用于呼出气体中肺癌标志物检测的声波传感器及仪器的研究
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摘要
肺癌的发病率及死亡率逐年上升,早发现、早治疗是提高肺癌患者生存率的关键。常规肺癌检测方法不能用于大范围高危人群筛查,急需一种无创、快捷、廉价的肺癌诊断方法。人体呼出气体中的挥发性有机化合物以及呼出气体冷凝物中都存在与肺癌相关的标志物,因此可通过呼吸检测来实现肺癌的无创诊断。
本文在实验室已有的呼出气体肺癌标志物研究的基础上,研制了分别用于呼出气体中挥发性有机化合物检测和呼出气体冷凝物中癌胚抗原检测的声波传感器及分析仪器,并进行了临床试验。本文研究工作得到了浙江省科技厅重大攻关项目和国家自然科学基金仪器专项的资助。本文的主要内容如下：
1.针对呼出气体中的痕量挥发性有机化合物标志物,设计了高灵敏度、低温度系数的谐振型瑞利波气体传感器。该传感器的质量灵敏度达到2.667kHz/ng,传感器的翻转温度为23.36℃,在20℃到40℃范围内,温度灵敏度小于365.9Hz/0C,温度系数小于114ppm/℃;
2.设计并优化了谐振型瑞利波气体传感器的配用电路。在原有振荡电路中增加了移相环节,使传感器工作在中心频率下,提高了振荡电路的输出稳定度及动态响应范围。增加了滤波及衰减等环节,减小了混频输出中频信号中的射频噪声；
3.针对呼出气体冷凝物中的肺癌标志物癌胚抗原检测,设计并对比了钽酸锂与石英两种不同基底的乐甫波传感器。钽酸锂基底传感器在液体介质中衰减小,但质量灵敏度低。石英基底传感器的质量灵敏度达到-1.208°/ng,更适用于呼出气体冷凝物中的癌胚抗原检测；
4.针对两种不同基底乐甫波传感器液相条件下的不同工作特性,设计了两种聚二甲基硅氧烷微流控芯片。提出了微流控芯片与乐甫波传感器之间压合式的结合方法,优化了微流控芯片的通道高度,实现了微流控芯片与乐甫波传感器的多次利用。微流控芯片结合动态进样加快了传感器表而的修饰和检测过程,并实现了实时监测；
5.采用谐振型瑞利波气体传感器结合气相色谱分离技术,实现了呼出气体中肺癌标志性挥发性有机化合物的检测仪器,对十三烷和十三酮的检测下限达到1ppb。采用乐甫波免疫传感器结合微流控技术实现了呼出气体冷凝物中癌胚抗原的检测仪器,对癌胚抗原的检测下限达到了56.44ng/mL；
6.采用呼出气体中挥发性有机化合物的检测仪器测试了89例临床样品,肺癌呼吸检测的灵敏度为95.74%,特异性为69.05%；采用呼出气体冷凝物中癌胚抗原的检测仪器初步测试了6例临床样本,在2例肺癌病人的呼出气体冷凝物样本中检测到了癌胚抗原。
The morbidity and mortality of lung cancer increased year by year, early detection and early treatment is the key to increase the survival rate of lung cancer patients. Conventional lung cancer detection methods can not be used in a wide range of high-risk population screening, so a non-invasive, fast and inexpensive diagnostic method for lung cancer is urgently needed. Volatile organic compounds (VOCs) in the breath gas and the exhaled gas condensate (EBC) contain markers which are associated with lung cancer, so non-invasive diagnosis of lung cancer can be achieved through breath test.
Based on the laboratory's existing research on the exhaled lung cancer markers, two kinds of acoustic sensors and analytical instruments were developed for the detection of VOCs in the exhaled breath and carcino-embryonic antigen (CEA) in EBC, and clinical experiments were carried out. The work of this paper has been funded by Key Scientific and Technological Research Project of Science and Technology Department of Zhejiang Province of China, and the instrument specific project of National Natural Science Foundation.
The major contents of this thesis are listed below:
1) Resonant Rayleigh Wave gas sensor with high sensitivity and low temperature coefficient was designed for the detection of trace VOCs markers in exhaled breath. The mass sensitivity of this sensor is2.667kHz/ng,the turnover temperature is23.36℃, the temperature sensitivity between20℃and40℃is less than365.9Hz/℃, and the temperature coefficient is less than1.4ppm/℃.
2) Circuits for Rayleigh Wave gas sensors were designed and optimized. Phase shifter is added into the oscillator, forcing the working frequency of the sensors close to the center frequency, so the stability and the dynamic range of the oscillation circuit are improved. RF noise in the IF output of mixer is reduced by additional filters and attenuators.
3) Two kinds of Love Wave sensors with different piezoelectric substrate were developed for the detection of CEA in EBC. Sensors based on LiTaO3have little loss in liquid, but the sensitivity is low. The sensors based on quartz are more suitable for detection of CEA in EBC, which have a high mass sensitivity of-1.208°/ng.
4) Two kinds of Polydimethylsiloxane (PDMS) microfluidic chips were designed, because the two kinds of Love Wave sensors have different working characteristics in liquid. A pressure method to combine the love wave sensor with PDMS chip was proposed, realizing reusability of the sensor chips and microfluidic chips. The height of the channel in PDMS chips was optimized. Besides, fast and realtime detection was achieved by dynamic injection mode.
5) Combining resonant Rayleigh Wave gas sensors with gas chromatography (GC), instrument for detection of exhaled VOCs lung cancer markers was developed. The limit of detection (LOD) to tridecane and tridecanone is lower than I ppb. Combining Love Wave immunosensors with microfluidics, instrument for detection of CEA in EBC was developed. The LOD to CEA is56.44ng/mL.
6)89clinical VOCs samples were test by the VOCs detection instrument, the sensitivity is95.74%and selectivity is69.05%;6clinical EBC samples were test by the EBC detection instrument, CEA was detected in EBC samples of two lung cancer patients.
本文在实验室已有的呼出气体肺癌标志物研究的基础上,研制了分别用于呼出气体中挥发性有机化合物检测和呼出气体冷凝物中癌胚抗原检测的声波传感器及分析仪器,并进行了临床试验。本文研究工作得到了浙江省科技厅重大攻关项目和国家自然科学基金仪器专项的资助。本文的主要内容如下：
1.针对呼出气体中的痕量挥发性有机化合物标志物,设计了高灵敏度、低温度系数的谐振型瑞利波气体传感器。该传感器的质量灵敏度达到2.667kHz/ng,传感器的翻转温度为23.36℃,在20℃到40℃范围内,温度灵敏度小于365.9Hz/0C,温度系数小于114ppm/℃;
2.设计并优化了谐振型瑞利波气体传感器的配用电路。在原有振荡电路中增加了移相环节,使传感器工作在中心频率下,提高了振荡电路的输出稳定度及动态响应范围。增加了滤波及衰减等环节,减小了混频输出中频信号中的射频噪声；
3.针对呼出气体冷凝物中的肺癌标志物癌胚抗原检测,设计并对比了钽酸锂与石英两种不同基底的乐甫波传感器。钽酸锂基底传感器在液体介质中衰减小,但质量灵敏度低。石英基底传感器的质量灵敏度达到-1.208°/ng,更适用于呼出气体冷凝物中的癌胚抗原检测；
4.针对两种不同基底乐甫波传感器液相条件下的不同工作特性,设计了两种聚二甲基硅氧烷微流控芯片。提出了微流控芯片与乐甫波传感器之间压合式的结合方法,优化了微流控芯片的通道高度,实现了微流控芯片与乐甫波传感器的多次利用。微流控芯片结合动态进样加快了传感器表而的修饰和检测过程,并实现了实时监测；
5.采用谐振型瑞利波气体传感器结合气相色谱分离技术,实现了呼出气体中肺癌标志性挥发性有机化合物的检测仪器,对十三烷和十三酮的检测下限达到1ppb。采用乐甫波免疫传感器结合微流控技术实现了呼出气体冷凝物中癌胚抗原的检测仪器,对癌胚抗原的检测下限达到了56.44ng/mL；
6.采用呼出气体中挥发性有机化合物的检测仪器测试了89例临床样品,肺癌呼吸检测的灵敏度为95.74%,特异性为69.05%；采用呼出气体冷凝物中癌胚抗原的检测仪器初步测试了6例临床样本,在2例肺癌病人的呼出气体冷凝物样本中检测到了癌胚抗原。
The morbidity and mortality of lung cancer increased year by year, early detection and early treatment is the key to increase the survival rate of lung cancer patients. Conventional lung cancer detection methods can not be used in a wide range of high-risk population screening, so a non-invasive, fast and inexpensive diagnostic method for lung cancer is urgently needed. Volatile organic compounds (VOCs) in the breath gas and the exhaled gas condensate (EBC) contain markers which are associated with lung cancer, so non-invasive diagnosis of lung cancer can be achieved through breath test.
Based on the laboratory's existing research on the exhaled lung cancer markers, two kinds of acoustic sensors and analytical instruments were developed for the detection of VOCs in the exhaled breath and carcino-embryonic antigen (CEA) in EBC, and clinical experiments were carried out. The work of this paper has been funded by Key Scientific and Technological Research Project of Science and Technology Department of Zhejiang Province of China, and the instrument specific project of National Natural Science Foundation.
The major contents of this thesis are listed below:
1) Resonant Rayleigh Wave gas sensor with high sensitivity and low temperature coefficient was designed for the detection of trace VOCs markers in exhaled breath. The mass sensitivity of this sensor is2.667kHz/ng,the turnover temperature is23.36℃, the temperature sensitivity between20℃and40℃is less than365.9Hz/℃, and the temperature coefficient is less than1.4ppm/℃.
2) Circuits for Rayleigh Wave gas sensors were designed and optimized. Phase shifter is added into the oscillator, forcing the working frequency of the sensors close to the center frequency, so the stability and the dynamic range of the oscillation circuit are improved. RF noise in the IF output of mixer is reduced by additional filters and attenuators.
3) Two kinds of Love Wave sensors with different piezoelectric substrate were developed for the detection of CEA in EBC. Sensors based on LiTaO3have little loss in liquid, but the sensitivity is low. The sensors based on quartz are more suitable for detection of CEA in EBC, which have a high mass sensitivity of-1.208°/ng.
4) Two kinds of Polydimethylsiloxane (PDMS) microfluidic chips were designed, because the two kinds of Love Wave sensors have different working characteristics in liquid. A pressure method to combine the love wave sensor with PDMS chip was proposed, realizing reusability of the sensor chips and microfluidic chips. The height of the channel in PDMS chips was optimized. Besides, fast and realtime detection was achieved by dynamic injection mode.
5) Combining resonant Rayleigh Wave gas sensors with gas chromatography (GC), instrument for detection of exhaled VOCs lung cancer markers was developed. The limit of detection (LOD) to tridecane and tridecanone is lower than I ppb. Combining Love Wave immunosensors with microfluidics, instrument for detection of CEA in EBC was developed. The LOD to CEA is56.44ng/mL.
6)89clinical VOCs samples were test by the VOCs detection instrument, the sensitivity is95.74%and selectivity is69.05%;6clinical EBC samples were test by the EBC detection instrument, CEA was detected in EBC samples of two lung cancer patients.
引文
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90. https://dspace.lib.cranfield.ac.uk/handle/1826/7861
91. http://www.lgpump.com.cn/product/product628.html
92. http://www.saw-instruments.de
93. http://www.anhuisafe.com/Show.asp?id=214
94. http://www.estcal.com
95.陈琳,非小细胞肺癌患者呼出气冷凝液CEA、ET-1测定的意义,复旦大学硕士学位论文,2009
96.董敬军,陶一江,陈建荣,蔡映云,陶国华,非小细胞肺癌患者呼出气冷凝液中CEA检测的临床意义,临床肺科杂志,2008,13(7)：828-830
97.马航,肺癌治疗前后呼出气冷凝液和血浆中CEA、ET-1水平观察,交通医学,2009,23(6)：656-657
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