快速检测农产品中大肠杆菌O157：H7的生物传感方法与仪器研究
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摘要
本博士论文针对现有农产品安全快速检测领域中关于致病菌检测的重大技术需求,结合分子生物学、纳米技术、材料科学、传感技术、仪器分析技术等领域的最新研究成果,探索用于农产品中致病菌快速检测的新型生物传感方法。以大肠杆菌O157：H7为研究对象,在充分分析与总结现有大肠杆菌O157：H7检测方法的基础上,提出以生物传感方法为研究主线,研究两种非标记型大肠杆菌O157：H7生物传感方法,即基于表面等离子体共振(Surface Plasmon Resonance, SPR)的生物传感方法和基于电化学阻抗谱(Electrochemical Impedance Spectroscopy, EIS)的生物传感方法。对这两种检测技术在大肠杆菌O157：H7检测领域的应用现状进行了研究,总结了其目前所存在的技术瓶颈。利用新型生物识别分子和纳米材料,解决这两种检测技术在大肠杆菌O157：H7快速、灵敏、方便检测中存在的问题,为寻求更为快捷、性能更高的大肠杆菌O157：H7检测方法提供理论依据。同时,结合仪器分析技术,开发便携式SPR检测仪和手持式阻抗仪,搭建用于现场样品中大肠杆菌O157：H7快速检测的平台。
主要研究内容、结果和结论如下：
(1)快速检测大肠杆菌O157：H7的SPR生物传感方法研究
基于消减抑制原理的SPR生物传感方法检测大肠杆菌O157：H7
分析和总结了直接SPR生物传感方法检测致病菌所存在的缺陷(致病菌体积超过了SPR分析方法300nm的最佳检测范围),提出了一种基于消减抑制原理的SPR生物传感方法,并验证了其检测大肠杆菌O157：H7的可行性。在消减抑制检测过程中,目标菌大肠杆菌O157：H7首先与抗体混合一段时间,再通过梯度离心过程来分离得到未与目标菌结合的剩余抗体,然后将分离得到的剩余抗体流动注射到修饰有二抗的SPR传感芯片表面,当抗体被二抗捕获后,便产生SPR信号。利用SPR信号与目标菌之间的关系,建立消减抑制SPR生物传感方法检测大肠杆菌O157：H7的模型。结果表明：该新型检测方法产生的SPR信号与浓度为3.0×104～3.0×108cfu mL-1的大肠杆菌O157：H7成反比。相比于直接SPR法和酶联免疫吸附测定法(检测限均为3.0×105cfu mL-1),消减抑制SPR生物传感方法有效提高了检测方法的灵敏度,其检测限整整下降了一个数量级(3.0×104cfu mL-1)。基于消减抑制原理的SPR生物传感方法有效地解决了直接SPR法存在的灵敏度低等缺陷,为SPR技术检测其它较大体积的分析物(细菌、真菌、细胞等)提供了新思路.
基于凝集素生物识别分子的SPR生物传感方法检测大肠杆菌O157：H7
针对抗体作为生物识别分子检测致病菌所存在的问题(抗体筛选过程复杂、成本高、易失活),提出了一种以凝集素作为生物识别分子的新型SPR生物传感方法,并验证了其检测农产品中大肠杆菌O157：H7的可行性。凝集素能与细菌表面的糖基进行特异性地结合,同时具有合成简便、成本低廉、体积小等优点。实验过程中,凝集素首先通过单分子自组装层固定在SPR芯片表面,当目标菌流过芯片表面时,凝集素分子就会将其捕获,利用SPR仪记录目标菌捕获后的SPR信号变化,从而达到检测目标菌的目的。选用五种凝集素(麦胚凝集素、刀豆凝集素、荆豆凝集素、花生凝集素、山槐凝集素)和三种细菌(大肠杆菌O157：H7、大肠杆菌DH5α、李斯特菌)进行筛选,得到对大肠杆菌O157：H7具有高亲和力、高特异性的凝集素。结果表明：当以麦胚凝集素修饰的SPR芯片来检测大肠杆菌O157：H7时,SPR信号最强,其与浓度范围为3×105～3×108cfu mL-1的目标菌成线性关系,检测限为3×103cfu mL-1。此外,将该新型SPR生物传感方法用于检测农产品样品(黄瓜和牛肉)中的大肠杆菌O157：H7,其检测限分别为3×104cfu mL-1和3×105cfu mL-1。基于凝集素生物识别分子的SPR生物传感方法有效地解决了抗体作为生物识别分子存在的成本高、稳定性差等缺陷,为SPR技术检测致病菌提供了新途径,同时也为开发低成本的SPR传感芯片用于致病菌的检测提供了借鉴。
(2)快速检测大肠杆菌O157：H7的EIS生物传感方法研究
基于纳米可抛弃传感器的EIS生物传感方法检测大肠杆菌O157：H7
针对现有EIS生物传感方法检测致病菌所存在的传感器昂贵、灵敏度不高等问题,提出了一种基于纳米可抛弃传感器的EIS生物传感方法,并验证了其检测大肠杆菌O157：H7的可行性。纳米可抛弃传感器是以商业化的丝网印刷电极为基底,采用电化学方法在其表面依次电沉积石墨烯和金纳米颗粒。其中石墨烯层采用先滴涂后还原的方法制备,即先电极表面滴涂一层氧化石墨烯,然后利用电流-时间曲线法将其还原为石墨烯。而金纳米颗粒层则是通过电流-时间曲线法一步电沉积在石墨烯层表面。大肠杆菌O157：H7抗体通过其与金纳米颗粒之间的静电吸附作用固定在电极表面,BSA用来封闭金颗粒表面的空余位点。当大肠杆菌O157：H7被电极表面的抗体捕获后,阻碍了电极表面的电子转移,从而使得电子转移电阻增大。结果表明：电子转移电阻的变化与浓度在1.5×103～1.5×107cfu mL-1之间的大肠杆菌O157：H7成正比,检测限为1.5×103cfu mL-1。特异性测试表明该纳米可抛弃传感器对非目标菌大肠杆菌DH5α、李斯特菌、金黄色葡萄球菌无明显响应信号。基于纳米可抛弃传感器的EIS生物传感方法具有分析速度快、灵敏度高、成本低等特点,为今后开发用于大规模样品筛选的低成本工具提供了可行的参考依据。
基于石墨烯纸传感器的EIS生物传感方法检测大肠杆菌O157：H7
结合当前传感技术领域的研究现状,提出了一种基于纸传感器的新型EIS生物传感方法,并验证了其快速检测农产品中大肠杆菌O157：H7的可行性。纸传感器采用石墨烯纸作为基底,并在其表面修饰金纳米颗粒层用以固定抗体。石墨烯纸通过真空抽滤和碘化氢还原两个步骤制取,金纳米颗粒修饰层则采用电流-时间曲线法一步电沉积在石墨烯纸表面。抗体通过生物素-亲和素法固定在金纳米颗粒表面位点,多余的位点则用BSA封闭,当大肠杆菌与纸传感器表面的抗体结合后,会引起纸传感器表面的界面特性变化。利用EIS来研究这种界面特性变化,发现EIS中的电子转移电阻值与纸传感器表面目标菌的捕获量成一定的正比关系。结果表明：该新型EIS生物传感方法可以检测浓度范围为1.5×102～1.5×107cfu mL-1的大肠杆菌O157：H7,检测限为1.5×102cfu mL-1。此外,该新型传感方法还具有较高的特异性、较好的重复性和稳定性及较高的机械强度。基于石墨烯纸传感器的EIS生物传感方法为研发低成本、高灵敏、快速简便的致病菌检测方法提供了新思路。
(3)检测大肠杆菌O157：H7的便携式仪器
基于SPR技术的小型化大肠杆菌O157：H7快速检测仪
探索搭建基于SPR技术的小型化大肠杆菌O157：H7快速检测仪的可行性。小型化SPR检测仪是由集成式SpreetaTM SPR传感系统和本实验室开发的传感控制硬件及软件构成。硬件部分主要包括主控部分设计,软件部分包括芯片程序和仪器上位机软件两部分。通过检测乙醇和葡萄糖溶液,验证了该仪器检测结果的稳定性和准确性。结合前期研究的SPR生物传感方法,将上述开发的小型化SPR检测仪用于大肠杆菌O157：H7的检测。结果显示,该小型化SPR检测仪能够用于样品中3.0×105～3.0×108cfu mL-1浓度范围内大肠杆菌O157：H7的检测。此外,该分析仪器还能实现多通道同时检测,且检测速度快、样品用量少、小型便携,可以作为一种用于大肠杆菌O157：H7现场快速检测的有效分析工具。
基于阻抗技术的手持式大肠杆菌O157：H7快速检测仪
探索搭建基于阻抗技术的手持式大肠杆菌O157：H7快速检测仪的可行性。手持式阻抗检测仪是由纳米材料修饰的丝网印刷电极和本实验室开发的小型阻抗仪构成。结合前期研究的纳米材料修饰方法来制备丝网印刷电极,首先在丝网印刷电极表面滴涂一层氧化石墨烯膜,然后使用电化学法进行还原,接着通过电化学方法沉积一层金纳米颗粒,最后抗体通过静电吸附固定在金纳米颗粒表面。研究表明,小型阻抗仪在1kHz频率下测试到的阻抗值与大肠杆菌O157：H7的浓度成正比。该小型阻抗传感器对大肠杆菌O157：H7的检测范围为1.5×104～1.5×107cfu mL-1,检测限为1.5×104cfumL-1。该小型阻抗仪具有测量结果准确,精度高,检测速度快等特点,同时小型便携,可以作为一种用于大肠杆菌O157：H7现场快速检测的有效分析工具。
This work aimed to develop novel biosensing methods for the detection of foodborne pathogens in agricultrual products by using the latest research achievements in the field of molecular biology, nanotechnology, material science, sensing technique, and instrumental analysis technique. The Escherichia coli O157:H7(E. coli O157:H7) was selected as the target bacteria. After the careful study on the current detection methods for E. coli O157:H7, we chose two kinds of unlabeled biosensing methods for E. coli O157:H7detection in our study, including surface plasmon resonance (SPR) based biosensing method and electrochemical impedance spectroscopy (EIS) based biosensing method. However, some limitations still remain as the challenges when dealing with large-scale applications. Here, we used novel detection mode, bio-recognition molecule (e.g. lectin) and nanomaterials (e.g. gold nanoparticles and graphene) to develop novel biosensing methods for rapid and sensitive detection of E. coli O157:H7. Furthermore, we developed a portable SPR instrument and a handheld impedance instrument for E. coli O157:H7detection. This work provides several effective and useful biosensing methods for the detection of E. coli O157:H7, which may hold great promising in routine sensing applications.
Main results and conclusions are as follows:
(1) SPR biosensing methods for E. coli O157:H7detection
Subtractive inhibition mechanism based SPR biosensing method for E. coli O157:H7detection
A novel SPR biosensing method by means of a subtractive inhibition assay was developed for the sensitive detection of E. coli O157:H7in order to solve the problems in direct SPR detection method. In the subtractive inhibition assay, E. coli O157:H7cells and goat polyclonal antibodies for E. coli O157:H7were firstly incubated for a while. Then, the E. coli O157:H7cells that bound with antibodies were removed by stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using BIAcore3000biosensor. We used the relationship between SPR signal and concentrations of E. coli O157:H7to establish the detection model. Results show that the signal was inversely correlated with the concentrations of E. coli O157:H7cells in a range from3.0x104to3.0x108cfu mL-1with the detection limit of3.0x104cfu mL-1. This detection limit was lower than those obtained by direct SPR method (in which the antibodies was firstly immobilized on the chip surface and then to capture E. coli O157:H7) and ELISA. The subtractive inhibition mechanism based SPR biosensing method could solve the drawbacks in current direct SPR method (e.g. low sensitivity), which may provide a useful way to implement SPR technique for other pathogens detection.
Lectin based SPR biosensing method for E. coli O157:H7detection
A novel SPR biosensing method using lectin as bio-recognition molecule was developed for the rapid and sensitive detection of E. coli O157:H7. The selective interaction of lectins with carbohydrate components on the surface of bacterial cells was used as the recognition principle for the detection of E. coli O157:H7. This novel bio-recognition molecule shows some intrinsic advantages over antibodies, such as inexpensive and physicochemical stable. In the detection procedure, lectin molecules were firstly immobilized onto the chip surface via molecular self-assembly layer. When the target bacteria was captured by the lectins immobilized on the chip, the SPR signal increased, which was correlated with the concentrations of bacteria cells. Five types of lectins from Triticum vulgaris, Canavailia ensiformis, Ulex europaeus, Arachis hypogaea, and Maackia amurensis, were employed to evaluate the selectivity of the approach for binding E. coli O157:H7effectively. A linear range of3×105-3×108cfu mL-1and a detection limit of3x103cfu mL-1were obtained for determination of E. coli O157:H7when using the lectin from Triticum vulgaris as the bio-recognition element. Furthermore, the proposed biosensor was used to detect E. coli O157:H7in agricultural products. The detection limits in case of E. coli O157:H7contaminated cucumber samples and ground beef samples were3.0×104and3.0×105cfu mL-1, respectively. This work firstly reveals that the lectin could be an effective and promising bio-recognition material for constructing SPR biosensors for pathogens detection, which may have wide applications in routine sensing applications.
(2) EIS biosensing methods for E. coli O157:H7detection
Disposable nanocomposite sensor based EIS biosensing method for E. coli O157:H7detection
A novel EIS biosensing method using disposable nanocomposite sensor for the low-cost and high-sensitive detection of E. coli O157:H7was developed. The commercial screen-printed electrode was used as the substrate in the proposed sensor. Then, a graphene oxide film was formed on the surface of screen-printed electrode using simple drop-coating method. The reduction of graphene oxide into graphene was performed by amperometric i-t curve method. Then, one-step electrodeposition method was employed to prepare gold nanoparticles on the graphene film surface. Antibody was immobilized on the surface of gold nanoparticles through electrostatic adsorption, and then BSA was used to block the redundant active sites on gold nanoparticles. When E. coli O157:H7was captured by the disposable sensor, the electron transfer kinetics on electrode surface was hindered, inducing the increase on electron transfer resistance. Results show that the change of electron transfer resistance is directly proportional to concentrations of E. coli O157:H7. A wide linear range of1.5×103-1.5×107cfu mL-1and a low detection limit of1.5×103cfu mL-1were obtained using the developed biosensing method. Additionally, nontarget bacteria, such as E. coli DH5a, Listeria monocytogenes, Staphylococcus aureus, were used to demonstrate the high selectivity and specificity of the biosensor. High-performance, low cost, and easy operation make the developed EIS biosensing method very attractive in routine sensing applications.
Graphene paper sensor based EIS biosensing method for E. coli O157:H7detection
In this study, a low-cost and robust impedimetric immunosensor based on gold nanoparticles modified free-standing graphene paper electrode for rapid and sensitive detection of E. coli O157:H7was developed. Graphene paper was prepared by chemical reduction of graphene oxide paper obtained from vacuum filtration method. Scanning electron microscope, Raman spectroscopy and X-ray diffraction techniques were employed to investigate the surface morphology and crystal structure of the prepared graphene paper. The gold nanoparticles were grown on the surface of graphene paper electrode by one-step electrodeposition technique. The immobilization of anti-E. coli O157:H7antibodies on paper electrode were performed via biotin-streptavidin system. Electrochemical impedance spectroscopy was used to detect E. coli O157:H7captured on the paper electrode. Results show that the developed paper immunosensor possesses greatly enhanced sensing performance, such as wide liner range (1.5×102-1.5×107cfu mL-1), low detection limit (1.5×102cfu mL-1), and excellent specificity. Furthermore, flexible test demonstrate the graphene paper based sensing device has high tolerability to mechanical stress. The strategy of structurally integrating metal nanomaterials, graphene paper, and biorecognition molecules would provide new insights into design of flexible immunosensors for routine sensing applications.
(3) Portable instruments for the detection of E. coli O157:H7
Construction of portable SPR instrument for rapid detection of E. coli O157:H7
The feasibility of the construction of portable SPR instrument for the rapid detection of E. coli O157:H7was investigated. This instrument is composed of commercial integrated SpreetaTM SPR sensor system and sensing control part developed by our laboratory. The sensing control part includes hardware and supported software. The stability and precision of the developed instrument was verified by testing the ethanol and glucose solutions. Furthermore, the developed instrument was employed to detection E. coli O157:H7by combining with the established SPR biosensing methods in previous work. Results show that the miniature SPR instrument could be used to detect E. coli O157:H7in the range of3.0×105-3.0×108cfu mL-1. Additionally, this instrument could achieve multi-channel simultaneous detection of various samples. Fast, less sample, and portable make the developed instrument as an effective tool for on-site and rapid detection of E. coli O157:H7.
Construction of handheld impedance instrument for rapid detection of E. coli O157:H7
The feasibility of the construction of handheld impedance instrument for the rapid detection of E. coli O157:H7was investigated. This instrument consists of nanomaterials modified screen-printed electrode and handheld impedance meter. The screen-printed electrode was prepared with the same method mentioned in the previous work. When E. coli O157:H7was captured by antibody modified electrode, the impedance at the frequency of1kHz measured by handheld impedance meter increased. The portable instrument was used for the detection of E. coli O157:H7at the range of1.5×104-1.5x107cfu mL-1and detection limit of1.5x104cfu mL-1. Excellent accuracy, precision, and portability make the developed instrument as an effective tool for on-site and rapid detection of E. coli O157:H7.
主要研究内容、结果和结论如下：
(1)快速检测大肠杆菌O157：H7的SPR生物传感方法研究
基于消减抑制原理的SPR生物传感方法检测大肠杆菌O157：H7
分析和总结了直接SPR生物传感方法检测致病菌所存在的缺陷(致病菌体积超过了SPR分析方法300nm的最佳检测范围),提出了一种基于消减抑制原理的SPR生物传感方法,并验证了其检测大肠杆菌O157：H7的可行性。在消减抑制检测过程中,目标菌大肠杆菌O157：H7首先与抗体混合一段时间,再通过梯度离心过程来分离得到未与目标菌结合的剩余抗体,然后将分离得到的剩余抗体流动注射到修饰有二抗的SPR传感芯片表面,当抗体被二抗捕获后,便产生SPR信号。利用SPR信号与目标菌之间的关系,建立消减抑制SPR生物传感方法检测大肠杆菌O157：H7的模型。结果表明：该新型检测方法产生的SPR信号与浓度为3.0×104～3.0×108cfu mL-1的大肠杆菌O157：H7成反比。相比于直接SPR法和酶联免疫吸附测定法(检测限均为3.0×105cfu mL-1),消减抑制SPR生物传感方法有效提高了检测方法的灵敏度,其检测限整整下降了一个数量级(3.0×104cfu mL-1)。基于消减抑制原理的SPR生物传感方法有效地解决了直接SPR法存在的灵敏度低等缺陷,为SPR技术检测其它较大体积的分析物(细菌、真菌、细胞等)提供了新思路.
基于凝集素生物识别分子的SPR生物传感方法检测大肠杆菌O157：H7
针对抗体作为生物识别分子检测致病菌所存在的问题(抗体筛选过程复杂、成本高、易失活),提出了一种以凝集素作为生物识别分子的新型SPR生物传感方法,并验证了其检测农产品中大肠杆菌O157：H7的可行性。凝集素能与细菌表面的糖基进行特异性地结合,同时具有合成简便、成本低廉、体积小等优点。实验过程中,凝集素首先通过单分子自组装层固定在SPR芯片表面,当目标菌流过芯片表面时,凝集素分子就会将其捕获,利用SPR仪记录目标菌捕获后的SPR信号变化,从而达到检测目标菌的目的。选用五种凝集素(麦胚凝集素、刀豆凝集素、荆豆凝集素、花生凝集素、山槐凝集素)和三种细菌(大肠杆菌O157：H7、大肠杆菌DH5α、李斯特菌)进行筛选,得到对大肠杆菌O157：H7具有高亲和力、高特异性的凝集素。结果表明：当以麦胚凝集素修饰的SPR芯片来检测大肠杆菌O157：H7时,SPR信号最强,其与浓度范围为3×105～3×108cfu mL-1的目标菌成线性关系,检测限为3×103cfu mL-1。此外,将该新型SPR生物传感方法用于检测农产品样品(黄瓜和牛肉)中的大肠杆菌O157：H7,其检测限分别为3×104cfu mL-1和3×105cfu mL-1。基于凝集素生物识别分子的SPR生物传感方法有效地解决了抗体作为生物识别分子存在的成本高、稳定性差等缺陷,为SPR技术检测致病菌提供了新途径,同时也为开发低成本的SPR传感芯片用于致病菌的检测提供了借鉴。
(2)快速检测大肠杆菌O157：H7的EIS生物传感方法研究
基于纳米可抛弃传感器的EIS生物传感方法检测大肠杆菌O157：H7
针对现有EIS生物传感方法检测致病菌所存在的传感器昂贵、灵敏度不高等问题,提出了一种基于纳米可抛弃传感器的EIS生物传感方法,并验证了其检测大肠杆菌O157：H7的可行性。纳米可抛弃传感器是以商业化的丝网印刷电极为基底,采用电化学方法在其表面依次电沉积石墨烯和金纳米颗粒。其中石墨烯层采用先滴涂后还原的方法制备,即先电极表面滴涂一层氧化石墨烯,然后利用电流-时间曲线法将其还原为石墨烯。而金纳米颗粒层则是通过电流-时间曲线法一步电沉积在石墨烯层表面。大肠杆菌O157：H7抗体通过其与金纳米颗粒之间的静电吸附作用固定在电极表面,BSA用来封闭金颗粒表面的空余位点。当大肠杆菌O157：H7被电极表面的抗体捕获后,阻碍了电极表面的电子转移,从而使得电子转移电阻增大。结果表明：电子转移电阻的变化与浓度在1.5×103～1.5×107cfu mL-1之间的大肠杆菌O157：H7成正比,检测限为1.5×103cfu mL-1。特异性测试表明该纳米可抛弃传感器对非目标菌大肠杆菌DH5α、李斯特菌、金黄色葡萄球菌无明显响应信号。基于纳米可抛弃传感器的EIS生物传感方法具有分析速度快、灵敏度高、成本低等特点,为今后开发用于大规模样品筛选的低成本工具提供了可行的参考依据。
基于石墨烯纸传感器的EIS生物传感方法检测大肠杆菌O157：H7
结合当前传感技术领域的研究现状,提出了一种基于纸传感器的新型EIS生物传感方法,并验证了其快速检测农产品中大肠杆菌O157：H7的可行性。纸传感器采用石墨烯纸作为基底,并在其表面修饰金纳米颗粒层用以固定抗体。石墨烯纸通过真空抽滤和碘化氢还原两个步骤制取,金纳米颗粒修饰层则采用电流-时间曲线法一步电沉积在石墨烯纸表面。抗体通过生物素-亲和素法固定在金纳米颗粒表面位点,多余的位点则用BSA封闭,当大肠杆菌与纸传感器表面的抗体结合后,会引起纸传感器表面的界面特性变化。利用EIS来研究这种界面特性变化,发现EIS中的电子转移电阻值与纸传感器表面目标菌的捕获量成一定的正比关系。结果表明：该新型EIS生物传感方法可以检测浓度范围为1.5×102～1.5×107cfu mL-1的大肠杆菌O157：H7,检测限为1.5×102cfu mL-1。此外,该新型传感方法还具有较高的特异性、较好的重复性和稳定性及较高的机械强度。基于石墨烯纸传感器的EIS生物传感方法为研发低成本、高灵敏、快速简便的致病菌检测方法提供了新思路。
(3)检测大肠杆菌O157：H7的便携式仪器
基于SPR技术的小型化大肠杆菌O157：H7快速检测仪
探索搭建基于SPR技术的小型化大肠杆菌O157：H7快速检测仪的可行性。小型化SPR检测仪是由集成式SpreetaTM SPR传感系统和本实验室开发的传感控制硬件及软件构成。硬件部分主要包括主控部分设计,软件部分包括芯片程序和仪器上位机软件两部分。通过检测乙醇和葡萄糖溶液,验证了该仪器检测结果的稳定性和准确性。结合前期研究的SPR生物传感方法,将上述开发的小型化SPR检测仪用于大肠杆菌O157：H7的检测。结果显示,该小型化SPR检测仪能够用于样品中3.0×105～3.0×108cfu mL-1浓度范围内大肠杆菌O157：H7的检测。此外,该分析仪器还能实现多通道同时检测,且检测速度快、样品用量少、小型便携,可以作为一种用于大肠杆菌O157：H7现场快速检测的有效分析工具。
基于阻抗技术的手持式大肠杆菌O157：H7快速检测仪
探索搭建基于阻抗技术的手持式大肠杆菌O157：H7快速检测仪的可行性。手持式阻抗检测仪是由纳米材料修饰的丝网印刷电极和本实验室开发的小型阻抗仪构成。结合前期研究的纳米材料修饰方法来制备丝网印刷电极,首先在丝网印刷电极表面滴涂一层氧化石墨烯膜,然后使用电化学法进行还原,接着通过电化学方法沉积一层金纳米颗粒,最后抗体通过静电吸附固定在金纳米颗粒表面。研究表明,小型阻抗仪在1kHz频率下测试到的阻抗值与大肠杆菌O157：H7的浓度成正比。该小型阻抗传感器对大肠杆菌O157：H7的检测范围为1.5×104～1.5×107cfu mL-1,检测限为1.5×104cfumL-1。该小型阻抗仪具有测量结果准确,精度高,检测速度快等特点,同时小型便携,可以作为一种用于大肠杆菌O157：H7现场快速检测的有效分析工具。
This work aimed to develop novel biosensing methods for the detection of foodborne pathogens in agricultrual products by using the latest research achievements in the field of molecular biology, nanotechnology, material science, sensing technique, and instrumental analysis technique. The Escherichia coli O157:H7(E. coli O157:H7) was selected as the target bacteria. After the careful study on the current detection methods for E. coli O157:H7, we chose two kinds of unlabeled biosensing methods for E. coli O157:H7detection in our study, including surface plasmon resonance (SPR) based biosensing method and electrochemical impedance spectroscopy (EIS) based biosensing method. However, some limitations still remain as the challenges when dealing with large-scale applications. Here, we used novel detection mode, bio-recognition molecule (e.g. lectin) and nanomaterials (e.g. gold nanoparticles and graphene) to develop novel biosensing methods for rapid and sensitive detection of E. coli O157:H7. Furthermore, we developed a portable SPR instrument and a handheld impedance instrument for E. coli O157:H7detection. This work provides several effective and useful biosensing methods for the detection of E. coli O157:H7, which may hold great promising in routine sensing applications.
Main results and conclusions are as follows:
(1) SPR biosensing methods for E. coli O157:H7detection
Subtractive inhibition mechanism based SPR biosensing method for E. coli O157:H7detection
A novel SPR biosensing method by means of a subtractive inhibition assay was developed for the sensitive detection of E. coli O157:H7in order to solve the problems in direct SPR detection method. In the subtractive inhibition assay, E. coli O157:H7cells and goat polyclonal antibodies for E. coli O157:H7were firstly incubated for a while. Then, the E. coli O157:H7cells that bound with antibodies were removed by stepwise centrifugation process. The remaining free unbound antibodies were detected through interaction with rabbit anti-goat IgG polyclonal antibodies immobilized on the sensor chip using BIAcore3000biosensor. We used the relationship between SPR signal and concentrations of E. coli O157:H7to establish the detection model. Results show that the signal was inversely correlated with the concentrations of E. coli O157:H7cells in a range from3.0x104to3.0x108cfu mL-1with the detection limit of3.0x104cfu mL-1. This detection limit was lower than those obtained by direct SPR method (in which the antibodies was firstly immobilized on the chip surface and then to capture E. coli O157:H7) and ELISA. The subtractive inhibition mechanism based SPR biosensing method could solve the drawbacks in current direct SPR method (e.g. low sensitivity), which may provide a useful way to implement SPR technique for other pathogens detection.
Lectin based SPR biosensing method for E. coli O157:H7detection
A novel SPR biosensing method using lectin as bio-recognition molecule was developed for the rapid and sensitive detection of E. coli O157:H7. The selective interaction of lectins with carbohydrate components on the surface of bacterial cells was used as the recognition principle for the detection of E. coli O157:H7. This novel bio-recognition molecule shows some intrinsic advantages over antibodies, such as inexpensive and physicochemical stable. In the detection procedure, lectin molecules were firstly immobilized onto the chip surface via molecular self-assembly layer. When the target bacteria was captured by the lectins immobilized on the chip, the SPR signal increased, which was correlated with the concentrations of bacteria cells. Five types of lectins from Triticum vulgaris, Canavailia ensiformis, Ulex europaeus, Arachis hypogaea, and Maackia amurensis, were employed to evaluate the selectivity of the approach for binding E. coli O157:H7effectively. A linear range of3×105-3×108cfu mL-1and a detection limit of3x103cfu mL-1were obtained for determination of E. coli O157:H7when using the lectin from Triticum vulgaris as the bio-recognition element. Furthermore, the proposed biosensor was used to detect E. coli O157:H7in agricultural products. The detection limits in case of E. coli O157:H7contaminated cucumber samples and ground beef samples were3.0×104and3.0×105cfu mL-1, respectively. This work firstly reveals that the lectin could be an effective and promising bio-recognition material for constructing SPR biosensors for pathogens detection, which may have wide applications in routine sensing applications.
(2) EIS biosensing methods for E. coli O157:H7detection
Disposable nanocomposite sensor based EIS biosensing method for E. coli O157:H7detection
A novel EIS biosensing method using disposable nanocomposite sensor for the low-cost and high-sensitive detection of E. coli O157:H7was developed. The commercial screen-printed electrode was used as the substrate in the proposed sensor. Then, a graphene oxide film was formed on the surface of screen-printed electrode using simple drop-coating method. The reduction of graphene oxide into graphene was performed by amperometric i-t curve method. Then, one-step electrodeposition method was employed to prepare gold nanoparticles on the graphene film surface. Antibody was immobilized on the surface of gold nanoparticles through electrostatic adsorption, and then BSA was used to block the redundant active sites on gold nanoparticles. When E. coli O157:H7was captured by the disposable sensor, the electron transfer kinetics on electrode surface was hindered, inducing the increase on electron transfer resistance. Results show that the change of electron transfer resistance is directly proportional to concentrations of E. coli O157:H7. A wide linear range of1.5×103-1.5×107cfu mL-1and a low detection limit of1.5×103cfu mL-1were obtained using the developed biosensing method. Additionally, nontarget bacteria, such as E. coli DH5a, Listeria monocytogenes, Staphylococcus aureus, were used to demonstrate the high selectivity and specificity of the biosensor. High-performance, low cost, and easy operation make the developed EIS biosensing method very attractive in routine sensing applications.
Graphene paper sensor based EIS biosensing method for E. coli O157:H7detection
In this study, a low-cost and robust impedimetric immunosensor based on gold nanoparticles modified free-standing graphene paper electrode for rapid and sensitive detection of E. coli O157:H7was developed. Graphene paper was prepared by chemical reduction of graphene oxide paper obtained from vacuum filtration method. Scanning electron microscope, Raman spectroscopy and X-ray diffraction techniques were employed to investigate the surface morphology and crystal structure of the prepared graphene paper. The gold nanoparticles were grown on the surface of graphene paper electrode by one-step electrodeposition technique. The immobilization of anti-E. coli O157:H7antibodies on paper electrode were performed via biotin-streptavidin system. Electrochemical impedance spectroscopy was used to detect E. coli O157:H7captured on the paper electrode. Results show that the developed paper immunosensor possesses greatly enhanced sensing performance, such as wide liner range (1.5×102-1.5×107cfu mL-1), low detection limit (1.5×102cfu mL-1), and excellent specificity. Furthermore, flexible test demonstrate the graphene paper based sensing device has high tolerability to mechanical stress. The strategy of structurally integrating metal nanomaterials, graphene paper, and biorecognition molecules would provide new insights into design of flexible immunosensors for routine sensing applications.
(3) Portable instruments for the detection of E. coli O157:H7
Construction of portable SPR instrument for rapid detection of E. coli O157:H7
The feasibility of the construction of portable SPR instrument for the rapid detection of E. coli O157:H7was investigated. This instrument is composed of commercial integrated SpreetaTM SPR sensor system and sensing control part developed by our laboratory. The sensing control part includes hardware and supported software. The stability and precision of the developed instrument was verified by testing the ethanol and glucose solutions. Furthermore, the developed instrument was employed to detection E. coli O157:H7by combining with the established SPR biosensing methods in previous work. Results show that the miniature SPR instrument could be used to detect E. coli O157:H7in the range of3.0×105-3.0×108cfu mL-1. Additionally, this instrument could achieve multi-channel simultaneous detection of various samples. Fast, less sample, and portable make the developed instrument as an effective tool for on-site and rapid detection of E. coli O157:H7.
Construction of handheld impedance instrument for rapid detection of E. coli O157:H7
The feasibility of the construction of handheld impedance instrument for the rapid detection of E. coli O157:H7was investigated. This instrument consists of nanomaterials modified screen-printed electrode and handheld impedance meter. The screen-printed electrode was prepared with the same method mentioned in the previous work. When E. coli O157:H7was captured by antibody modified electrode, the impedance at the frequency of1kHz measured by handheld impedance meter increased. The portable instrument was used for the detection of E. coli O157:H7at the range of1.5×104-1.5x107cfu mL-1and detection limit of1.5x104cfu mL-1. Excellent accuracy, precision, and portability make the developed instrument as an effective tool for on-site and rapid detection of E. coli O157:H7.
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