无线磁弹性化学/生物传感器的研制及应用研究
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摘要
本论文致力于无线磁弹性化学/生物传感器的研制及其在生化分析中的应用研究。在外加交变磁场中,磁性膜片受磁场激励产生磁矩,将磁能转换为机械能,并产生沿长度方向的伸缩振动,即磁致伸缩(magnetostrictive)。当外加交变磁场的频率与磁性膜片的机械振动频率相等时,磁片产生共振,此时振幅最大,对应的振动频率为磁性膜片的共振频率。当磁性膜片传感器的表面性质(质量负载、粘弹性等)发生变化时,其共振频率随之发生改变。由于传感器材料本身是磁性的,其伸缩振动产生磁通,产生的磁通可由检测线圈探测到,信号经放大后由外部仪器测定。磁弹性传感器中信号的激发与传送是通过磁场进行的,传感器与检测仪器之间不需任何的物理连接,属于无线无源(wireless, passive)传感器。磁弹性传感器无线无源的特征使其在活体、在体及无损检测等领域具有广泛的应用前景。基于上述原理,本论文主要进行了以下几方面的研究工作:
(1)测定体液pH用无线磁弹性传感器研制:基于pH敏感聚合物,制备了无线磁弹性pH传感器;研究了pH聚合物组成对传感器稳定性和响应灵敏度的影响,考察了体液中干扰物对传感器响应的影响。随溶液pH变化,pH敏感聚合物在碱性溶液中膨胀,酸性溶液中收缩,从而引起传感器表面质量负载和共振频率发生变化。基于此构建的磁弹性pH传感器的共振频率随体液pH升高而下降,在pH 5 ~ 8之间呈近似线性响应,响应灵敏度为200 Hz/pH,可准确测定0.1 pH的变化。对体液pH体外测试表明,制备的pH传感器有望开发为可进行活体在体分析的无线磁弹性传感装置。
(2)测定血糖用无线磁弹性传感器研制:基于上一节磁弹性pH传感器,进一步制备了无线磁弹性葡萄糖生物传感器;研究了传感器对体外血糖测定的稳定性、生物相容性及响应灵敏度。采用pH敏感聚合物膜对酶催化反应进行放大以提高传感器的灵敏度。在葡萄糖氧化酶(GOD)和过氧化氢酶(catalase)的共同存在下,催化氧化葡萄糖生成葡萄糖酸,引起pH聚合物收缩,导致传感器表面质量负载减小、传感器共振频率增加。制备的无线磁弹性葡萄糖传感器的共振频移随血糖浓度的升高而上升,在血糖浓度为2.5 ~ 20 mM之间,传感器的响应是线性、可逆的,对血糖的检测限为1.2 mM。传感器对血糖体外测定结果表明,制备的无线磁弹性葡萄糖生物传感器有望应用于活体在体分析,有潜力作为植入传感器实现对血糖无线监测。
(3)无线磁弹性双酶葡萄糖传感器研制:基于辣根过氧化物酶(HRP)和葡萄糖氧化酶(GOD)双酶层,制备了无线磁弹性双酶葡萄糖生物传感器。利用酶的催化沉淀反应对传感器的响应进行质量放大以提高其灵敏度。葡萄糖氧化酶催化氧化葡萄糖产生葡萄糖酸和过氧化氢(H2O2),产生的过氧化氢在辣根过氧化物酶存在下,催化氧化3,3′,5,5′-四甲基联苯氨(TMB)生成沉淀。产生的沉淀沉积在传感器表面,导致传感器表面质量负载增加,传感器共振频率下降。基于此构建的无线磁弹性双酶葡萄糖传感器的共振频移随葡萄糖浓度的升高而上升,在葡萄糖浓度为5 ~ 50 mM范围内呈线性响应,对葡萄糖的检测限为2 mM。
(4)无线磁弹性微生物传感器研制:以预聚合了聚氨酯膜的磁性膜片为敏感元件,制备了无线磁弹性微生物传感器;研究了微生物生长导致培养基性质(如黏度、密度等)改变对传感器共振频率的影响,考察了细菌在传感器表面吸附对传感器响应的贡献。基于药物对微生物生长的抑制,利用无线磁弹性微生物传感器进行菌株药敏试验研究,依据抗菌药物对细菌的抑制作用可进行抗生素的药效评测。微生物生长时消耗培养基,将大分子的蛋白质分解为小分子导致培养液粘弹性发生变化,同时微生物在传感器表面的吸附引起质量变化。通过测定微生物培养时传感器共振频率和振幅的变化对微生物进行快速检测,获得了绿脓杆菌(P. areuginosa)和结核杆菌(M. tuberculosis)的生长曲线,考察了不同细菌特征生长曲线的差异,据此可望对细菌进行初步的分型。利用石英晶体微天平(QCM)、显微成像等手段研究了培养液性质变化和细菌吸附对传感器共振频率的影响。传感器对绿脓杆菌和结核杆菌检测浓度范围分别为103 ~ 108和104 ~ 109 cells/mL,相应检测限分别为103和104 cells/mL。基于此,研究了可用于细菌早期诊断和快速分析的无线磁弹性传感检测新方法。
(5)无线磁弹性传感器用于细菌生物被膜形成机制的研究。基于绿脓杆菌(P. areuginosa)易于在固体界面粘附且形成细菌生物被膜(biofilms)的特性,利用无线磁弹性传感器初步研究了细菌生物被膜的形成机制。预聚合了聚氨酯膜的磁性膜片作为敏感元件,同时测定了细菌生物被膜形成过程中传感器共振频率和振幅的变化。细菌生物被膜的形成改变了传感器表面的粘弹性和质量负载。由磁弹性传感器共振信号的变化,可探测细菌生物被膜的形成过程及生物被膜的成熟。无线磁弹性传感器是基于磁致伸缩原理设计的,通过改变激励信号的强度,考察了细菌生物被膜在传感器表面的粘附强度,形成的细菌生物被膜在传感器表面粘附牢固。
This dissertation is devoted to the fabrication of wireless magnetoelastic bio/chemical sensors and their applications in bio/chemical analysis. In response to a time-varying AC magnetic field, a magnetoelastic ribbon efficiently couples and converts magnetic energy into mechanical energy. The elastic energy mechanically deforms the sensor, causing it mechanically vibrate along its length. When the frequency of the applied AC magnetic field is equal to the mechanical resonance frequency of the ribbon, the vibration amplitude is maximal and the sensor vibrates at its characteristic resonance frequency that shifts in response to mass loading. Since the magnetoelastic material is magnetostrictive, the vibration of the sensor in turn generates a time varying magnetic flux, which can be remotely measured with a set of pick-up coils. The excitation and transmission of signals of magnetoelastic sensor are carried out remotely by magnetic field. No direct physical connections between the sensor and the detection system are required, nor is any internal power required. The wireless nature of the magnetoelastic sensor makes it a powerful candidate for in situ and in vivo analysis. The details of work are summarized as follows:
(1) A wireless magnetoelastic sensor for determination of body fluid acidity is developed. The magnetoelastic pH sensor was fabricated by coating a layer of pH-sensitive polymer on a magnetostrictive ribbon, which pre-coated with a layer of polyurethane film. The effects of composition of pH-sensitive polymer on sensor stability and sensitivity, and the interferents existing in physiological fluid on sensors responses were investigated. The sensor transduction signal is derived from the pH-polymer mass differences between its relatively shrunken state in acidic solution and relatively swollen state in alkaline solution. The shift in the resonant frequency of magnetoelastic pH sensor is linear and reversible between pH 5 and 8 with a sensitivity of 200 Hz/pH and a measurement resolution of 0.1 pH. The proposed magnetoelastic pH sensor platform offers a great opportunity for developing a useful in vivo and in situ physiological pH measurement technology.
(2) A wireless magnetoelastic glucose biosensor in blood is developed. The glucose biosensor was fabricated based on magnetoelastic pH sensor (pH-polymer used as sensing film). The magnetoelastic biosensor was fabricated by coating the ribbon-like, magnetoelastic sensor with a pH-sensitive polymer and a biolayer of glucose oxidase (GOD) and catalase. pH-sensitive polymer is used as a sensing film to amplify the mass change associated with enzyme biocatalytic reaction and to increase the sensor sensitivity. The GOD-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the sensor mass loading and increases the resonant frequency. At glucose concentration range of 2.5 ~ 20.0 mM, the biosensor responses are reversible and linear, with a detection limit of 1.2 mM. The proposed magnetoelastic glucose biosensor can potentially be used as a planted sensor and applied to in vivo and in situ measurement of glucose concentrations in body blood.
(3) A wireless magnetoelastic bienzyme glucose biosensor is described. The bienzyme biosensor was fabricated by first coating the magnetoelastic-ribbon with horseradish peroxidase (HRP) and upon it a layer of glucose oxidase (GOD). Sensor sensitivity is increased by amplifying the mass change associated with enzyme reaction by biocatalytic precipitation. The GOD catalyzed oxidation of glucose produces gluconic acid and H2O2, and the generated H2O2 biocatalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to insoluble product in the presence of HRP. The insoluble product accumulated on the sensor surface, which resulted in the increasing in sensor mass loading and decreasing in resonant frequency. And the extent of resulting sensor resonant frequency changing, correlated with the amount of glucose. The biosensor response is linear in the range of glucose concentrations of 5 ~ 50 mM, with a detection limit of 2 mM. The biosensor is applied to determine glucose concentration in urine sample.
(4) A wireless magnetoleastic microorganism sensor is developed for the early diagnosis and rapid detection of bacteria. The microorganism sensor is fabricated by coating a magnetoelastic-ribbon with a polyurethane protecting film. Bacteria consume nutrients from the culture medium in growing and reproducing process, and produces small molecules, with a corresponding change in viscosity of culture medium. The resonant frequency changes of magnetoelastic sensor resulted from the properties changes of a liquid culture medium and bacteria adhesion to the sensor surface. The bacteria concentration can be quantified based on the changes in resonant frequency and amplitude during culture course. Pseudomonas aeruginosa (P. aeruginosa) and Mycobacterium Tuberculosis (M. Tuberculosis) were selected for the analytical objects. Bacteria can be identified from their characteristic growth curve since different microorganisms show different response profiles to their culture medium. The effects of change in culture medium properties and bacteria adhesion on sensor resonant frequency were investigated with quartz crystal microbalance (QCM), microscopy imaging. The drug-resistance on bacteria growth in culture medium was evaluated based on this proposed method. Using the described technique we are able to directly quantify P. aeruginosa and M. Tuberculosis concentrations in the range of 103 to 108 and 104 to 109 cells/mL, and with a detection limit of 103 and 104 cells/mL, respectively.
(5) A wireless, passive magnetoelastic sensing device is presented for the in situ, continuous, and real-time evaluation of the formation of Pseudomonas aeruginosa (P. aeruginosa) biofilms. The polyurethane-coated magnetoelastic-ribbon is used as a transducer for monitoring of P. aeruginosa biofilm formation. In a flowing system, both the resonant frequency and amplitude of the sensor are wirelessly monitored through magnetic field telemetry. Changes in the resonant characteristics of the sensor provide information on the biofilm growth characteristics. The adhesion strength of the biofilm was evaluated by increasing the applied excitation voltage, showing that a tightly attached film was formed.
(1)测定体液pH用无线磁弹性传感器研制:基于pH敏感聚合物,制备了无线磁弹性pH传感器;研究了pH聚合物组成对传感器稳定性和响应灵敏度的影响,考察了体液中干扰物对传感器响应的影响。随溶液pH变化,pH敏感聚合物在碱性溶液中膨胀,酸性溶液中收缩,从而引起传感器表面质量负载和共振频率发生变化。基于此构建的磁弹性pH传感器的共振频率随体液pH升高而下降,在pH 5 ~ 8之间呈近似线性响应,响应灵敏度为200 Hz/pH,可准确测定0.1 pH的变化。对体液pH体外测试表明,制备的pH传感器有望开发为可进行活体在体分析的无线磁弹性传感装置。
(2)测定血糖用无线磁弹性传感器研制:基于上一节磁弹性pH传感器,进一步制备了无线磁弹性葡萄糖生物传感器;研究了传感器对体外血糖测定的稳定性、生物相容性及响应灵敏度。采用pH敏感聚合物膜对酶催化反应进行放大以提高传感器的灵敏度。在葡萄糖氧化酶(GOD)和过氧化氢酶(catalase)的共同存在下,催化氧化葡萄糖生成葡萄糖酸,引起pH聚合物收缩,导致传感器表面质量负载减小、传感器共振频率增加。制备的无线磁弹性葡萄糖传感器的共振频移随血糖浓度的升高而上升,在血糖浓度为2.5 ~ 20 mM之间,传感器的响应是线性、可逆的,对血糖的检测限为1.2 mM。传感器对血糖体外测定结果表明,制备的无线磁弹性葡萄糖生物传感器有望应用于活体在体分析,有潜力作为植入传感器实现对血糖无线监测。
(3)无线磁弹性双酶葡萄糖传感器研制:基于辣根过氧化物酶(HRP)和葡萄糖氧化酶(GOD)双酶层,制备了无线磁弹性双酶葡萄糖生物传感器。利用酶的催化沉淀反应对传感器的响应进行质量放大以提高其灵敏度。葡萄糖氧化酶催化氧化葡萄糖产生葡萄糖酸和过氧化氢(H2O2),产生的过氧化氢在辣根过氧化物酶存在下,催化氧化3,3′,5,5′-四甲基联苯氨(TMB)生成沉淀。产生的沉淀沉积在传感器表面,导致传感器表面质量负载增加,传感器共振频率下降。基于此构建的无线磁弹性双酶葡萄糖传感器的共振频移随葡萄糖浓度的升高而上升,在葡萄糖浓度为5 ~ 50 mM范围内呈线性响应,对葡萄糖的检测限为2 mM。
(4)无线磁弹性微生物传感器研制:以预聚合了聚氨酯膜的磁性膜片为敏感元件,制备了无线磁弹性微生物传感器;研究了微生物生长导致培养基性质(如黏度、密度等)改变对传感器共振频率的影响,考察了细菌在传感器表面吸附对传感器响应的贡献。基于药物对微生物生长的抑制,利用无线磁弹性微生物传感器进行菌株药敏试验研究,依据抗菌药物对细菌的抑制作用可进行抗生素的药效评测。微生物生长时消耗培养基,将大分子的蛋白质分解为小分子导致培养液粘弹性发生变化,同时微生物在传感器表面的吸附引起质量变化。通过测定微生物培养时传感器共振频率和振幅的变化对微生物进行快速检测,获得了绿脓杆菌(P. areuginosa)和结核杆菌(M. tuberculosis)的生长曲线,考察了不同细菌特征生长曲线的差异,据此可望对细菌进行初步的分型。利用石英晶体微天平(QCM)、显微成像等手段研究了培养液性质变化和细菌吸附对传感器共振频率的影响。传感器对绿脓杆菌和结核杆菌检测浓度范围分别为103 ~ 108和104 ~ 109 cells/mL,相应检测限分别为103和104 cells/mL。基于此,研究了可用于细菌早期诊断和快速分析的无线磁弹性传感检测新方法。
(5)无线磁弹性传感器用于细菌生物被膜形成机制的研究。基于绿脓杆菌(P. areuginosa)易于在固体界面粘附且形成细菌生物被膜(biofilms)的特性,利用无线磁弹性传感器初步研究了细菌生物被膜的形成机制。预聚合了聚氨酯膜的磁性膜片作为敏感元件,同时测定了细菌生物被膜形成过程中传感器共振频率和振幅的变化。细菌生物被膜的形成改变了传感器表面的粘弹性和质量负载。由磁弹性传感器共振信号的变化,可探测细菌生物被膜的形成过程及生物被膜的成熟。无线磁弹性传感器是基于磁致伸缩原理设计的,通过改变激励信号的强度,考察了细菌生物被膜在传感器表面的粘附强度,形成的细菌生物被膜在传感器表面粘附牢固。
This dissertation is devoted to the fabrication of wireless magnetoelastic bio/chemical sensors and their applications in bio/chemical analysis. In response to a time-varying AC magnetic field, a magnetoelastic ribbon efficiently couples and converts magnetic energy into mechanical energy. The elastic energy mechanically deforms the sensor, causing it mechanically vibrate along its length. When the frequency of the applied AC magnetic field is equal to the mechanical resonance frequency of the ribbon, the vibration amplitude is maximal and the sensor vibrates at its characteristic resonance frequency that shifts in response to mass loading. Since the magnetoelastic material is magnetostrictive, the vibration of the sensor in turn generates a time varying magnetic flux, which can be remotely measured with a set of pick-up coils. The excitation and transmission of signals of magnetoelastic sensor are carried out remotely by magnetic field. No direct physical connections between the sensor and the detection system are required, nor is any internal power required. The wireless nature of the magnetoelastic sensor makes it a powerful candidate for in situ and in vivo analysis. The details of work are summarized as follows:
(1) A wireless magnetoelastic sensor for determination of body fluid acidity is developed. The magnetoelastic pH sensor was fabricated by coating a layer of pH-sensitive polymer on a magnetostrictive ribbon, which pre-coated with a layer of polyurethane film. The effects of composition of pH-sensitive polymer on sensor stability and sensitivity, and the interferents existing in physiological fluid on sensors responses were investigated. The sensor transduction signal is derived from the pH-polymer mass differences between its relatively shrunken state in acidic solution and relatively swollen state in alkaline solution. The shift in the resonant frequency of magnetoelastic pH sensor is linear and reversible between pH 5 and 8 with a sensitivity of 200 Hz/pH and a measurement resolution of 0.1 pH. The proposed magnetoelastic pH sensor platform offers a great opportunity for developing a useful in vivo and in situ physiological pH measurement technology.
(2) A wireless magnetoelastic glucose biosensor in blood is developed. The glucose biosensor was fabricated based on magnetoelastic pH sensor (pH-polymer used as sensing film). The magnetoelastic biosensor was fabricated by coating the ribbon-like, magnetoelastic sensor with a pH-sensitive polymer and a biolayer of glucose oxidase (GOD) and catalase. pH-sensitive polymer is used as a sensing film to amplify the mass change associated with enzyme biocatalytic reaction and to increase the sensor sensitivity. The GOD-catalyzed oxidation of glucose produces gluconic acid, inducing the pH-responsive polymer to shrink, which in turn decreases the sensor mass loading and increases the resonant frequency. At glucose concentration range of 2.5 ~ 20.0 mM, the biosensor responses are reversible and linear, with a detection limit of 1.2 mM. The proposed magnetoelastic glucose biosensor can potentially be used as a planted sensor and applied to in vivo and in situ measurement of glucose concentrations in body blood.
(3) A wireless magnetoelastic bienzyme glucose biosensor is described. The bienzyme biosensor was fabricated by first coating the magnetoelastic-ribbon with horseradish peroxidase (HRP) and upon it a layer of glucose oxidase (GOD). Sensor sensitivity is increased by amplifying the mass change associated with enzyme reaction by biocatalytic precipitation. The GOD catalyzed oxidation of glucose produces gluconic acid and H2O2, and the generated H2O2 biocatalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to insoluble product in the presence of HRP. The insoluble product accumulated on the sensor surface, which resulted in the increasing in sensor mass loading and decreasing in resonant frequency. And the extent of resulting sensor resonant frequency changing, correlated with the amount of glucose. The biosensor response is linear in the range of glucose concentrations of 5 ~ 50 mM, with a detection limit of 2 mM. The biosensor is applied to determine glucose concentration in urine sample.
(4) A wireless magnetoleastic microorganism sensor is developed for the early diagnosis and rapid detection of bacteria. The microorganism sensor is fabricated by coating a magnetoelastic-ribbon with a polyurethane protecting film. Bacteria consume nutrients from the culture medium in growing and reproducing process, and produces small molecules, with a corresponding change in viscosity of culture medium. The resonant frequency changes of magnetoelastic sensor resulted from the properties changes of a liquid culture medium and bacteria adhesion to the sensor surface. The bacteria concentration can be quantified based on the changes in resonant frequency and amplitude during culture course. Pseudomonas aeruginosa (P. aeruginosa) and Mycobacterium Tuberculosis (M. Tuberculosis) were selected for the analytical objects. Bacteria can be identified from their characteristic growth curve since different microorganisms show different response profiles to their culture medium. The effects of change in culture medium properties and bacteria adhesion on sensor resonant frequency were investigated with quartz crystal microbalance (QCM), microscopy imaging. The drug-resistance on bacteria growth in culture medium was evaluated based on this proposed method. Using the described technique we are able to directly quantify P. aeruginosa and M. Tuberculosis concentrations in the range of 103 to 108 and 104 to 109 cells/mL, and with a detection limit of 103 and 104 cells/mL, respectively.
(5) A wireless, passive magnetoelastic sensing device is presented for the in situ, continuous, and real-time evaluation of the formation of Pseudomonas aeruginosa (P. aeruginosa) biofilms. The polyurethane-coated magnetoelastic-ribbon is used as a transducer for monitoring of P. aeruginosa biofilm formation. In a flowing system, both the resonant frequency and amplitude of the sensor are wirelessly monitored through magnetic field telemetry. Changes in the resonant characteristics of the sensor provide information on the biofilm growth characteristics. The adhesion strength of the biofilm was evaluated by increasing the applied excitation voltage, showing that a tightly attached film was formed.
引文
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