后用示差脉冲伏安法测定固定在电极表面上的二茂铁。通过分析检测不同浓度大肠杆菌溶液获得的电流信号,从而实现了对大肠杆菌的定量检测。实验结果表明,在1.0×102 cfu/ML～1.0×106 cfu/mL浓度范围内,二茂铁的电流信号与大肠杆菌浓度的对数呈线性关系,检出限为50cfu/mL。通过对实际水样进行富集浓缩,该电化学免疫技术可以检测到5 cfu/10mL大肠杆菌。
第四部分：巯基糖衍生物功能化的电化学生物传感器用于活体肿瘤细胞表面多聚糖的竞争检测
本文合成了巯基糖衍生物用于构建电化学生物传感器,采用竞争策略检测活体肿瘤细胞表面多聚糖的表达水平。该传感器利用纳米金/碳纳米管修饰玻碳电极(GCE/MWNT/AuNP)为基底,通过Au-S键固定巯基糖衍生物。此外,采用碳纳米管为载体固定辣根过氧化酶(HRP)和刀豆球蛋白(Con A)制备了{ConA-MWNT-HRP}生物复合物。以人体肺、肝、前列腺组织中活体肿瘤细胞表面甘露糖为研究对象,利用甘露糖和刀豆球蛋白的特异性结合,传感器表面的巯基糖衍生物与细胞表面的甘露糖竞争结合{Con A-MWNT-HRP}生物复合物。以对苯二酚为电子媒介体,通过测定辣根过氧化酶催化过氧化氢产生的响应电流对活细胞表面的多聚糖进行检测。结果表明,在优化的实验条件下,本方法用于肿瘤细胞的定量检测具有宽的线性范围和低的检测限。同时,我们计算了每个细胞表面甘露糖的数目：每个肺癌细胞A549含有甘露糖的数目为5.8×1010个,每个肝癌细胞QGY-7703含有甘露糖的数目为1.3×1010个,每个前列腺癌细胞LNCaP含有甘露糖的数目为1.9×1010个。本文所研制的巯基糖衍生物功能化的电化学生物传感器用于甘露糖的测定具有灵敏度高、选择性好、响应信号快等优点,为活细胞表面多聚糖的检测提供了新的方法。
第五部分：凝集素电化学生物传感器用于活体肿瘤细胞表面多聚糖的检测研究
本文制备了一种高灵敏度和高选择性的凝集素(lectins)生物传感器并将其用于检测甘露糖(mannose)和唾液酸(sialic acid)在人体肺、肝、前列腺组织中正常细胞和肿瘤细胞表面的表达水平。采用碳纳米管/纳米金(MWNT/AuNP)修饰电极固定凝集素,利用细胞表面的多聚糖(glycans)和凝集素之间的特异性相互作用将细胞吸附在传感器表面,然后结合硫堇(Th)包被金纳米粒子标记的凝集素{lectin-Au-Th}构建“三明治”夹心结构。最后用示差脉冲伏安法(DPV)定量检测硫堇,实现了对细胞表面多聚糖的测定。实验结果表明,甘露糖在正常细胞和肿瘤细胞表面的表达水平普遍较高；唾液酸在肿瘤细胞表面的表达水平要明显高于其在正常细胞表面的表达,研究结果对于揭示肿瘤细胞的生物学行为具有一定的指导意义。同时,依据肿瘤细胞表面唾液酸与接骨木凝集素(SNA)的特异性相互作用,将该电化学生物传感器定量检测了人体肺、肝、前列腺组织中的肿瘤细胞和每个肿瘤细胞表面唾液酸的含量并获得了满意的结果。本方法具有灵敏度高、选择性好的优点,在肿瘤疾病的早期诊断中具有很好的应用前景。
Electrochemical biosensors, combining informatics and bio techno to gy, serve as an interdisciplinary frontier related to chemistry, biology, physics and electronics. Owing to their high sensitivity and selectivity, fast responses and advantages in miniaturization and online detection potentials, electrochemical biosensors have been extensively studied and applied in clinical medicine, food inspection and environment protection. Recently, nanomaterials, due to their unique properties, are widely used for developing electrochemical biosensors. The application of nanomaterials has brought a great momentum to electrochemical biosensors and opens new horizons for highly sensitive detection, which provides an avenue for high-throughput analysis of biological components on living cells.
In this dissertation, we studied on the development of the novel electrochemical biosensors based on nanomaterials, and their applications in the analysis of E. coli in water and glycans on living cells. The details are listed below:
Part 1. Development of an Electrochemical Immunoassay for Rapid Detection of E. coli Using Anodic Stripping Voltammetry Based on Cu@Au Nanoparticles as Antibody Labels
A sensitive electrochemical immunoassay for rapid detection of E. coli has been developed by Anodic Stripping Voltammetry (ASV) based on Cu@Au nanoparticles (NPs) as anti-E. coli antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM). After Cu@Au-labeled antibody reacted with the immobilized E. coli on PS-modified ITO chip, Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu2+ were determined at Nafion/Hg-modified glassy carbon electrode (GCE/Nafion/Hg) by ASV. The utilization of GCE/Nafion/Hg could enhance the sensitivity for Cu2+ detection with a concentration as low as 9.0×10-12 M. Since Cu@Au NPs labels were only present when antibody reacted with E. coli, the amount of Cu2+ directly reflected the number of E. coli. The technique could detect E. coli with a detection limit of 30 cfu/mL and the overall analysis could be completed in 2 h. By introducing a pre-enrichment step, a concentration of 3 cfu/10 mL E. coli in surface water was detected by the electrochemical immunoassay.
Part 2. A Supersensitive DNA Electrochemical Biosensor Based on Magnetic Beads for E. coli Detection by DNA Hybridization
A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of E. coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH2) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzymeβ-D-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 0.30 nM and denatured PCR products for 0.50 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×102 to 2.0×103 cfu/mL with a detection limit of 50 cfu/mL.
Part 3. Optimized Ferrocene-Functionalized ZnO Nanorods for Signal Amplification in Electrochemical Immunoassay of E. coli
A novel strategy using ferrocene (Fc)-functionalized ZnO nanorods (NRs) for the amplified electrochemical immunoassay was developed in the present work. The detection antibody (dAb) and Fc were immobilized onto the surface of ZnO NRs, denoted as{dAb-ZnO-Fc} bioconjugates. The amount of dAb and Fc in the bioconjugates was investigated using the copper reduction/bicinchoninic acid reaction (BCA protein assay) and inductive coupled plasma-atomic emission spectroscopy (ICP-AES), respectively. Greatly amplified signal was achieved in the sandwich-type immunoassay when the dAb and Fc linked to ZnO NRs at a proper ratio. Using E. coli as a model antigen, the designed immunoassay showed an excellent analytical performance, and exhibited a wide dynamic response range of E. coli concentration from 1.0×102 to 1.0×106 cfu/mL with a detection limit of 50 cfu/mL (S/N=3). By introducing a pre-enrichment step, the detection of 5 cfu/10 mL E. coli in hospital sewage water was realized.
Part 4. Carbohydrate Derivative-Functionalized Electrochemical Biosensor for Competitive Assay of Glycan Expression on Living Cancer Cells
A novel carbohydrate derivative-functionalized electrochemical biosensor was developed for competitive analysis of glycan expression on living cancer cells. Mannose present on cancer cells derived from human lung, liver, and prostate was used as a model glycan. The biosensor was designed by employing the multiwalled carbon nanotube/Au nanoparticle (MWNT/AuNP) composite film to modify an electrode surface for thiomannosyl dimer ([mannose-S]2) assembling through Au-S bond. The{Con A-MWNT-HRP} bioconjugates, as recognition elements, were prepared by exploiting amplification effect of MWNTs for loading enormous horseradish peroxidase (HRP) labels and mannose-specific concanavalin A (Con A). Using a rapid and one-step competitive assay, the biosensor surface-confined thiomannosyl dimer competed with cell surface mannose to specifically recognize the {Con A-MWNT-HRP} bioconjugates. The proposed biosensor exhibited attractive performances for the analysis of cancer cells with wide linear ranges and low detection limits. The average amount of mannose on single cell surface was also detected to be 5.8×1010 molecules for A549 (lung cancer),1.3×1010 molecules for QGY-7703 (liver cancer), and 1.9×1010 molecules for LNCaP (prostate cancer). The carbohydrate derivative-functionalized electrochemical biosensor shows high sensitivity, selectivity and rapid response, and possesses promising application in the study of glycan changes on living cancer cells.
Part 5. Lectin-Based Biosensor Strategy for Electrochemical Assay of Glycan Expression on Living Cancer Cells
We report a novel lectin-based biosensor for electrochemical assay of cancer-associated glycosylation by comparative study of mannose and sialic acid expression on normal and cancer cells derived from human lung, liver and prostate. Using a sandwich format, high sensitivity and selectivity were achieved by combining the lectin-based biosensor with the{lectin-Au-Th} bioconjugates featuring lectin and thionine (Th) labels linked to gold nanoparticles (AuNPs) for signal amplification. The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells compared to normal ones. The differences in the two glycan expression indicated that sialic acid could serve as a potential bio marker for early cancer detection. The lectin-based biosensor was also successfully used to quantify cancer cells and evaluate the average amount of sialic acid on single cell surface, which could supply significant information on glycan functions in cancer progression. Overall, the lectin-based electrochemical biosensor provides an effective pathway to analyze glycan expression on living cells, and may greatly facilitate the medical diagnosis and treatment in early process of cancer.
本论文的工作主要集中在将纳米技术和电化学传感技术相结合,开发了基于纳米材料的新型电化学生物传感器并将其用于检测水体中大肠杆菌和细胞表面的多聚糖。该传感技术为水体中大肠杆菌的快速检测提供了新方法,同时也为肿瘤疾病的早期诊断及治疗提供了新途径。具体研究内容如下：
第一部分：Cu@Au复合纳米粒子标记抗体的电化学免疫方法用于水体中大肠杆菌的快速检测
本文制备了Cu@Au复合纳米粒子,并将其用于标记大肠杆菌抗体,利用电化学免疫技术实现了对水体中大肠杆菌的快速检测。Cu@Au复合纳米粒子具有优良的生物相容性、电化学活性和稳定性。与单独的金纳米颗粒相比,Cu@Au复合纳米粒子作为抗体标记物大幅提高了电化学检测的灵敏度。在实验过程中,首先将大肠杆菌吸附在聚苯乙烯修饰的ITO导电玻璃表面,利用抗体和大肠杆菌之间的免疫反应把Cu@Au复合纳米粒子标记的抗体结合在ITO导电玻璃表面。将Cu@Au复合纳米粒子在溴氢酸中氧化为离子形式,然后用阳极溶出伏安法定量检测溶液中的Cu2+。为了提高检测灵敏度,采用Nafion/汞膜修饰的玻碳电极(GCE/Nafion/Hg)作为工作电极,Cu2+的检测限可达9.0×10-12 M。结果表明,在50 cfu/mL～5.0×104 cfu/mL浓度范围内,铜的响应电流与大肠杆菌浓度的对数呈线性关系,检测限为30 cfu/mL,总的分析时间为2 h。将研究的电化学免疫方法用于地表水中大肠杆菌的测定,通过对实际水样进行预富集,能够检测到大肠杆菌的浓度为3 cfu/10 mL。
第二部分：基于磁性高分子微球的电化学DNA生物传感器用于水体中大肠杆菌的检测
本文研制了一种新型的基于磁性高分子微球的电化学DNA生物传感器,并将其用于水体中大肠杆菌的检测。以海藻酸包裹钴的磁性高分子微球作为DNA探针的固体基质,根据大肠杆菌细胞体内uid A基因合成了特异性的DNA序列,制备了用于大肠杆菌检测的DNA探针。利用透射电镜技术对制备的磁性高分子微球进行了表征,并通过红外光谱法证实了特定DNA序列与磁性高分子微球的成功连接。在DNA杂交前后,分别对嵌入式杂交指示剂柔红霉素进行电化学测定,根据电化学信号的变化对目标DNA进行检测。采用非互补DNA序列、三个碱基错配的DNA序列及完全互补DNA序列验证了DNA探针的选择性。实验过程中利用聚合酶链反应(PCR)技术提取了大肠杆菌细胞体内uid A基因片断,并用电化学DNA生物传感器对经过热处理后的PCR产物和水体中大肠杆菌进行了测定。结果表明,本文研制的电化学DNA生物传感器可以检测到0.30 nM完全互补DNA序列和0.50 ng/μL的PCR产物,对大肠杆菌的检出限为50 cfu/mL。
第三部分：基于二茂铁修饰氧化锌纳米棒的信号放大策略用于水体中大肠杆菌的电化学免疫检测
本文将大肠杆菌检测抗体(dAb)和二茂铁(Fc)共同修饰于氧化锌纳米棒(ZnO NRs)表面制备了{dAb-ZnO-Fc}生物复合物,并将其用于水体中大肠杆菌的电化学免疫检测。采用BCA蛋白测定法(BCA protein assay)和电感耦合等离子体原子发射光谱法(ICP-AES)分别对检测抗体和二茂铁在氧化锌纳米棒上的最优配比进行了研究。该生物复合物利用二茂铁作为电活性物质产生电信号,检测抗体用于免疫结合大肠杆菌。采用“三明治”夹心结构,首先将捕获抗体固定在巯基乙酸修饰的金电极表面,然后通过免疫反应结合大肠杆菌,进而吸附{dAb-ZnO-Fc}生物复合物,最后用示差脉冲伏安法测定固定在电极表面上的二茂铁。通过分析检测不同浓度大肠杆菌溶液获得的电流信号,从而实现了对大肠杆菌的定量检测。实验结果表明,在1.0×102 cfu/ML～1.0×106 cfu/mL浓度范围内,二茂铁的电流信号与大肠杆菌浓度的对数呈线性关系,检出限为50cfu/mL。通过对实际水样进行富集浓缩,该电化学免疫技术可以检测到5 cfu/10mL大肠杆菌。
第四部分：巯基糖衍生物功能化的电化学生物传感器用于活体肿瘤细胞表面多聚糖的竞争检测
本文合成了巯基糖衍生物用于构建电化学生物传感器,采用竞争策略检测活体肿瘤细胞表面多聚糖的表达水平。该传感器利用纳米金/碳纳米管修饰玻碳电极(GCE/MWNT/AuNP)为基底,通过Au-S键固定巯基糖衍生物。此外,采用碳纳米管为载体固定辣根过氧化酶(HRP)和刀豆球蛋白(Con A)制备了{ConA-MWNT-HRP}生物复合物。以人体肺、肝、前列腺组织中活体肿瘤细胞表面甘露糖为研究对象,利用甘露糖和刀豆球蛋白的特异性结合,传感器表面的巯基糖衍生物与细胞表面的甘露糖竞争结合{Con A-MWNT-HRP}生物复合物。以对苯二酚为电子媒介体,通过测定辣根过氧化酶催化过氧化氢产生的响应电流对活细胞表面的多聚糖进行检测。结果表明,在优化的实验条件下,本方法用于肿瘤细胞的定量检测具有宽的线性范围和低的检测限。同时,我们计算了每个细胞表面甘露糖的数目：每个肺癌细胞A549含有甘露糖的数目为5.8×1010个,每个肝癌细胞QGY-7703含有甘露糖的数目为1.3×1010个,每个前列腺癌细胞LNCaP含有甘露糖的数目为1.9×1010个。本文所研制的巯基糖衍生物功能化的电化学生物传感器用于甘露糖的测定具有灵敏度高、选择性好、响应信号快等优点,为活细胞表面多聚糖的检测提供了新的方法。
第五部分：凝集素电化学生物传感器用于活体肿瘤细胞表面多聚糖的检测研究
本文制备了一种高灵敏度和高选择性的凝集素(lectins)生物传感器并将其用于检测甘露糖(mannose)和唾液酸(sialic acid)在人体肺、肝、前列腺组织中正常细胞和肿瘤细胞表面的表达水平。采用碳纳米管/纳米金(MWNT/AuNP)修饰电极固定凝集素,利用细胞表面的多聚糖(glycans)和凝集素之间的特异性相互作用将细胞吸附在传感器表面,然后结合硫堇(Th)包被金纳米粒子标记的凝集素{lectin-Au-Th}构建“三明治”夹心结构。最后用示差脉冲伏安法(DPV)定量检测硫堇,实现了对细胞表面多聚糖的测定。实验结果表明,甘露糖在正常细胞和肿瘤细胞表面的表达水平普遍较高；唾液酸在肿瘤细胞表面的表达水平要明显高于其在正常细胞表面的表达,研究结果对于揭示肿瘤细胞的生物学行为具有一定的指导意义。同时,依据肿瘤细胞表面唾液酸与接骨木凝集素(SNA)的特异性相互作用,将该电化学生物传感器定量检测了人体肺、肝、前列腺组织中的肿瘤细胞和每个肿瘤细胞表面唾液酸的含量并获得了满意的结果。本方法具有灵敏度高、选择性好的优点,在肿瘤疾病的早期诊断中具有很好的应用前景。
Electrochemical biosensors, combining informatics and bio techno to gy, serve as an interdisciplinary frontier related to chemistry, biology, physics and electronics. Owing to their high sensitivity and selectivity, fast responses and advantages in miniaturization and online detection potentials, electrochemical biosensors have been extensively studied and applied in clinical medicine, food inspection and environment protection. Recently, nanomaterials, due to their unique properties, are widely used for developing electrochemical biosensors. The application of nanomaterials has brought a great momentum to electrochemical biosensors and opens new horizons for highly sensitive detection, which provides an avenue for high-throughput analysis of biological components on living cells.
In this dissertation, we studied on the development of the novel electrochemical biosensors based on nanomaterials, and their applications in the analysis of E. coli in water and glycans on living cells. The details are listed below:
Part 1. Development of an Electrochemical Immunoassay for Rapid Detection of E. coli Using Anodic Stripping Voltammetry Based on Cu@Au Nanoparticles as Antibody Labels
A sensitive electrochemical immunoassay for rapid detection of E. coli has been developed by Anodic Stripping Voltammetry (ASV) based on Cu@Au nanoparticles (NPs) as anti-E. coli antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM). After Cu@Au-labeled antibody reacted with the immobilized E. coli on PS-modified ITO chip, Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu2+ were determined at Nafion/Hg-modified glassy carbon electrode (GCE/Nafion/Hg) by ASV. The utilization of GCE/Nafion/Hg could enhance the sensitivity for Cu2+ detection with a concentration as low as 9.0×10-12 M. Since Cu@Au NPs labels were only present when antibody reacted with E. coli, the amount of Cu2+ directly reflected the number of E. coli. The technique could detect E. coli with a detection limit of 30 cfu/mL and the overall analysis could be completed in 2 h. By introducing a pre-enrichment step, a concentration of 3 cfu/10 mL E. coli in surface water was detected by the electrochemical immunoassay.
Part 2. A Supersensitive DNA Electrochemical Biosensor Based on Magnetic Beads for E. coli Detection by DNA Hybridization
A new type of DNA sequence-specific electrochemical biosensor based on magnetic beads for the detection of E. coli is reported in the present work. Alginic acid-coated cobalt magnetic beads, capped with 5'-(NH2) oligonucleotide and employed not only for magnetic separation but also as the solid adsorbent, were used as DNA probes to hybridize with the target E. coli DNA sequence. This assay was specific for E. coli detection depending on the uid A gene, which encodes for the enzymeβ-D-glucuronidase produced by E. coli strains. When daunomycin (DNR) was used as DNA hybridization indicator, the target sequences of E. coli hybridized with the probes resulted in the decrease of DNR reduction peak current, which was proportional to the E. coli concentration. The optimization of the hybridization detection was carried out and the specificity of the probes was also demonstrated. This DNA biosensor can be employed to detect a complementary target sequence for 0.30 nM and denatured PCR products for 0.50 ng/μL. The linear range of the developed biosensor for the detection of E. coli cells was from 1.0×102 to 2.0×103 cfu/mL with a detection limit of 50 cfu/mL.
Part 3. Optimized Ferrocene-Functionalized ZnO Nanorods for Signal Amplification in Electrochemical Immunoassay of E. coli
A novel strategy using ferrocene (Fc)-functionalized ZnO nanorods (NRs) for the amplified electrochemical immunoassay was developed in the present work. The detection antibody (dAb) and Fc were immobilized onto the surface of ZnO NRs, denoted as{dAb-ZnO-Fc} bioconjugates. The amount of dAb and Fc in the bioconjugates was investigated using the copper reduction/bicinchoninic acid reaction (BCA protein assay) and inductive coupled plasma-atomic emission spectroscopy (ICP-AES), respectively. Greatly amplified signal was achieved in the sandwich-type immunoassay when the dAb and Fc linked to ZnO NRs at a proper ratio. Using E. coli as a model antigen, the designed immunoassay showed an excellent analytical performance, and exhibited a wide dynamic response range of E. coli concentration from 1.0×102 to 1.0×106 cfu/mL with a detection limit of 50 cfu/mL (S/N=3). By introducing a pre-enrichment step, the detection of 5 cfu/10 mL E. coli in hospital sewage water was realized.
Part 4. Carbohydrate Derivative-Functionalized Electrochemical Biosensor for Competitive Assay of Glycan Expression on Living Cancer Cells
A novel carbohydrate derivative-functionalized electrochemical biosensor was developed for competitive analysis of glycan expression on living cancer cells. Mannose present on cancer cells derived from human lung, liver, and prostate was used as a model glycan. The biosensor was designed by employing the multiwalled carbon nanotube/Au nanoparticle (MWNT/AuNP) composite film to modify an electrode surface for thiomannosyl dimer ([mannose-S]2) assembling through Au-S bond. The{Con A-MWNT-HRP} bioconjugates, as recognition elements, were prepared by exploiting amplification effect of MWNTs for loading enormous horseradish peroxidase (HRP) labels and mannose-specific concanavalin A (Con A). Using a rapid and one-step competitive assay, the biosensor surface-confined thiomannosyl dimer competed with cell surface mannose to specifically recognize the {Con A-MWNT-HRP} bioconjugates. The proposed biosensor exhibited attractive performances for the analysis of cancer cells with wide linear ranges and low detection limits. The average amount of mannose on single cell surface was also detected to be 5.8×1010 molecules for A549 (lung cancer),1.3×1010 molecules for QGY-7703 (liver cancer), and 1.9×1010 molecules for LNCaP (prostate cancer). The carbohydrate derivative-functionalized electrochemical biosensor shows high sensitivity, selectivity and rapid response, and possesses promising application in the study of glycan changes on living cancer cells.
Part 5. Lectin-Based Biosensor Strategy for Electrochemical Assay of Glycan Expression on Living Cancer Cells
We report a novel lectin-based biosensor for electrochemical assay of cancer-associated glycosylation by comparative study of mannose and sialic acid expression on normal and cancer cells derived from human lung, liver and prostate. Using a sandwich format, high sensitivity and selectivity were achieved by combining the lectin-based biosensor with the{lectin-Au-Th} bioconjugates featuring lectin and thionine (Th) labels linked to gold nanoparticles (AuNPs) for signal amplification. The proposed strategy demonstrated that mannose exhibited high expression levels in both normal and cancer cells, while sialic acid was more abundant in cancer cells compared to normal ones. The differences in the two glycan expression indicated that sialic acid could serve as a potential bio marker for early cancer detection. The lectin-based biosensor was also successfully used to quantify cancer cells and evaluate the average amount of sialic acid on single cell surface, which could supply significant information on glycan functions in cancer progression. Overall, the lectin-based electrochemical biosensor provides an effective pathway to analyze glycan expression on living cells, and may greatly facilitate the medical diagnosis and treatment in early process of cancer.
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